Robin Ketteler

Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3K27M and Trp53 loss alone are sufficient for neoplastic transformation if introduced in utero. H3.3K27M-driven lesions are clonal, H3K27me3 depleted, Olig2 positive, highly proliferative, and diffusely spreading, thus recapitulating hallmark molecular and histopathological features of pHGG. Addition of wild-type PDGFRA decreases latency and increases tumor invasion, while ATRX knockdown is associated with more circumscribed tumors. H3.3K27M-tumor cells serially engraft in recipient mice, and preliminary drug screening reveals mutation-specific vulnerabilities. Overall, we provide a faithful H3.3K27M-pHGG model which enables insights into oncohistone pathogenesis and investigation of future therapies.

Macroautophagy/autophagy is a cellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane organelles called autophagosomes. Lipidation of ubiquitin-like LC3/GABARAP proteins on the autophagosome membrane is important for autophagy. The cysteine protease ATG4 executes 2 LC3/GABARAP processing events: priming of newly synthesized pro-LC3/GABARAP to enable subsequent lipidation, and delipidation/deconjugation of lipidated LC3/GABARAP (the exact purpose of which is unclear in mammals). Four ATG4 isoforms (ATG4A to ATG4D) exist in mammals; however, the functional redundancy of these proteins in cells is poorly understood. Here we show that human HAP1 and HeLa cells lacking ATG4B exhibit a severe but incomplete defect in LC3/GABARAP processing and autophagy. By further genetic depletion of ATG4 isoforms using CRISPR-Cas9 and siRNA we uncover that ATG4A, ATG4C and ATGD all contribute to residual priming activity, which is sufficient to enable lipidation of endogenous GABARAPL1 on autophagic structures. We also demonstrate that expressing high levels of pre-primed LC3B in ATG4-deficient cells can rescue a defect in autophagic degradation of the cargo receptor SQSTM1/p62, suggesting that delipidation by human ATG4 is not essential for autophagosome formation and fusion with lysosomes. Overall, our study provides a comprehensive characterization of ATG4 isoform function during autophagy in human cells. Abbreviations: Atg: autophagy-related; baf A1: bafilomycin A1; CASP3: caspase 3; CLEM: correlative light and electron microscopy; CMV: cytomegalovirus; CRISPR: clustered regularly interspaced short palindromic repeats; DKO: double knockout; EGFP: enhanced green fluorescent protein; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor-associated protein like 1; GABARAPL2: GABA type A receptor-associated protein like 2; GFP: green fluorescent protein; HB: homogenization buffer; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LIR: LC3 interacting region; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN2: mitofusin 2; N.A.: numerical aperture; NEM: N-ethylmaleimide; PDHA1: pyruvate dehydrogenase E1 alpha 1 subunit; PLD: phospholipase D; PE: phosphatidylethanolamine; RLUC: Renilla luciferase; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TKO: triple knockout; ULK1: unc-51 like autophagy activating kinase 1; VCL: vinculin; WT: wild-type.

Upon induction of autophagy, the ubiquitin-like protein LC3 is conjugated to phosphatidylethanolamine (PE) on the inner and outer membrane of autophagosomes to allow cargo selection and autophagosome formation. LC3 undergoes two processing steps, the proteolytic cleavage of pro-LC3 and the de-lipidation of LC3-PE from autophagosomes, both executed by the same cysteine protease ATG4. How ATG4 activity is regulated to co-ordinate these events is currently unknown. Here we find that ULK1, a protein kinase activated at the autophagosome formation site, phosphorylates human ATG4B on serine 316. Phosphorylation at this residue results in inhibition of its catalytic activity in vitro and in vivo. On the other hand, phosphatase PP2A-PP2R3B can remove this inhibitory phosphorylation. We propose that the opposing activities of ULK1-mediated phosphorylation and PP2A-mediated dephosphorylation provide a phospho-switch that regulates the cellular activity of ATG4B to control LC3 processing.Upon autophagy induction, LC3 is cleaved by the protease ATG4 and conjugated to the autophagosomal membrane; however, its removal is mediated by the same protease. Here the authors show that ULK1-mediated phosphorylation and PP2A-mediated dephosphorylation of ATG4 regulates its cellular activity to control LC3 processing.

Llamas (Lama glama) naturally produce heavy chain-only antibodies (Abs) in addition to conventional Abs. The variable regions (VHH) in these heavy chain-only Abs demonstrate comparable affinity and specificity for antigens to conventional immunoglobulins despite their much smaller size. To date, immunizations in humans and animal models have yielded only Abs with limited ability to neutralize HIV-1. In this study, a VHH phagemid library generated from a llama that was multiply immunized with recombinant trimeric HIV-1 envelope proteins (Envs) was screened directly for HIV-1 neutralization. One VHH, L8CJ3 (J3), neutralized 96 of 100 tested HIV-1 strains, encompassing subtypes A, B, C, D, BC, AE, AG, AC, ACD, CD, and G. J3 also potently neutralized chimeric simian-HIV strains with HIV subtypes B and C Env. The sequence of J3 is highly divergent from previous anti-HIV-1 VHH and its own germline sequence. J3 achieves broad and potent neutralization of HIV-1 via interaction with the CD4-binding site of HIV-1 Env. This study may represent a new benchmark for immunogens to be included in B cell-based vaccines and supports the development of VHH as anti-HIV-1 microbicides.

CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.

<h2>Summary</h2> Weibel-Palade bodies (WPBs), endothelial-specific secretory granules that are central to primary hemostasis and inflammation, occur in dimensions ranging between 0.5 and 5 μm. How their size is determined and whether it has a functional relevance are at present unknown. Here, we provide evidence for a dual role of the Golgi apparatus in controlling the size of these secretory carriers. At the ministack level, cisternae constrain the size of nanostructures ("quanta") of von Willebrand factor (vWF), the main WPB cargo. The ribbon architecture of the Golgi then allows copackaging of a variable number of vWF quanta within the continuous lumen of the <i>trans</i>-Golgi network, thereby generating organelles of different sizes. Reducing the WPB size abates endothelial cell hemostatic function by drastically diminishing platelet recruitment, but, strikingly, the inflammatory response (the endothelial capacity to engage leukocytes) is unaltered. Size can thus confer functional plasticity to an organelle by differentially affecting its activities.

Mitochondrial quality control is essential for maintaining a healthy population of mitochondria. Two proteins associated with Parkinson disease, the kinase PINK1 and the E3 ubiquitin ligase PRKN, play a central role in the selective degradation of heavily damaged mitochondria (mitophagy), thus avoiding their toxic accumulation. Most of the knowledge on PINK1-PRKN mitophagy comes from in vitro experiments involving the treatment of mammalian cells with high concentrations of mitochondrial uncouplers, such as CCCP. These chemicals have been shown to mediate off target effects, other than mitochondrial depolarization. A matter of controversy between mitochondrial physiologists and cell biologists is the discrepancy between concentrations of CCCP needed to activate mitophagy (usually >10 μM), when compared to the much lower concentrations used to depolarize mitochondria (<1 μM). Thus, there is an urgent need for optimizing the current methods to assess PINK1-PRKN mitophagy in vitro. In this study, we address the utilization of high CCCP concentrations commonly used to activate mitophagy. Combining live fluorescence microscopy and biochemistry, we show that the FBS/BSA in the cell culture medium reduces the ability of CCCP to induce PINK1 accumulation at depolarized mitochondria, subsequent PRKN recruitment and ubiquitin phosphorylation, and ultimately mitochondrial clearance. As a result, high concentrations of CCCP are required to induce mitophagy in FBS/BSA containing media. These data unite mitochondrial physiology and mitophagy studies and are a first step toward a consensus on optimal experimental conditions for PINK1-PRKN mitophagy and mitochondrial physiology investigations to be carried out in parallel.Abbreviations: BSA: bovine serum albumin; CCCP: carbonyl cyanide m-chlorophenylhydrazone; DMEM: dulbecco’s Modified Eagle’s Medium; DNP: 2,4-dinitrophenol; FBS: fetal bovine serum; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GSH: glutathione; HBSS: Hanks‘ balanced salt solution; mtKeima: mitochondria-targeted monomeric keima-red; PBS: phosphate buffered saline; PD: Parkinson disease; PINK1: PTEN induced kinase 1; POE SHSY5Ys: FLAG-PRKN over-expressing SHSY5Y cells; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; TMRM: tetramethylrhodamine methyl ester; WB: western blot; WT: wild-type; ΔΨm: mitochondrial membrane potential

Research in signal transduction aims to identify the functions of different signaling pathways in physiological and pathological states. Traditional techniques using biochemical, genetic or cell biological approaches have made important contributions to our understanding of cellular signaling. However, the single-gene approach does not take into account the full complexity of cell signaling. With the availability of omics techniques, great progress has been made in understanding signaling networks. Omics approaches can be classified into two categories: 'molecular profiling', including genomic, proteomic, post-translational modification and interactome profiling; and 'molecular perturbation', including genetic and functional perturbations.

Mitochondrial dysfunction is implicated in many neurodegenerative diseases including Parkinson's disease (PD). Induced pluripotent stem cells (iPSCs) provide a unique cell model for studying neurological diseases. We have established a high-content assay that can simultaneously measure mitochondrial function, morphology and cell viability in iPSC-derived dopaminergic neurons. iPSCs from PD patients with mutations in SNCA and unaffected controls were differentiated into dopaminergic neurons, seeded in 384-well plates and stained with the mitochondrial membrane potential dependent dye TMRM, alongside Hoechst-33342 and Calcein-AM. Images were acquired using an automated confocal screening microscope and single cells were analysed using automated image analysis software. PD neurons displayed reduced mitochondrial membrane potential and altered mitochondrial morphology compared to control neurons. This assay demonstrates that high content screening techniques can be applied to the analysis of mitochondria in iPSC-derived neurons. This technique could form part of a drug discovery platform to test potential new therapeutics for PD and other neurodegenerative diseases.

Viruses are a major threat to human health and economic well-being. In recent years Ebola, Zika, influenza, and chikungunya virus epidemics have raised awareness that infections can spread rapidly before vaccines or specific antagonists can be made available. Broad-spectrum antivirals are drugs with the potential to inhibit infection by viruses from different groups or families, which may be deployed during outbreaks when specific diagnostics, vaccines or directly acting antivirals are not available. While pathogen-directed approaches are generally effective against a few closely related viruses, targeting cellular pathways used by multiple viral agents can have broad-spectrum efficacy. Virus entry, particularly clathrin-mediated endocytosis, constitutes an attractive target as it is used by many viruses. Using a phenotypic screening strategy where the inhibitory activity of small molecules was sequentially tested against different viruses, we identified 12 compounds with broad-spectrum activity, and found a subset blocking viral internalisation and/or fusion. Importantly, we show that compounds identified with this approach can reduce viral replication in a mouse model of Zika infection. This work provides proof of concept that it is possible to identify broad-spectrum inhibitors by iterative phenotypic screenings, and that inhibition of host-pathways critical for viral life cycles can be an effective antiviral strategy.

Autophagy is a major cellular recycling process that delivers cellular material and entire organelles to lysosomes for degradation, in a selective or non-selective manner. This process is essential for the maintenance of cellular energy levels, components and metabolites, as well as the elimination of cellular molecular damage, thereby playing an important role in numerous cellular activities. An important function of autophagy is to enable survival under starvation conditions and other stresses. The majority of factors implicated in ageing are modifiable through the process of autophagy, including the accumulation of oxidative damage and loss of proteostasis, genomic instability and epigenomic alteration. These primary causes of damage could lead to mitochondrial dysfunction, deregulation of nutrient sensing pathways and cellular senescence, finally causing a variety of ageing phenotypes. Remarkably, advances in the biology of ageing have revealed that ageing is a malleable process: a mild decrease in signalling through nutrient-sensing pathways can improve health and extend lifespan in all model organisms tested. Consequently, autophagy is implicated in both ageing and age-related disease. Enhancement of the autophagy process is a common characteristic of all principal, evolutionary conserved anti-ageing interventions, including dietary restriction, as well as inhibition of target of rapamycin (TOR) and insulin/IGF-1 signalling (IIS). As an emerging and critical process in ageing, this review will highlight how autophagy can be modulated for health improvement.

The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ entry, plays important roles in physiology and pathology. However, only few pharmacological compounds directly and selectively modulate its activity. Here, we perform high-throughput screening on a US Food and Drug Administration (FDA)-approved drug library comprising 1,600 compounds to identify molecules modulating mitochondrial Ca2+ uptake. We find amorolfine and benzethonium to be positive and negative MCU modulators, respectively. In agreement with the positive effect of MCU in muscle trophism, amorolfine increases muscle size, and MCU silencing is sufficient to blunt amorolfine-induced hypertrophy. Conversely, in the triple-negative breast cancer cell line MDA-MB-231, benzethonium delays cell growth and migration in an MCU-dependent manner and protects from ceramide-induced apoptosis, in line with the role of mitochondrial Ca2+ uptake in cancer progression. Overall, we identify amorolfine and benzethonium as effective MCU-targeting drugs applicable to a wide array of experimental and disease conditions.

Genetic and pharmacological inhibition of mitochondrial fission induced by acute myocardial ischaemia/reperfusion injury (IRI) has been shown to reduce myocardial infarct size. The clinically used anti-hypertensive and heart failure medication, hydralazine, is known to have anti-oxidant and anti-apoptotic effects. Here, we investigated whether hydralazine confers acute cardioprotection by inhibiting Drp1-mediated mitochondrial fission.Pre-treatment with hydralazine was shown to inhibit both mitochondrial fission and mitochondrial membrane depolarisation induced by oxidative stress in HeLa cells. In mouse embryonic fibroblasts (MEFs), pre-treatment with hydralazine attenuated mitochondrial fission and cell death induced by oxidative stress, but this effect was absent in MEFs deficient in the mitochondrial fission protein, Drp1. Molecular docking and surface plasmon resonance studies demonstrated binding of hydralazine to the GTPase domain of the mitochondrial fission protein, Drp1 (KD 8.6±1.0 µM), and inhibition of Drp1 GTPase activity in a dose-dependent manner. In isolated adult murine cardiomyocytes subjected to simulated IRI, hydralazine inhibited mitochondrial fission, preserved mitochondrial fusion events, and reduced cardiomyocyte death (hydralazine 24.7±2.5% vs. control 34.1±1.5%, P=0.0012). In ex vivo perfused murine hearts subjected to acute IRI, pre-treatment with hydralazine reduced myocardial infarct size (as % left ventricle: hydralazine 29.6±6.5% vs. vehicle control 54.1±4.9%, P=0.0083), and in the murine heart subjected to in vivo IRI, the administration of hydralazine at reperfusion, decreased myocardial infarct size (as % area-at-risk: hydralazine 28.9±3.0% vs. vehicle control 58.2±3.8%, P<0.001).We show that, in addition to its antioxidant and anti-apoptotic effects, hydralazine, confers acute cardioprotection by inhibiting IRI-induced mitochondrial fission, raising the possibility of repurposing hydralazine as a novel cardioprotective therapy for improving post-infarction outcomes.

Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism, probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterisation of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 on the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared with wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.

<h2>Abstract</h2> Hematopoietic cytokine receptors, such as the erythropoietin receptor (EpoR), are single membrane-spanning proteins. Signal transduction through EpoR is crucial for the formation of mature erythrocytes [1]. Structural evidence shows that in the unliganded form EpoR exists as a preformed homodimer in an open scissor–like conformation [2] precluding the activation of signaling. In contrast to the extracellular domain of the growth hormone receptor (GHR) [3], the structure of the agonist-bound EpoR extracellular region shows only minimal contacts between the membrane-proximal regions [4]. This evidence suggests that the domains facilitating receptor dimerization may differ between cytokine receptors. We show that the EpoR transmembrane domain (TM) has a strong potential to self interact in a bacterial reporter system. Abolishing self assembly of the EpoR TM by a double point mutation (Leu 240–Leu 241 mutated to Gly-Pro) impairs signal transduction by EpoR in hematopoietic cells and the formation of erythroid colonies upon reconstitution in erythroid progenitor cells from EpoR^−/− mice. Interestingly, inhibiting TM self assembly in the constitutively active mutant EpoR R129C abrogates formation of disulfide-linked receptor homodimers and consequently results in the loss of ligand-independent signal transduction. Thus, efficient signal transduction through EpoR and possibly other preformed receptor oligomers may be determined by the dynamics of TM self assembly.

Frameshifting results from two main mechanisms: genomic insertions or deletions (indels) or programmed ribosomal frameshifting. Whereas indels can disrupt normal protein function, programmed ribosomal frameshifting can result in dual-coding genes, each of which can produce multiple functional products. Here, I summarize technical advances that have made it possible to identify programmed ribosomal frameshifting events in a systematic way. The results of these studies suggest that such frameshifting occurs in all genomes, and I will discuss methods that could help characterize the resulting alternative proteomes.

Cardiomyocytes from human embryonic stem cells (hESC-CMs) and induced pluripotent stem cells (hiPSC-CMs) represent new models for drug discovery. Although hypertrophy is a high-priority target, we found that hiPSC-CMs were systematically unresponsive to hypertrophic signals such as the α-adrenoceptor (αAR) agonist phenylephrine (PE) compared to hESC-CMs. We investigated signaling at multiple levels to understand the underlying mechanism of this differential responsiveness. The expression of the normal α1AR gene, ADRA1A, was reversibly silenced during differentiation, accompanied by ADRA1B upregulation in either cell type. ADRA1B signaling was intact in hESC-CMs, but not in hiPSC-CMs. We observed an increased tonic activity of inhibitory kinase pathways in hiPSC-CMs, and inhibition of antihypertrophic kinases revealed hypertrophic increases. There is tonic suppression of cell growth in hiPSC-CMs, but not hESC-CMs, limiting their use in investigation of hypertrophic signaling. These data raise questions regarding the hiPSC-CM as a valid model for certain aspects of cardiac disease.

We are increasingly accumulating molecular data about a cell. The challenge is how to integrate them within a unified conceptual and computational framework enabling new discoveries. Hence, we propose a novel, data-driven concept of an integrated cell, iCell. Also, we introduce a computational prototype of an iCell, which integrates three omics, tissue-specific molecular interaction network types. We construct iCells of four cancers and the corresponding tissue controls and identify the most rewired genes in cancer. Many of them are of unknown function and cannot be identified as different in cancer in any specific molecular network. We biologically validate that they have a role in cancer by knockdown experiments followed by cell viability assays. We find additional support through Kaplan-Meier survival curves of thousands of patients. Finally, we extend this analysis to uncover pan-cancer genes. Our methodology is universal and enables integrative comparisons of diverse omics data over cells and tissues.

Induced pluripotent stem cells and their derivatives have become an important tool for researching disease mechanisms. It is hoped that they could be used to discover new therapies by providing the most reliable and relevant human in vitro disease models for drug discovery. This review will summarize recent efforts to use stem cell–derived neurons for drug screening. We also explain the current hurdles to using these cells for high-throughput pharmaceutical screening and developments that may help overcome these hurdles. Finally, we critically discuss whether induced pluripotent stem cell–derived neurons will come to fruition as a model that is regularly used to screen for drugs to treat neurological diseases.

Microtubule-associated protein 1 light chain 3 α (LC3)/GABA type A receptor-associated protein (GABARAP) comprises a family of ubiquitin-like proteins involved in (macro)autophagy, an important intracellular degradation pathway that delivers cytoplasmic material to lysosomes via double-membrane vesicles called autophagosomes. The only currently known cellular molecules covalently modified by LC3/GABARAP are membrane phospholipids such as phosphatidylethanolamine in the autophagosome membrane. Autophagy-related 4 cysteine peptidase (ATG4) proteases process inactive pro-LC3/GABARAP before lipidation, and the same proteases can also deconjugate LC3/GABARAP from lipids. To determine whether LC3/GABARAP has other molecular targets, here we generated a pre-processed LC3B mutant (Q116P) that is resistant to ATG4-mediated deconjugation. Upon expression in human cells and when assessed by immunoblotting under reducing and denaturing conditions, deconjugation-resistant LC3B accumulated in multiple forms and at much higher molecular weights than free LC3B. We observed a similar accumulation when pre-processed versions of all mammalian LC3/GABARAP isoforms were expressed in ATG4-deficient cell lines, suggesting that LC3/GABARAP can attach also to other larger molecules. We identified ATG3, the E2-like enzyme involved in LC3/GABARAP lipidation, as one target of conjugation with multiple copies of LC3/GABARAP. We show that LC3B-ATG3 conjugates are distinct from the LC3B-ATG3 thioester intermediate formed before lipidation, and we biochemically demonstrate that ATG4B can cleave LC3B-ATG3 conjugates. Finally, we determined ATG3 residue Lys-243 as an LC3B modification site. Overall, we provide the first cellular evidence that mammalian LC3/GABARAP post-translationally modifies proteins akin to ubiquitination ("LC3ylation"), with ATG4 proteases acting like deubiquitinating enzymes to counteract this modification ("deLC3ylation").

Loss of telomeric repeats has been causally linked to replicative senescence and aging in human cells. In contrast to normal somatic cells, which are telomerase‐negative, hematopoietic stem cells have low levels of telomerase, which can be transiently upregulated upon cytokine stimulation. To examine whether ectopic expression of telomerase can overcome telomere erosion in hematopoietic progenitor cells, we overexpressed telomerase in CD34+ and AC133+ cord blood (CB) cells using retroviral vectors containing hTERT, the catalytic component of telomerase. Although the hTERT‐transduced CB cells exhibited significantly elevated telomerase activity (approximately 10‐fold), the mean telomere length was only increased up to 600 bp, which was in contrast to hTERT‐transduced fibroblast cells gaining more than 2‐kb telomeric repeats. Moreover, ectopic telomerase activity did not prevent overall telomere shortening, which was in the range of 1.3 kb in serum‐free expansion culture. We also blocked endogenous telomerase activity by ectopic expression of dominant‐negative hTERT. Whereas CB cells with absent telomerase activity showed reduced absolute numbers of colony‐forming cells, we observed increased rates only for burst‐forming units erythroid when the enzyme was overexpressed. These results suggest that telomere shortening in human hematopoietic progenitor cells cannot be compensated by increased levels of telomerase alone and is likely to be dependent on other factors, such as telomere binding proteins. Furthermore, telomerase function seems to be directly associated with the proliferative capacity of stem cells and may exert an additional role in lineage differentiation.

Changes in the size of cellular organelles are often linked to modifications in their function. Endothelial cells store von Willebrand Factor (vWF), a glycoprotein essential to haemostasis in Weibel-Palade bodies (WPBs), cigar-shaped secretory granules that are generated in a wide range of sizes. We recently showed that forcing changes in the size of WPBs modifies the activity of this cargo. We now find that endothelial cells treated with statins produce shorter WPBs and that the vWF they release at exocytosis displays a reduced capability to recruit platelets to the endothelial cell surface. Investigating other functional consequences of size changes of WPBs, we also report that the endothelial surface-associated vWF formed at exocytosis recruits soluble plasma vWF and that this process is reduced by treatments that shorten WPBs, statins included. These results indicate that the post-exocytic adhesive activity of vWF towards platelets and plasma vWF at the endothelial surface reflects the size of their storage organelle. Our findings therefore show that changes in WPB size, by influencing the adhesive activity of its vWF cargo, may represent a novel mode of regulation of platelet aggregation at the vascular wall.

<h2>Summary</h2> The canine transmissible venereal tumor (CTVT) is a clonally transmissible cancer that regresses spontaneously or after treatment with vincristine, but we know little about the regression mechanisms. We performed global transcriptional, methylation, and functional pathway analyses on serial biopsies of vincristine-treated CTVTs and found that regression occurs in sequential steps; activation of the innate immune system and host epithelial tissue remodeling followed by immune infiltration of the tumor, arrest in the cell cycle, and repair of tissue damage. We identified <i>CCL5</i> as a possible driver of CTVT regression. Changes in gene expression are associated with methylation changes at specific intragenic sites. Our results underscore the critical role of host innate immunity in triggering cancer regression.

The discovery of RNA interference (RNAi) has enabled several breakthrough discoveries in the area of functional genomics. The RNAi technology has emerged as one of the major tools for drug target identification and has been steadily improved to allow gene manipulation in cell lines, tissues, and whole organisms. One of the major hurdles for the use of RNAi in high-throughput screening has been delivery to cells and tissues. Some cell types are refractory to high-efficiency transfection with standard methods such as lipofection or calcium phosphate precipitation and require different means. Electroporation is a powerful and versatile method for delivery of RNA, DNA, peptides, and small molecules into cell lines and primary cells, as well as whole tissues and organisms. Of particular interest is the use of electroporation for delivery of small interfering RNA oligonucleotides and clustered regularly interspaced short palindromic repeats/Cas9 plasmid vectors in high-throughput screening and for therapeutic applications. Here, we will review the use of electroporation in high-throughput screening in cell lines and tissues.

Autophagy is a cellular process that has been defined and analyzed almost entirely by qualitative measures. In no small part, this is attributable to the absence of robust quantitative assays that can easily and reliably permit the progress of key steps in autophagy to be assessed. We have recently developed a cell-based assay that specifically measures proteolytic cleavage of a tripartite sensor protein by the autophagy protease ATG4B. Activation of ATG4B results in release of Gaussia luciferase from cells that can be non-invasively harvested from cellular supernatants. Here, we compare this technique to existing methods and propose that this type of assay will be suitable for genome-wide functional screens and in vivo analysis of autophagy.

Manipulation of gene expression on a genome-wide level is one of the most important systematic tools in the post-genome era. Such manipulations have largely been enabled by expression cloning approaches using sequence-verified cDNA libraries, large-scale RNA interference libraries (shRNA or siRNA) and zinc finger nuclease technologies. More recently, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas)9-mediated gene editing technology has been described that holds great promise for future use of this technology in genomic manipulation. It was suggested that the CRISPR system has the potential to be used in high-throughput, large-scale loss of function screening. Here we discuss some of the challenges in engineering of CRISPR/Cas genomic libraries and some of the aspects that need to be addressed in order to use this technology on a high-throughput scale.

Deregulation of cyclin-dependent kinases 4 and 6 (CDK4/6) is highly prevalent in cancer; yet, inhibitors against these kinases are currently used only in restricted tumour contexts. The extent to which cancers depend on CDK4/6 and the mechanisms that may undermine such dependency are poorly understood. Here, we report that signalling engaging the MET proto-oncogene receptor tyrosine kinase/focal adhesion kinase (FAK) axis leads to CDK4/6-independent CDK2 activation, involving as critical mechanistic events loss of the CDKI p21CIP1 and gain of its regulator, the ubiquitin ligase subunit SKP2. Combined inhibition of MET/FAK and CDK4/6 eliminates the proliferation capacity of cancer cells in culture, and enhances tumour growth inhibition in vivo. Activation of the MET/FAK axis is known to arise through cancer extrinsic and intrinsic cues. Our work predicts that such cues support cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases and identifies MET/FAK as a tractable route to broaden the utility of CDK4/6 inhibitor-based therapies in the clinic.

HIV-1 integrates more frequently into transcribed genes, however the biological significance of HIV-1 integration targeting has remained elusive. Using a selective high-throughput chemical screen, we discovered that the cardiac glycoside digoxin inhibits wild-type HIV-1 infection more potently than HIV-1 bearing a single point mutation (N74D) in the capsid protein. We confirmed that digoxin repressed viral gene expression by targeting the cellular Na+/K+ ATPase, but this did not explain its selectivity. Parallel RNAseq and integration mapping in infected cells demonstrated that digoxin inhibited expression of genes involved in T-cell activation and cell metabolism. Analysis of >400,000 unique integration sites showed that WT virus integrated more frequently than N74D mutant within or near genes susceptible to repression by digoxin and involved in T-cell activation and cell metabolism. Two main gene networks down-regulated by the drug were CD40L and CD38. Blocking CD40L by neutralizing antibodies selectively inhibited WT virus infection, phenocopying digoxin. Thus the selectivity of digoxin depends on a combination of integration targeting and repression of specific gene networks. The drug unmasked a functional connection between HIV-1 integration and T-cell activation. Our results suggest that HIV-1 evolved integration site selection to couple its early gene expression with the status of target CD4+ T-cells, which may affect latency and viral reactivation.

The formation of myelinating Schwann cells (mSCs) involves the remarkable biogenic process, which rapidly generates the myelin sheath. Once formed, the mSC transitions to a stable homeostatic state, with loss of this stability associated with neuropathies. The histone deacetylases histone deacetylase 1 (HDAC1) and HDAC2 are required for the myelination transcriptional program. Here, we show a distinct role for HDAC3, in that, while dispensable for the formation of mSCs, it is essential for the stability of the myelin sheath once formed—with loss resulting in progressive severe neuropathy in adulthood. This is associated with the prior failure to downregulate the biogenic program upon entering the homeostatic state leading to hypertrophy and hypermyelination of the mSCs, progressing to the development of severe myelination defects. Our results highlight distinct roles of HDAC1/2 and HDAC3 in controlling the differentiation and homeostatic states of a cell with broad implications for the understanding of this important cell-state transition.

Robust cellular models are key in determining pathological mechanisms that lead to neurotoxicity in Huntington's disease (HD) and for high throughput pre-clinical screening of potential therapeutic compounds. Such models exist but mostly comprise non-human or non-neuronal cells that may not recapitulate the correct biochemical milieu involved in pathology. We have developed a new human neuronal cell model of HD, using neural stem cells (ReNcell VM NSCs) stably transduced to express exon 1 huntingtin (HTT) fragments with variable length polyglutamine (polyQ) tracts. Using a system with matched expression levels of exon 1 HTT fragments, we investigated the effect of increasing polyQ repeat length on HTT inclusion formation, location, neuronal survival, and mitochondrial function with a view to creating an in vitro screening platform for therapeutic screening. We found that expression of exon 1 HTT fragments with longer polyQ tracts led to the formation of intra-nuclear inclusions in a polyQ length-dependent manner during neurogenesis. There was no overt effect on neuronal viability, but defects of mitochondrial function were found in the pathogenic lines. Thus, we have a human neuronal cell model of HD that may recapitulate some of the earliest stages of HD pathogenesis, namely inclusion formation and mitochondrial dysfunction.

Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies followed by ciliated differentiation at the air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique's broader utility, including in pre-clinical PCD research, has been restricted by the limited number of basal cells that can be expanded from such biopsies.We describe an immunofluorescence screening method, enabled by extensive expansion of basal cells from PCD patients and the directed differentiation of these cells into ciliated epithelium in miniaturised 96-well transwell format ALI cultures. As proof-of-principle, we performed a personalised investigation in a patient with a rare and severe form of PCD (reduced generation of motile cilia), in this case caused by a homozygous nonsense mutation in the MCIDAS gene.Initial analyses of ciliary ultrastructure, beat pattern and beat frequency in the 96-well transwell format ALI cultures indicate that a range of different PCD defects can be retained in these cultures. The screening system in our proof-of-principal investigation allowed drugs that induce translational readthrough to be evaluated alone or in combination with nonsense-mediated decay inhibitors. We observed restoration of basal body formation but not the generation of cilia in the patient's nasal epithelial cells in vitro. CONCLUSION: Our study provides a platform for higher throughput analyses of airway epithelia that is applicable in a range of settings and suggests novel avenues for drug evaluation and development in PCD caused by nonsense mutations.

Induced pluripotent stem cells (iPSCs) have great potential as physiological disease models for human disorders where access to primary cells is difficult, such as neurons. In recent years, many protocols have been developed for the generation of iPSCs and the differentiation into specialised cell subtypes of interest. More recently, these models have been modified to allow large-scale phenotyping and high-content screening of small molecule compounds in iPSC-derived neuronal cells. Here, we describe the automated seeding of day 11 ventral midbrain progenitor cells into 96-well plates, administration of compounds, automated staining for immunofluorescence, the acquisition of images on a high-content screening platform and workflows for image analysis.

Protein cleavage is a central event in many regulated biological processes. We describe a system for detecting intracellular proteolysis based on non-conventional secretion of Gaussia luciferase (GLUC). GLUC exits the cell without benefit of a secretory leader peptide, but can be anchored in the cell by fusion to beta-actin. By including protease cleavage sites between GLUC and beta-actin, proteolytic cleavage can be detected. Using this assay, we have identified regulators of autophagy, apoptosis and beta-actin cleavage.

In 2014, Tralau-Stewart et al. published an overview of academic drug discovery efforts in the United Kingdom based on a survey they conducted (UK academic drug discovery. Nature Rev. Drug Discov. 13, 15–16 (2014))1. They observed that academic screening within the United Kingdom is comparable to that in the United States (discussed in Ref. 2) with regard to primary therapeutic focus (with cancer, infectious disease and cardiovascular disease constituting the most highly prioritized therapeutic indications) and areas of unmet medical need. Parallels were also drawn between the motivational drivers and annual operating costs of screening in both countries. However, regarding infrastructure, it was reported that most drug discovery programmes in academic screening groups (ASGs) in the United Kingdom were conducted in a traditional research group (that is, a team of postdoctoral researchers, Ph.D. students and technicians led by a single principal investigator), with only 13% of groups operating in centres dedicated to drug discovery. The most surprising finding was that “access to high-throughput screening (HTS) facilities and associated compound libraries were not reported by any UK group” (Ref. 1). Tralau-Stewart et al. clearly stated that the responses provided a “snapshot” of academic research at the time of the survey (2013) and not a comprehensive analysis, as some groups may not have received or responded to the survey1. However, it seems to us that the presence of centre-led ASGs and industry-standard drug discovery programmes conducted within an academic environment in the United Kingdom was substantially understated by the survey results. Therefore, we conducted another investigation of the academic drug discovery landscape in the United Kingdom by identifying academic drug discovery units and facilities and analysing their characteristics, which we discuss here.

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase (TK) that—once activated upon ligand binding—leads to receptor dimerization, recruitment of protein complexes, and activation of multiple signaling cascades. The EGFR is frequently overexpressed or mutated in various cancers leading to aberrant signaling and tumor growth. Hence, identification of interaction partners that bind to mutated EGFR can help identify novel targets for drug discovery. Here, we used a systematic approach to identify novel proteins that are involved in cancerous EGFR signaling. Using a combination of high-content imaging and a mammalian membrane two-hybrid protein–protein interaction method, we identified eight novel interaction partners of EGFR, of which half strongly interacted with oncogenic, hyperactive EGFR variants. One of these, transforming acidic coiled-coil proteins (TACC) 3, stabilizes EGFR on the cell surface, which results in an increase in downstream signaling via the mitogen-activated protein kinase and AKT pathway. Depletion of TACC3 from cells using small hairpin RNA (shRNA) knockdown or small-molecule targeting reduced mitogenic signaling in non-small cell lung cancer cell lines, suggesting that targeting TACC3 has potential as a new therapeutic approach for non-small cell lung cancer.

The cellular stress response autophagy has been implicated in various diseases including neuro-degeneration and cancer.The role of autophagy in cancer is not clearly understood and both tumour promoting and tumour suppressive effects of autophagy have been reported, which complicates the design of therapeutic strategies based on targeting the autophagy pathway.Here, we have systematically analyzed gene expression data for 47 autophagy genes for deletions, amplifications and mutations in various cancers.We found that several cancer types have frequent autophagy gene amplifications, whereas deletions are more frequent in prostate adenocarcinomas.Other cancer types such as glioblastoma and thyroid carcinoma show very few alterations in any of the 47 autophagy genes.Overall, individual autophagy core genes are altered at low frequency in cancer, suggesting that cancer cells require functional autophagy.Some autophagy genes show frequent single base mutations, such as members of the ULK family of protein kinases.Furthermore, we found hotspot mutations in the arginine-rich stretch in MAP1LC3A resulting in reduced cleavage of MAP1LC3A by ATG4B both in vitro and in vivo, suggesting a functional implication of this gene mutation in cancer development.

Dystroglycan, an extracellular matrix receptor, has essential functions in various tissues. Loss of α-dystroglycan-laminin interaction due to defective glycosylation of α-dystroglycan underlies a group of congenital muscular dystrophies often associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic human dystroglycanopathy cell models has limited our ability to test potential drugs in a human- and neural-specific context. Here, we generated induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous FKRP (fukutin-related protein gene) mutation. We showed that CRISPR/Cas9-mediated gene correction of FKRP restored glycosylation of α-dystroglycan in iPSC-derived cortical neurons, whereas targeted gene mutation of FKRP in wild-type cells disrupted this glycosylation. In parallel, we screened 31,954 small molecule compounds using a mouse myoblast line for increased glycosylation of α-dystroglycan. Using human FKRP-iPSC-derived neural cells for hit validation, we demonstrated that compound 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile (4BPPNit) significantly augmented glycosylation of α-dystroglycan, in part through upregulation of LARGE1 glycosyltransferase gene expression. Together, isogenic human iPSC-derived cells represent a valuable platform for facilitating dystroglycanopathy drug discovery and therapeutic development.

It is long established that von Willebrand factor (VWF) is central to hemostasis and thrombosis. Endothelial VWF is stored in cell-specific secretory granules, Weibel-Palade bodies (WPBs), organelles generated in a wide range of lengths (0.5-5.0 µm). WPB size responds to physiological cues and pharmacological treatment, and VWF secretion from shortened WPBs dramatically reduces platelet and plasma VWF adhesion to an endothelial surface.We hypothesized that WPB-shortening represented a novel target for antithrombotic therapy. Our objective was to determine whether compounds exhibiting this activity do exist.Using a microscopy approach coupled to automated image analysis, we measured the size of WPB bodies in primary human endothelial cells treated with licensed compounds for 24 hours.A novel approach to identification of antithrombotic compounds generated a significant number of candidates with the ability to shorten WPBs. In vitro assays of two selected compounds confirm that they inhibit the pro-hemostatic activity of secreted VWF. This set of compounds acting at a very early stage of the hemostatic process could well prove to be a useful adjunct to current antithrombotic therapeutics. Further, in the current SARS-CoV-2 pandemic, with a considerable fraction of critically ill COVID-19 patients affected by hypercoagulability, these WPB size-reducing drugs might also provide welcome therapeutic leads for frontline clinicians and researchers.

Secreted luciferases are highly useful bioluminescent reporters for cell-based assays and drug discovery. A variety of secreted luciferases from marine organisms have been described that harbor an N-terminal signal peptide for release along the classical secretory pathway. Here, we have characterized the secretion of Gaussia luciferase in more detail.We describe three basic mechanisms by which GLUC can be released from cells: first, classical secretion by virtue of the N-terminal signal peptide; second, internal signal peptide-mediated secretion and third, non-conventional secretion in the absence of an N-terminal signal peptide. Non-conventional release of dNGLUC is not stress-induced, does not require autophagy and can be enhanced by growth factor stimulation. Furthermore, we have identified the golgi-associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1) as a suppressor of release of dNGLUC.Due to its secretion via multiple secretion pathways GLUC can find multiple applications as a research tool to study classical and non-conventional secretion. As GLUC can also be released from a reporter construct by internal signal peptide-mediated secretion it can be incorporated in a novel bicistronic secretion system.

Autophagy protease ATG4B is a key regulator of the LC3/GABARAP conjugation system required for autophagosome formation, maturation and closure. Members of the ATG4 and the LC3/GABARAP family have been implicated in various diseases including cancer, and targeting the ATG4B protease has been suggested as a potential therapeutic anti-cancer strategy. Recently, it has been demonstrated that ATG4B is regulated by multiple post-translational modifications, including phosphorylation and de-phosphorylation. In order to identify regulators of ATG4B activity, we optimized a cell-based luciferase assay based on ATG4B-dependent release of Gaussia luciferase. We applied this assay in a proof-of-concept small molecule compound screen and identified activating compounds that increase cellular ATG4B activity. Next, we performed a high-throughput screen to identify kinases and phosphatases that regulate cellular ATG4B activity using siRNA mediated knockdown and cDNA overexpression. Of these, we provide preliminary evidence that the kinase AKT2 enhances ATG4B activity in cells. We provide all raw and processed data from the screens as a resource for further analysis. Overall, our findings provide novel insights into the regulation of ATG4B and highlight the importance of post-translational modifications of ATG4B.

Beta-Propeller Protein-Associated Neurodegeneration (BPAN) is one of the commonest forms of Neurodegeneration with Brain Iron Accumulation, caused by mutations in the gene encoding the autophagy-related protein, WDR45. The mechanisms linking autophagy, iron overload and neurodegeneration in BPAN are poorly understood and, as a result, there are currently no disease-modifying treatments for this progressive disorder. We have developed a patient-derived, induced pluripotent stem cell (iPSC)-based midbrain dopaminergic neuronal cell model of BPAN (3 patient, 2 age-matched controls and 2 isogenic control lines) which shows defective autophagy and aberrant gene expression in key neurodegenerative, neurodevelopmental and collagen pathways. A high content imaging-based medium-throughput blinded drug screen using the FDA-approved Prestwick library identified 5 cardiac glycosides that both corrected disease-related defective autophagosome formation and restored BPAN-specific gene expression profiles. Our findings have clear translational potential and emphasise the utility of iPSC-based modelling in elucidating disease pathophysiology and identifying targeted therapeutics for early-onset monogenic disorders.

Beta-Propeller Protein-Associated Neurodegeneration (BPAN) is one of the commonest forms of Neurodegeneration with Brain Iron Accumulation, caused by mutations in the gene encoding the autophagy-related protein, WDR45. The mechanisms linking autophagy, iron overload and neurodegeneration in BPAN are poorly understood and, as a result, there are currently no disease-modifying treatments for this progressive disorder. We have developed a patient-derived, induced pluripotent stem cell (iPSC)-based midbrain dopaminergic neuronal cell model of BPAN (3 patient, 2 age-matched controls and 2 isogenic control lines) which shows defective autophagy and aberrant gene expression in key neurodegenerative, neurodevelopmental and collagen pathways. A high content imaging-based medium-throughput blinded drug screen using the FDA-approved Prestwick library identified 5 cardiac glycosides that both corrected disease-related defective autophagosome formation and restored BPAN-specific gene expression profiles. Our findings have clear translational potential and emphasise the utility of iPSC-based modelling in elucidating disease pathophysiology and identifying targeted therapeutics for early-onset monogenic disorders.

ABSTRACT Targeted protein degradation (TPD) has opened new opportunities to investigate signalling pathways as a research tool, and as a unique therapeutic strategy using bifunctional chimeric small molecules, with candidate molecules in clinical trials for the treatment of breast cancer and prostate cancer. Most current TPD approaches use the 26S proteasomal machinery via PROteolysis TArgeting Chimeras (PROTACs), however, new emerging strategies using the autophagy system, termed AUtophagy TArgeting Chimeras (AUTACs) expand on the degrader arsenal and repertoire of targets that can be degraded. This includes non-protein molecules such as lipid droplets, organelles, insoluble protein aggregates as well as typical TPD targets, soluble intracellular proteins. AUTACs were proposed to operate by binding the target of interest (TOI) and linking it to an autophagy cargo protein (LC3 or p62), tethering the TOI into forming autophagosomes. In this study, we designed an alternative strategy for AUTACs, reasoning that the local recruitment and activation of ULK1 is sufficient to induce the formation of an autophagosome at the site of recruitment. As a proof of concept, we used an ULK1 agonist linked to a mitochondrial targeting ligand and termed these chimeric molecules ULK1-Recruiting Chimeras (ULKRECs). We show that local activation of ULK1 by ULKRECs at the outer mitochondrial membrane (OMM) induces mitophagy, further enhanced by mitochondrial insult. Using Parkinson’s disease (PD) patient-derived fibroblasts, we show the ULKRECs induce mitophagy independently of the PRKN/PINK axis, components required to signal for canonical mitophagy in response to stressors and often dysfunctional in many neurological diseases. We propose that ULKRECs are a novel class of degraders that have potential as unique therapeutics for diseases where dysfunctional mitophagy plays a key role in disease pathology and progression.

Autophagy and cellular metabolism are tightly linked processes, but how individual metabolic enzymes regulate the process of autophagy is not well understood. This study implicates ribose-5-phosphate isomerase (RPIA), a key regulator of the pentose phosphate pathway, in the control of autophagy. We used a dual gene deletion strategy, combining shRNA-mediated knockdown studies with CRISPR/Cas9 genome editing. Knockdown of RPIA by shRNA or genomic deletion by CRISPR/Cas9 genome editing, results in an increase of ATG4B-mediated LC3 processing and in the appearance of LC3-positive autophagosomes in cells. Increased LC3 processing upon knockdown of RPIA can be reversed by treatment with the antioxidant N-acetyl cysteine. The results are consistent with a model in which RPIA suppresses autophagy and LC3 processing by modulation of redox signaling.

Protein kinases are essential regulators of most cellular processes and are involved in the etiology and progression of multiple diseases. The cdc2-like kinases (CLKs) have been linked to various neurodegenerative disorders, metabolic regulation, and virus infection, and the kinases have been recognized as potential drug targets. Here, we have developed a screening workflow for the identification of potent CLK2 inhibitors and identified compounds with a novel chemical scaffold structure, the benzobisthiazoles, that has not been previously reported for kinase inhibitors. We propose models for binding of these compounds to CLK family proteins and key residues in CLK2 that are important for the compound interactions and the kinase activity. We identified structural elements within the benzobisthiazole that determine CLK2 and CLK3 inhibition, thus providing a rationale for selectivity assays. In summary, our results will inform structure-based design of CLK family inhibitors based on the novel benzobisthiazole scaffold.

The small cytokine-inducible SH2 domain-containing protein (CIS) has been implicated in the negative regulation of signaling through cytokine receptors. CIS reduces growth of erythropoietin receptor (EpoR)-dependent cell lines, but its role in proliferation, differentiation, and survival of erythroid progenitor cells has not been resolved. To dissect the function of CIS in cell lines and erythroid progenitor cells, we generated green fluorescent protein (GFP)-tagged versions of wild type CIS, a mutant harboring an inactivated SH2 domain (CIS R107K), and a mutant with a deletion of the SOCS Box (CISΔBox). Retroviral expression of the GFP fusion proteins in BaF3-EpoR cells revealed that both Tyr-401 in the EpoR and an intact SH2 domain within CIS are prerequisites for receptor recruitment. As a consequence, both are essential for the growth inhibitory effect of CIS, whereas the CIS SOCS box is dispensable. Accordingly, the retroviral expression of GFP-CIS but not GFP-CIS R107K impaired proliferation of erythroid progenitor cells in colony assays. Erythroid differentiation was unaffected by either protein. Interestingly, apoptosis of erythroid progenitor cells was increased upon GFP-CIS expression and this required the presence both of an intact SH2 domain and the SOCS box. Thus, CIS negatively regulates signaling at two levels, apoptosis and proliferation, and thereby sets a threshold for signal transduction. The small cytokine-inducible SH2 domain-containing protein (CIS) has been implicated in the negative regulation of signaling through cytokine receptors. CIS reduces growth of erythropoietin receptor (EpoR)-dependent cell lines, but its role in proliferation, differentiation, and survival of erythroid progenitor cells has not been resolved. To dissect the function of CIS in cell lines and erythroid progenitor cells, we generated green fluorescent protein (GFP)-tagged versions of wild type CIS, a mutant harboring an inactivated SH2 domain (CIS R107K), and a mutant with a deletion of the SOCS Box (CISΔBox). Retroviral expression of the GFP fusion proteins in BaF3-EpoR cells revealed that both Tyr-401 in the EpoR and an intact SH2 domain within CIS are prerequisites for receptor recruitment. As a consequence, both are essential for the growth inhibitory effect of CIS, whereas the CIS SOCS box is dispensable. Accordingly, the retroviral expression of GFP-CIS but not GFP-CIS R107K impaired proliferation of erythroid progenitor cells in colony assays. Erythroid differentiation was unaffected by either protein. Interestingly, apoptosis of erythroid progenitor cells was increased upon GFP-CIS expression and this required the presence both of an intact SH2 domain and the SOCS box. Thus, CIS negatively regulates signaling at two levels, apoptosis and proliferation, and thereby sets a threshold for signal transduction. erythropoietin erythropoietin receptor Src homology signal transducers and activators of transcription cytokine-inducible SH2 domain-containing protein suppressor of cytokine signaling interleukin green fluorescent protein hemagglutinin phycoerythrin colony-forming unit Janus kinase 2 7-amino-actinomycin D TdT-mediated dUTP-X nick-end labeling Survival, proliferation, and differentiation of hematopoietic cells are regulated by multiple cytokines (1Metcalf D. Nature. 1989; 339: 27-30Crossref PubMed Scopus (928) Google Scholar), and erythropoietin (Epo)1 is essential for erythropoiesis. By binding to the erythropoietin receptor (EpoR), a member of the hematopoietic cytokine receptor superfamily, Epo prevents apoptosis of erythroid progenitor cells and promotes their proliferation and erythroid maturation (2Wu H. Liu X. Jaenisch R. Lodish H.F. Cell. 1995; 83: 59-67Abstract Full Text PDF PubMed Scopus (870) Google Scholar). The formation of erythrocytes is tightly controlled by the coordinated activation of several signal-promoting and signal-terminating cascades activated by the EpoR (3Klingmüller U. Eur. J. Biochem. 1997; 249: 637-647Crossref PubMed Scopus (99) Google Scholar). The receptor-associated tyrosine kinase JAK2 is essential for surface appearance of the EpoR (4Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) and becomes activated after Epo binding. JAK2 in turn phosphorylates several tyrosine residues on the EpoR-cytosolic domain and probably on JAK2 itself that serve as docking sites for SH2 or protein tyrosine binding domains of downstream signal transduction proteins such as STAT5, phosphatidylinositol 3-kinase, Shc, and tyrosine phosphatases SHP1 and SHP2 (5Klingmüller U. Bergelson S. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8324-8328Crossref PubMed Scopus (165) Google Scholar, 6Damen J.E. Wakao H. Miyajima A. Krosl J. Humphries R.K. Cutler R.L. Krystal G. EMBO J. 1995; 14: 5557-5568Crossref PubMed Scopus (264) Google Scholar, 7Gobert S. Chretien S. Gouilleux F. Muller O. Pallard C. Dusanter-Fourt I. Groner B. Lacombe C. Gisselbrecht S. Mayeux P. EMBO J. 1996; 15: 2434-2441Crossref PubMed Scopus (193) Google Scholar, 8Klingmüller U., Wu, H. Hsiao J.G. Toker A. Duckworth B.C. Cantley L.C. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3016-3021Crossref PubMed Scopus (150) Google Scholar, 9Klingmüller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (846) Google Scholar, 10Tauchi T. Damen J.E. Toyama K. Feng G.S. Broxmeyer H.E. Krystal G. Blood. 1996; 87: 4495-44501Crossref PubMed Google Scholar). EpoR (phospho)tyrosine residues 343 and 401 serve as docking sites for the latent transcription factor STAT5 (5Klingmüller U. Bergelson S. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8324-8328Crossref PubMed Scopus (165) Google Scholar, 6Damen J.E. Wakao H. Miyajima A. Krosl J. Humphries R.K. Cutler R.L. Krystal G. EMBO J. 1995; 14: 5557-5568Crossref PubMed Scopus (264) Google Scholar, 7Gobert S. Chretien S. Gouilleux F. Muller O. Pallard C. Dusanter-Fourt I. Groner B. Lacombe C. Gisselbrecht S. Mayeux P. EMBO J. 1996; 15: 2434-2441Crossref PubMed Scopus (193) Google Scholar). STAT5 becomes tyrosine-phosphorylated upon receptor recruitment, homodimerizes, and migrates to the nucleus where it promotes the activation of target genes. STAT5 activates transcription of the Bcl-x Lgene (11Silva M. Benito A. Sanz C. Prosper F. Ekhterae D. Nunez G. Fernandez-Luna J. J. Biol. Chem. 1999; 274: 22165-22169Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 12Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). Bcl-xL has an essential role in preventing apoptosis of primitive and definitive erythrocytes at the end of maturation (13Motoyama N. Kimura T. Takahashi T. Watanabe T. Nakano T. J. Exp. Med. 1999; 189: 1691-1698Crossref PubMed Scopus (120) Google Scholar), and the STAT5 Bcl-xL signaling pathway has been suggested to protect cells from apoptosis and to promote cell proliferation (11Silva M. Benito A. Sanz C. Prosper F. Ekhterae D. Nunez G. Fernandez-Luna J. J. Biol. Chem. 1999; 274: 22165-22169Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 12Socolovsky M. Fallon A.E. Wang S. Brugnara C. Lodish H.F. Cell. 1999; 98: 181-191Abstract Full Text Full Text PDF PubMed Scopus (621) Google Scholar). A STAT5a/bnull mutation in fetal and neonatal mice leads to defects in erythroid maturation accompanied by increased apoptosis of erythroid progenitors (14Socolovsky M. Nam H. Fleming M.D. Haase V.H. Brugnara C. Lodish H.F. Blood. 2001; 98: 3261-3273Crossref PubMed Scopus (589) Google Scholar), supporting the notion that the STAT5Bcl-xL pathway mediates anti-apoptotic effects in erythroid cells. For the controlled production of erythroid cells, it is critical that the activation of signal-promoting cascades is counter-balanced by terminating events. In erythroid cells, these include activation of the tyrosine phosphatase SHP1 that dephosphorylates JAK2 (9Klingmüller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (846) Google Scholar) and induction of the negative regulatory protein CIS (cytokine-inducible SH2 domain-containing protein) (15Yoshimura A. Ohkubo T. Kiguchi T. Jenkins N.A. Gilbert D.J. Copeland N.G. Hara T. Miyajima A. EMBO J. 1995; 14: 2816-2826Crossref PubMed Scopus (636) Google Scholar). CIS lacks enzymatic activity and belongs to the family of suppressor of cytokine signaling (SOCS) proteins. These proteins possess a SH2 domain in the middle that mediates binding to phosphotyrosine residues and a SOCS Box at the C terminus that has been implicated in proteasomal degradation. By the use of a limited set of mutated EpoRs, tyrosine 401 in the cytoplasmic domain has been identified as the binding site for CIS (16Verdier F. Chretien S. Muller O. Varlet P. Yoshimura A. Gisselbrecht S. Lacombe C. Mayeux P. J. Biol. Chem. 1998; 273: 28185-28190Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). However, Tyr-401 is a multi-task residue that binds (besides CIS) other SOCS family members such as SOCS2 (17Eyckerman S. Verhee A. Van der Heyden J. Lemmens I. Van Ostade X. Vandekerckhove J. Tavernier J. Nat. Cell Biol. 2001; 3: 1114-1119Crossref PubMed Scopus (173) Google Scholar) and SOCS3 (18Sasaki A. Yasukawa H. Shouda T. Kitamura T. Dikic I. Yoshimura A. J. Biol. Chem. 2000; 275: 29338-29347Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 19Hortner M. Nielsch U. Mayr L.M. Heinrich P.C. Haan S. Eur. J. Biochem. 2002; 269: 2516-2526Crossref PubMed Scopus (69) Google Scholar) as well as the phosphatidylinositol phosphatase SHIP, the tyrosine phosphatase SHP2 (10Tauchi T. Damen J.E. Toyama K. Feng G.S. Broxmeyer H.E. Krystal G. Blood. 1996; 87: 4495-44501Crossref PubMed Google Scholar), and STAT5 (5Klingmüller U. Bergelson S. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8324-8328Crossref PubMed Scopus (165) Google Scholar, 6Damen J.E. Wakao H. Miyajima A. Krosl J. Humphries R.K. Cutler R.L. Krystal G. EMBO J. 1995; 14: 5557-5568Crossref PubMed Scopus (264) Google Scholar, 7Gobert S. Chretien S. Gouilleux F. Muller O. Pallard C. Dusanter-Fourt I. Groner B. Lacombe C. Gisselbrecht S. Mayeux P. EMBO J. 1996; 15: 2434-2441Crossref PubMed Scopus (193) Google Scholar). Forced expression of CIS inhibits proliferation of cell lines in response to Epo or interleukin-3 (IL-3) and results in reduced activation of STAT5 (20Matsumoto A. Masuhara M. Mitsui K. Yokouchi M. Ohtsubo M. Misawa H. Miyajima A. Yoshimura A. Blood. 1997; 89: 3148-3154Crossref PubMed Google Scholar, 21Jegalian A.G. Wu H. J. Interferon Cytokine Res. 2002; 22: 853-860Crossref PubMed Scopus (42) Google Scholar). The molecular mechanism of this effect is poorly understood because CIS lacks the kinase inhibitory region that mediates JAK2 inactivation by SOCS1 and SOCS3 (18Sasaki A. Yasukawa H. Shouda T. Kitamura T. Dikic I. Yoshimura A. J. Biol. Chem. 2000; 275: 29338-29347Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). It has been suggested that the CIS SH2 domain competes with STAT5 for binding to the EpoR (18Sasaki A. Yasukawa H. Shouda T. Kitamura T. Dikic I. Yoshimura A. J. Biol. Chem. 2000; 275: 29338-29347Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar), and the CIS SOCS Box has been proposed to mediate proteasome-dependent degradation of the EpoR (16Verdier F. Chretien S. Muller O. Varlet P. Yoshimura A. Gisselbrecht S. Lacombe C. Mayeux P. J. Biol. Chem. 1998; 273: 28185-28190Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The physiological role of CIS for the regulation of erythropoiesis remains to be determined since analyses of the two transgenic animal models harboring CIS under the control of the β-casein promoter (22Matsumoto A. Seki Y. Kubo M. Ohtsuka S. Suzuki A. Hayashi I. Tsuji K. Nakahata T. Okabe M. Yamada S. Yoshimura A. Mol. Cell. Biol. 1999; 19: 6396-6407Crossref PubMed Scopus (225) Google Scholar) or the CD4 promoter (23Li S. Chen S., Xu, X. Sundstedt A. Paulsson K.M. Anderson P. Karlsson S. Sjörgren H.O. Wang P. J. Exp. Med. 2000; 191: 985-994Crossref PubMed Scopus (90) Google Scholar) were focused on development of the mammary gland and on T-cell signaling, respectively. During embryonic development, CIS is first expressed in the fetal liver at embryonic day 12.5, the same stage as the EpoR (24Marine J.C. McKay C. Wang D. Topham D.J. Parganas E. Nakajima H. Pendeville H. Yasukawa H. Sasaki A. Yoshimura A. Ihle J.N. Cell. 1999; 98: 617-627Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). The role of CIS in fetal erythropoiesis has not been addressed yet since most studies on growth and survival have been undertaken in cell lines. Here we show that CIS overexpression mimics STAT5 loss-of-function in erythroid progenitor cells, and we determine which CIS domains are essential for this effect. To this end, we expressed GFP-tagged versions of the CIS protein in erythroid progenitor cells under the control of a retroviral promoter, which uncoupled CIS synthesis from activation of the JAK/STAT pathway. We show that elevated levels of CIS inhibit proliferation of erythroid progenitor cells. Although erythroid differentiation is unaffected, an intact CIS SH2 domain is essential for inhibition of proliferation. Similar to bone marrow-derived erythroid progenitor cells in STAT5 knock-out mice (14Socolovsky M. Nam H. Fleming M.D. Haase V.H. Brugnara C. Lodish H.F. Blood. 2001; 98: 3261-3273Crossref PubMed Scopus (589) Google Scholar), erythroid progenitor cells from fetal liver overexpressing CIS show an increased tendency to undergo apoptosis, which requires both the CIS SH2 domain and the SOCS box. Thus, we propose that CIS negatively regulates signaling through the EpoR by two mechanisms. 1) Recruitment of CIS to the EpoR via its SH2 domain is sufficient to repress proliferative responses. 2) In addition, the SOCS Box mediates apoptotic effects of CIS expression in fetal liver erythroid progenitor cells. Single or double Tyr to Phe mutant EpoRs were generated by PCR mutagenesis and inserted into the eukaryotic expression vector pXM as described previously (5Klingmüller U. Bergelson S. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8324-8328Crossref PubMed Scopus (165) Google Scholar, 9Klingmüller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (846) Google Scholar). EpoR Y401F was subcloned in frame as a ApaI- andEcoRI-digested fragment into the appropriate restriction sites of the retroviral expression vector pMX (puro)-EpoR. GFP-CIS fusion proteins were established by introducing an in-frameBglII restriction site at the 5′-end and a EcoRI restriction site at the 3′-end of the CIS cDNA and subcloning the DNA fragment in frame into the BamHI and EcoRI restriction sites of the retroviral expression vector pMX-enhanced GFP or pOSdSV (25Ketteler R. Glaser S. Sandra O. Martens U.M. Klingmuller U. Gene Ther. 2002; 9: 477-487Crossref PubMed Scopus (74) Google Scholar). The SOCS Box from CIS was deleted at amino acid 182, and the fragment was cloned via BglII and EcoRI into the BamHI and EcoRI restriction sites of pMOWS-GFP (25Ketteler R. Glaser S. Sandra O. Martens U.M. Klingmuller U. Gene Ther. 2002; 9: 477-487Crossref PubMed Scopus (74) Google Scholar). GFP-SOCS3 was generated by introducing BamHI and NotI restriction sites at the 5′ and 3′ ends and subcloned in-frame into pMOWS-GFP. Arginine 107 localized within the SH2 domain of CIS was replaced by lysine using overlap extension PCR and subcloned into the BamHI and EcoRI restriction sites of pMX-enhanced GFP. BaF3 cell lines expressing single or double Tyr to Phe mutant EpoRs in pXM were generated as described previously (5Klingmüller U. Bergelson S. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8324-8328Crossref PubMed Scopus (165) Google Scholar, 9Klingmüller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (846) Google Scholar). By retroviral transduction, pMX-EpoR or pMX-EpoR Y401F was introduced into the IL-3-dependent pro-B cell line BaF3. BaF3 cells were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and 10% WEHI-conditioned medium. Pools of stable transfectants were selected in 1.5 μg/ml puromycin (Sigma). The retroviral packaging cell line Phoenix-eco was maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum. Fetal livers from 13.5-day-old BALB/c mouse embryos were prepared as described elsewhere (25Ketteler R. Glaser S. Sandra O. Martens U.M. Klingmuller U. Gene Ther. 2002; 9: 477-487Crossref PubMed Scopus (74) Google Scholar). For Ter119 expression kinetics, the fetal liver cell preparation was incubated with anti-Ter119 (provided by Dr. Albrecht Müller) and subjected to AutoMACS depletion (Miltenyi Biotech, Bergisch-Gladbach, Germany). For TUNEL assay, the cells were depleted for hematopoietic lineages with an antibody mixture as described previously (25Ketteler R. Glaser S. Sandra O. Martens U.M. Klingmuller U. Gene Ther. 2002; 9: 477-487Crossref PubMed Scopus (74) Google Scholar). Retroviral expression vectors were transiently transfected into Phoenix-eco cells using the Calcium-phosphate method (25Ketteler R. Glaser S. Sandra O. Martens U.M. Klingmuller U. Gene Ther. 2002; 9: 477-487Crossref PubMed Scopus (74) Google Scholar). Twenty-four hours after transfection of Phoenix cells, the medium was changed to Iscove's modified Eagle's medium containing 50 μm β-mercaptoethanol and 30% fetal calf serum. Twenty-four hours later, 1 ml of virus-containing supernatant was harvested and filtered through a 0.45-μm filter. For spin infection, the supernatant was mixed with 1 × 104 (for colony assays) or 5 × 104 (for liquid culture) freshly prepared fetal liver cells and spun for 2 h in an Eppendorf centrifuge at 1800 rpm at room temperature. The cells were supplemented with 0.4 unit/ml Epo (Cilag-Jansen, Bad Hamburg, Germany) and plated in 0.8% methylcellulose (StemCell Technologies, Vancouver, Canada) or seeded in Iscove's modified Eagles's medium, 30% fetal calf serum, 50 μm β-mercaptoethanol supplemented with 0.4 unit/ml Epo. GFP-positive and hemoglobinized erythroid colonies were identified by benzidine staining. BaF3 cells expressing the indicated EpoRs in the context of pMX were washed three times in medium and plated at a density of 5 × 104 cells/well in 24-well plates. After 3 days in culture in the indicated concentrations of Epo or in 10% WEHI-conditioned medium, cell numbers were determined using a Coulter counter. For BaF3 cells expressing the wild-type EpoR or EpoR, Y401F 1 × 107cells were used per immunoprecipitation. Lysis and immunoprecipitation experiments were carried out as described previously (9Klingmüller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (846) Google Scholar) using the following antibodies: crude rabbit antiserum raised against the extracellular domain of the EpoR (9Klingmüller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (846) Google Scholar), rabbit antiserum raised against CIS (kindly provided by A. Yoshimura), and rabbit antiserum raised against a GST-GFP fusion protein. Proteins were detected by immunoblotting with anti-phosphotyrosine antiserum 4G10 (Upstate Biotechnology, Palo Alto, CA) followed by enhanced chemiluminescence (Amersham Biosciences). Membranes were denatured with β-mercaptoethanol/SDS before reprobing for control of equal protein loading. To determine surface expression of HA-tagged EpoR (26Ketteler R. Heinrich A. Offe J. Becker V. Cohen J. Neumann D. Klingmüller U. J. Biol. Chem. 2002; 277: 26547-26552Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), BaF3 cells were transduced with retroviral supernatants and sorted for GFP-expression using MoFlo (Cytomation, Colorado). The sorted cells were incubated with rat anti-HA (Roche Molecular Biochemicals) as primary antibody and anti-rat IgG coupled to Cy5 (Dianova) as secondary antibody and analyzed for green and red fluorescence by flow cytometry. For apoptosis assay, the cells were stained with 5 μl of VIAprobe 7-AAD (BD Biosciences) and 5 μl of annexin V coupled to phycoerythrin (PE) or Cy5 (BD Biosciences) according to the manufacturer's instructions. For TUNEL assay, lineage negative fetal liver cells were cultivated for 24 h in serum-free medium PANserin401 (PANbiotech) supplemented with 0.05 unit/ml Epo. The cells were washed with phosphate-buffered saline, fixed with 2% paraformaldehyde, and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline. The TUNEL assay was performed using the TMR-Red in situ cell death kit (Roche Molecular Biochemicals) according to the manufacturer's instructions. Ter119 staining of fetal liver cells as a marker for erythroid differentiation (27Kina T. Ikuta K. Takayama E. Wada K. Majumdar A.S. Weissman I.L. Katsura Y. Brit. J. Haematol. 2000; 109: 280-287Crossref PubMed Scopus (292) Google Scholar) was performed with a rat anti-Ter119 antiserum (kindly provided by Dr. Albrecht Müller, Julius-Maximilian University, Würzburg, Germany) and a secondary anti-rat IgG coupled to Cy5. GFP, PE, Cy5, and TMR-Red fluorescence were detected with a Becton Dickinson FACScan (BD Biosciences) using the CellQuest software. To confirm that Tyr-401 in the EpoR cytoplasmic domain represents the major CIS binding site, we expressed the wild type EpoR or a panel of EpoRs containing specific single or double tyrosine to phenylalanine mutations in the IL-3-dependent pro-B cell line BaF3 and tested in co-immunoprecipitation experiments for their ability to bind CIS. Despite lacking one or two tyrosine residues, the mutant EpoRs were tyrosine-phosphorylated upon Epo stimulation to the same extent as the wild type receptor as judged by immunoprecipitation with antiserum recognizing the EpoR followed by immunoblotting with an anti-phosphotyrosine antibody (Fig. 1 A). In agreement with the observation of others, tyrosine-phosphorylated mutant EpoR Y401F was greatly impaired in its ability to associate with CIS (Fig. 1 A, lower panel, lane 3). Because a faint band representing the tyrosine-phosphorylated EpoR Y401F was consistently observed in these co-immunoprecipitation experiments, we conclude that Tyr-401 represents the major binding site for CIS on the EpoR but that other minor binding sites exist. Since Tyr-401 has been identified as the binding site for multiple signal-promoting and signal-terminating molecules including CIS, we asked whether the absence of Tyr-401 increased or decreased biological responsiveness of the EpoR. To examine proliferative signaling, BaF3 cells stably expressing the wild type EpoR or the mutant EpoR Y401F were cultivated in the presence of increasing concentrations of Epo ranging from 0.1 to 10 units/ml. After 3 days, the cell number was determined and normalized to the cell growth obtained in the presence of WEHI-conditioned medium. All cell pools analyzed showed comparable growth rates in WEHI-conditioned medium. When grown in Epo, half-maximal proliferation of BaF3 cells expressing the wild type EpoR was achieved at 1 unit/ml Epo and equaled 17% of the growth obtained in the presence of WEHI-conditioned medium (Fig. 1 B). At 10 units/ml Epo, these cells grew to 45% of the number achieved in WEHI-conditioned medium. In contrast, although proliferation of BaF3 cells expressing EpoR Y401F remained Epo-dependent, these cells grew better at all Epo concentrations. At the highest Epo concentration employed, 76% of the growth observed in WEHI-conditioned medium was achieved compared with 45% obtained upon expression of the wild type EpoR. The expression of mutant EpoR Y401F allows cells to proliferate in Epo 2–4-fold less than normal and enables cells to grow to higher density. To confirm that the growth-promoting effects of EpoR Y401F are not caused by increased surface prevalence, we expressed HA-tagged forms of the wild type EpoR and EpoR Y401F in BaF3 cells and analyzed the extent of surface expression by flow cytometry (Fig. 1 C). This analysis revealed that in comparison to the HA-tagged wild type receptor, the amount of HA-EpoR Y401F detectable on the cell surface is not enhanced but rather slightly reduced. To determine whether the increased yield of BaF3 cells expressing EpoR Y401F correlates with increased cell survival, we performed viability staining of BaF3-EpoR and BaF3 EpoR Y401F cells. The cells were cultivated in the absence of growth factor or in the presence of 0.1 or 1 unit/ml Epo or, as a positive control, 2 ng/ml IL-3. The cells were stained with 7-AAD, a dye that stains dead cells. (Fig. 1 D). As expected, in the absence of cytokine, the majority of cells (90.7% of BaF3-EpoR and 85.9% of BaF3-EpoR Y401F cells) underwent death within 24 h, whereas in the presence of IL-3, death was reduced to ∼13% of the cells. Similarly, in 1 unit/ml Epo, only 20.5% BaF3-EpoR cells and 10.8% BaF3-EpoR Tyr-401 cells were 7-AAD-positive. At a lower Epo concentration (0.1 unit/ml), the number of 7-AAD-positive BaF3-EpoR cells increased to 50.0% but only 16.5% BaF3-EpoR Y401F cells were dead. Thus, the absence of Tyr-401 from the EpoR decreases sensitivity to undergo cell death and increases the proliferative yield, suggesting that EpoR Tyr-401 is important for activating a down-modulating signal. To determine the domains in CIS required for binding to the EpoR and for exerting biological functions, we generated GFP-tagged versions of wild type CIS and a mutant CIS harboring an inactivated SH2 domain (CIS R107K). To examine binding to (phospho)tyrosine 401 of the EpoR, GFP, GFP-CIS, and GFP-CIS R107K were stably expressed in parental BaF3, BaF3-EpoR, and BaF3-EpoR Y401F cells. The cells were stimulated with Epo, lysed, and subjected to immunoprecipitation using antisera against the EpoR or GFP. Tyrosine-phosphorylated EpoR complexed with GFP fusion proteins were subsequently identified by immunoblotting with anti-phosphotyrosine antiserum (Fig. 2). As expected, GFP alone was unable to bind either one of tyrosine-phosphorylated receptors (Fig. 2,lanes 5 and 6). However, compared with the total amount of phosphorylated EpoR (Fig. 2, lanes 2 and3), GFP-CIS preferentially bound the tyrosine-phosphorylated wild type EpoR and to a lesser extent tyrosine-phosphorylated EpoR Y401F (Fig. 2, lanes 8 and 9). By testing a panel of tyrosine-to-phenylalanine mutant EpoRs (Fig. 1 A), we could demonstrate that, despite the residual association of GFP-CIS with EpoR Y401F, Tyr-401 represents the major binding site for CIS in the EpoR (Fig. 2, lane 8). Conversely, mutating the critical arginine 107 in the CIS SH2 domain completely abrogated the ability of CIS to bind the tyrosine-phosphorylated EpoR (Fig. 2, lanes 11 and 12). This finding confirms the importance of the SH2 domain for receptor recruitment of CIS. To elucidate whether binding of CIS to the EpoR negatively regulates erythropoiesis, GFP-CIS and GFP-CIS R107K were transduced by retroviral infection into fetal liver cells and tested for their effect on CFU-E colony formation. The transduction efficiency ranged from 30 to 50% and was the same for transduction of GFP-CIS and GFP-CIS R107K. As shown in Fig. 3 A, the total number of CFU-E colonies expressing GFP-CIS was not significantly reduced compared with colonies expressing GFP-CIS R107K or GFP. Colonies grown in methylcellulose supplemented with 0.4 unit/ml Epo, thus favoring growth of CFU-E colonies, were inspected by fluorescence microscopy to monitor GFP-positive CFU-E colonies. As shown in Fig. 3 B, the expression of GFP-CIS and GFP-CIS R107K in erythroid progenitors is comparable with each other and is predominantly localized to the cytoplasm. To determine the effect of unregulated CIS expression on proliferation of erythroid progenitor cells, we investigated the cell proliferation rate within each GFP-positive colony. After 20–25 h of culture in Epo, colonies containing 4–16 cells predominated. To detect alterations in colony size, the formed erythroid colonies were classified into three categories: 4 cell, 4–8 cell, and 9–16 cell. After 23 h in culture, the percentage of the largest (9–16 cell) colonies expressing GFP-CIS (25%) was reduced compared with the percentage of the 9–16 cell colonies expressing GFP-CIS R107K (71%) (Fig. 3 C). The latter result was identical to the distribution of colony sizes after transduction of the control GFP protein (data not shown). Thus, the expression of GFP-CIS but not GFP-CIS R107K reduces proliferation of erythroid progenitor cells, indicating the importance of the CIS SH2 domain in this process. To determine whether these erythroid colonies were defective in differentiation, we directly examined all GFP-positive colonies after 24 h of cultivation for the extent of benzidine staining and detected no difference among the fusion proteins (data not shown). We conclude that the observed colonies are of erythroid origin and that maturation of erythroid progenitors is normal even though proliferation is impaired as a consequence of CIS expression. Accordingly, benzidine staining of CFU-E colonies after 3 days in culture with Epo did not show a difference between cells expressing GFP-CIS, GFP-CIS R107K, or GFP (Fig. 3 B, right panel). Similarly, as judged by fluorescence-activated cell sorter analysis, Ter119 expression occurred at normal rates in GFP-CIS, GFP-CIS R107K, and GFP-ex

G protein-coupled receptors (GP-CRs) are a major family of signaling molecules, central to the regulation of inflammatory responses. Their activation upon agonist binding is attenuated by GPCR kinases (GRKs), which desensitize the receptors through phosphorylation. G protein-coupled receptor kinase 2(GRK2) down-regulation in leukocytes has been closely linked to the progression of chronic inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. Because leukocytes must interact with the endothelium to infiltrate inflamed tissues, we hypothesized that GRK2 down-regulation in endothelial cells would also be pro-inflammatory.To determine whether GRK2 down-regulation in endothelial cells is pro-inflammatory.siRNA-mediated ablation of GRK2 in human umbilical vein endothelial cells (HUVECs) was used in analyses of the role of this kinase. Microscopic and biochemical analyses of Weibel-Palade body (WPB) formation and functioning, live cell imaging of calcium concentrations and video analyses of adhesion of monocyte-like THP-1 cells provide clear evidence of GRK2 function in histamine activation of endothelial cells.G protein-coupled receptor kinase 2 depletion in HUVECs increases WPB exocytosis and P-selectin-dependent adhesion of THP-1 cells to the endothelial surface upon histamine stimulation, relative to controls. Further, live imaging of intracellular calcium concentrations reveals amplified histamine receptor signaling in GRK2-depleted cells, suggesting GRK2 moderates WPB exocytosis through receptor desensitization.G protein-coupled receptor kinase 2 deficiency in endothelial cells results in increased pro-inflammatory signaling and enhanced leukocyte recruitment to activated endothelial cells. The ability of GRK2 to modulate initiation of inflammatory responses in endothelial cells as well as leukocytes now places GRK2 at the apex of control of this finely balanced process.

Signaling through hematopoietic cytokine receptors such as the erythropoietin receptor (EpoR) depends on the activation of a receptor-bound Janus kinase (JAK) and tyrosine phosphorylation of the cytoplasmic domain. To visualize the EpoR and elucidate structural requirements coordinating signal transduction, we probed the EpoR by inserting the green fluorescent protein (GFP) at various positions. We show that insertion of GFP in proximity to the transmembrane domain, either in the extracellular or the cytoplasmic domain, results in EpoR-GFP receptors incompetent to elicit biological responses in a factor-dependent cell line or in erythroid progenitor cells. Surprisingly, a receptor harboring GFP insertion in the middle of the cytoplasmic domain, and thereby separating the JAK2 binding site from the tyrosine residues, is capable of supporting signal transduction in response to ligand binding. Comparable with the wild type EpoR, but more efficient than a C-terminal EpoR-GFP fusion, this chimeric receptor promotes the maturation of erythroid progenitor cells and is localized in punctated endosome-like structures. We conclude that the extracellular, transmembrane, and membrane-proximal segment of the cytoplasmic domain form a rigid structural entity whose precise orientation is essential for the initiation of signal transduction, whereas the cytoplasmic domain possesses flexibility in adopting an activated conformation. Signaling through hematopoietic cytokine receptors such as the erythropoietin receptor (EpoR) depends on the activation of a receptor-bound Janus kinase (JAK) and tyrosine phosphorylation of the cytoplasmic domain. To visualize the EpoR and elucidate structural requirements coordinating signal transduction, we probed the EpoR by inserting the green fluorescent protein (GFP) at various positions. We show that insertion of GFP in proximity to the transmembrane domain, either in the extracellular or the cytoplasmic domain, results in EpoR-GFP receptors incompetent to elicit biological responses in a factor-dependent cell line or in erythroid progenitor cells. Surprisingly, a receptor harboring GFP insertion in the middle of the cytoplasmic domain, and thereby separating the JAK2 binding site from the tyrosine residues, is capable of supporting signal transduction in response to ligand binding. Comparable with the wild type EpoR, but more efficient than a C-terminal EpoR-GFP fusion, this chimeric receptor promotes the maturation of erythroid progenitor cells and is localized in punctated endosome-like structures. We conclude that the extracellular, transmembrane, and membrane-proximal segment of the cytoplasmic domain form a rigid structural entity whose precise orientation is essential for the initiation of signal transduction, whereas the cytoplasmic domain possesses flexibility in adopting an activated conformation. Epo receptor signal transducers and activators of transcription green fluorescent protein hemagglutinin transmembrane colony-forming unit-erythroid endoplasmic reticulum Ligand binding to membrane-spanning receptors supports signaling networks within cells. The specific structural requirements that enable conversion of ligand binding to the extracellular domain to an activated conformation of the cytoplasmic domain are poorly understood. Hematopoietic cytokine receptors share common features in the extracellular domain such as four spaced cysteines near the N terminus and a Trp-Ser-X-Trp-Ser (WSXWS) motif located proximal to the cell membrane (for a review, see Refs. 1Constantinescu S.N. Ghaffari S. Lodish H.F. Trends Endocrinol. Metab. 1999; 10: 18-23Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar and 2Klingmüller U. Eur. J. Biochem. 1997; 249: 637-647Crossref PubMed Scopus (99) Google Scholar). The cytoplasmic domain of hematopoietic cytokine receptors lack intrinsic enzymatic activity and therefore require recruitment of cytoplasmic kinases to promote signal transduction. A simple prototype of the hematopoietic cytokine receptor family is the erythropoietin receptor (EpoR)1 that is essential for the development of mature erythrocytes. Crystallographic evidence suggests that in the absence of ligand, the EpoR exists as a preformed dimer in an open scissors-like conformation (3Livnah O. Stura E.A. Middleton S.A. Johnson D.L. Jolliffe L.K. Wilson I.A. Science. 1999; 283: 987-990Crossref PubMed Scopus (545) Google Scholar). Upon ligand binding, a conformational switch facilitated by self-interaction of the transmembrane domains is induced, permitting the activation of an intracellular signal transduction cascade (4Kubatzky K.F. Ruan W. Gurezka R. Cohen J. Ketteler R. Watowich S.S. Neumann D. Langosch D. Klingmuller U. Curr. Biol. 2001; 11: 110-115Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). This process is supported by a conserved hydrophobic motif localized in the cytoplasmic juxtamembrane domain of the EpoR (5Constantinescu S.N. Huang L.J. Nam H. Lodish H.F. Mol. Cell. 2001; 7: 377-385Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). A continuous stretch of residues in the membrane-proximal domain of the EpoR mediates binding of the Janus kinase JAK2 and ensures transport of the EpoR from the endoplasmic reticulum to the cell surface (6Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The precise orientation of critical residues in the juxtamembrane motif is essential for JAK2 activation. Negative inhibitory molecules including the suppressor of cytokine signaling family of proteins (7Nicholson S.E. Hilton D.J. J. Leukocyte Biol. 1998; 63: 665-668Crossref PubMed Scopus (116) Google Scholar) and tyrosine phosphatases such as SHP-1 (8Klingmüller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (846) Google Scholar), PTP-1B (9Myers M.P. Andersen J.N. Cheng A. Tremblay M.L. Horvath C.M. Parisien J.P. Salmeen A. Barford D. Tonks N.K. J. Biol. Chem. 2001; 276: 47771-47774Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar), and CD45 (10Irie-Sasaki J. Sasaki T. Matsumoto W. Opavsky A. Cheng M. Welstead G. Griffiths E. Krawczyk C. Richardson C.D. Aitken K. Iscove N. Koretzky G. Johnson P. Liu P. Rothstein D.M. Penninger J.M. Nature. 2001; 409: 349-354Crossref PubMed Scopus (463) Google Scholar) tightly regulate JAK2. In addition, JAK2 is involved in activation of signal transducer and activator of transcription protein 1 (STAT1) and STAT3 by the EpoR, as shown by the use of the JAK2 inhibitor AG490 (11Kirito K. Nakajima K. Watanabe T. Uchida M. Tanaka M. Ozawa K. Komatsu N. Blood. 2002; 99: 102-110Crossref PubMed Scopus (63) Google Scholar). The cytoplasmic domain of the activated EpoR mediates the recruitment of secondary signaling molecules including the lipid kinase phosphoinositide 3-kinase (12Klingmüller U., Wu, H. Hsiao J.G. Toker A. Duckworth B.C. Cantley L.C. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3016-3021Crossref PubMed Scopus (150) Google Scholar, 13Damen J.E. Cutler R.L. Jiao H., Yi, T. Krystal G. J. Biol. Chem. 1995; 270: 23402-23408Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and activation of STATs that promote signal transmission from the cell surface to the nucleus. STAT1, STAT3, and STAT5 are involved in EpoR signal transduction (11Kirito K. Nakajima K. Watanabe T. Uchida M. Tanaka M. Ozawa K. Komatsu N. Blood. 2002; 99: 102-110Crossref PubMed Scopus (63) Google Scholar, 14Klingmüller U. Bergelson S. Hsiao J.G. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8324-8328Crossref PubMed Scopus (165) Google Scholar, 15Kirito K. Uchida M. Yamada M. Miura Y. Komatsu N. J. Biol. Chem. 1997; 272: 16507-16513Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 16Kirito K. Uchida M. Takatoku M. Nakajima K. Hirano T. Miura Y. Komatsu N. Blood. 1998; 92: 462-471Crossref PubMed Google Scholar). Docking of the tyrosine phosphatase SHP-1 leads to termination of signal transduction (8Klingmüller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (846) Google Scholar, 17Gobert S. Chretien S. Gouilleux F. Muller O. Pallard C. Dusanter-Fourt I. Groner B. Lacombe C. Gisselbrecht S. Mayeux P. EMBO J. 1996; 15: 2434-2441Crossref PubMed Scopus (193) Google Scholar). Signaling pathways activated in response to ligand binding to the EpoR have been studied in detail, but it is unresolved how activation of JAK2 is communicated to phosphorylation of the eight tyrosine residues localized in the membrane-distal cytoplasmic domain. Here we present a set of EpoR-GFP fusion proteins that are 1) ER-retained and signaling-incompetent, 2) surface-expressed but signaling-incompetent, and 3) surface-expressed and signaling-competent. Our analysis shows that the cytoplasmic domain of the EpoR can tolerate a large insertion separating the JAK2-activating segment from the respective tyrosine residues and yet coordinate biological responses supporting proliferation and differentiation of erythroid progenitor cells. Primers used are summarized in TableI. Thermostabilizing amino acid exchanges V163A, I167T, and S175G were introduced into the cDNA of enhanced green fluorescent protein (CLONTECH, Palo Alto, CA) by overlap extension PCR using as general 5′-primer primer number 1 that introduces a BglII restriction site and as general 3′-primer primer 2 encoding an EcoRI restriction site. V163A and I167T were introduced concomitantly using the primers 3-U and 3-L. Using the resulting cDNA as a template, S175G was introduced with the primers 4-U and 4-L. The EpoR-GFP receptors were generated by overlap extension PCR including the following steps. To generate EpoR-GFP1, first a shortened EpoR was established harboring aBamHI restriction site at amino acid position 224 in the EpoR without altering the amino acid sequence. The shortened EpoR fragment flanked by BclI/SalI (5′) andBglII/EcoRI (3′) restriction sites was generated using primers 5, 6-L, 6-U, and 7 and was cloned into theBamHI and EcoRI restriction sites in the retroviral vector pBABE (pBABE-EpoR-BamHI). Second, an in-frame fusion of the EpoR extracellular domain and GFP was established using primers 5, 8-L, 8-U, and 9. The joined fragment was subcloned via the SalI and BamHI in pBABE-EpoR-BamHI. Finally, inserting the EpoRBglII/EcoRI restriction fragment into pBABE-EpoR-BamHI, resulting in pBABE-EpoR-GFP1, completed the EpoR cDNA.Table IPrimers used for construction of EpoR-GFP chimeras Open table in a new tab To generate EpoR-GFP2, an EpoR subfragment (amino acids 1–304) encompassing the EpoR extracellular domain and transmembrane (TM) domain was produced using primers 5 and 10. The PCR fragment was digested with BclI and cloned into pBABE cut withBamHI and SalI (blunt), resulting in pBABE-EpoR-NotI. By PCR amplification, BglII (5′) and NotI (3′) restriction sites were introduced at the respective ends of the GFP cDNA using primers 11 and 12. The PCR fragment was subcloned via BglII and NotI in pBABE-NotI. The EpoR cDNA was completed by PCR amplification using primers 13 and 14 and inserting the PCR fragmentNotI and EcoRI in pBABE-EpoR-NotI, resulting in pBABE-EpoR-GFP2. EpoR-GFP3 bearing GFP inserted at amino acid position 336 in the EpoR cDNA was generated by using primers 15, 16-L, 16-U, and 17 and subcloned via BglII andSphI into the respective restriction sites in pBS-EpoR that harbors the EpoR cDNA inserted into BamHI andEcoRI in pBluescript II KS (Stratagene). EpoR-GFP4 harbors GFP fused to the C terminus of the EpoR and was generated by introducing NotI and EcoRI restriction sites at the 3′-end of the EpoR cDNA. The EpoR cytoplasmic domain was amplified using primers 13 and 18 and subcloned via BamHI and EcoRI into pBABE-EpoR-NotI, resulting in pBABE-EpoR-NotIcyto. NotI andEcoRI restriction sites flanking the GFP cDNA were amplified by PCR with primers 19 and 20 and inserted viaNotI and EcoRI in pBABE-EpoR-NotIcyto. The resulting EpoR-GFP cDNAs were verified by automated sequencing and inserted via SalI and EcoRI digestion into pMOWS (18Ketteler R. Glaser S. Sandra O. Martens U.M. Klingmüller U. Gene Ther. 2002; 9: 477-487Crossref PubMed Scopus (74) Google Scholar). The HA tag was inserted into wild type EpoR by excising the HA-containing fragment with EcoRI and BamHI from pMX-HA-EpoR-IRES-GFP (kindly provided by Dr. Stefan Constantinescu, Ludwig Institute for Cancer Research, Brussels, Belgium) and subcloning it into the EcoRI and PacI restriction sites of pMOWS-EpoR, yielding pMOWS-HA-EpoR. HA-EpoR-GFP1 and HA-EpoR-GFP2 were generated by subcloning the PmlI and BamHI fragment from pMOWS-EpoR-GFP1 or pMOWS-EpoR-GFP2 into the corresponding sites of pMOWS-HA-EpoR. The retroviral vectors were transiently transfected in Phoenix-Eco cells by calcium phosphate precipitation (18Ketteler R. Glaser S. Sandra O. Martens U.M. Klingmüller U. Gene Ther. 2002; 9: 477-487Crossref PubMed Scopus (74) Google Scholar) and visualized after 24 h or used for the production of transducing supernatants as described (18Ketteler R. Glaser S. Sandra O. Martens U.M. Klingmüller U. Gene Ther. 2002; 9: 477-487Crossref PubMed Scopus (74) Google Scholar). Transducing supernatants were applied to introduce the cDNA for the EpoR or EpoR-GFP chimera into the interleukin-3-dependent pro-B cell line BaF3 and fetal liver cells. Pools of BaF3 cells expressing the wild type EpoR or the EpoR-GFP chimera were selected in 1.5 μg/ml puromycin (Sigma) 48 h after transduction. Cell pools expressing comparable amounts of the receptors were identified by immunoblotting and used for further experiments. The selected cells were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum (Invitrogen) and 10% WEHI-conditioned medium in the presence of 1.5 μg/ml puromycin. Fetal liver cells derived from 12.5-day-old embryos from EpoR−/− mice (19Wu H. Liu X., R., J. Lodish H.F. Cell. 1995; 83: 59-67Abstract Full Text PDF PubMed Scopus (870) Google Scholar) were prepared and transduced as described (18Ketteler R. Glaser S. Sandra O. Martens U.M. Klingmüller U. Gene Ther. 2002; 9: 477-487Crossref PubMed Scopus (74) Google Scholar). The transduced cells were plated in 0.8% methylcellulose (StemCell Technologies, Vancouver, Canada) supplemented with 4 units/ml Epo (Cilag-Jansen, Bad Homburg, Germany). Colony-forming unit-erythroid (CFU-E) colony formation was monitored by benzidine staining of hemoglobinized cells. To ensure comparable transduction rates, GFP expression was assessed by fluorescence-activated cell sorting analysis (FACScan; Becton Dickinson, Palo Alto, CA) in transduced fetal liver cells of wild type mice after 20 h of cultivation in Iscove's modified Eagle's medium supplemented with Epo. Three independent pools of BaF3 cells expressing the wild type EpoR or EpoR-GFP chimera were analyzed by saturation binding of 125I-labeled Epo as described (20Levin I. Cohen J. Supino-Rosin L. Yoshimura A. Watowich S.S. Neumann D. FEBS Lett. 1998; 427: 164-170Crossref PubMed Scopus (27) Google Scholar). Surface expression of HA-tagged EpoR was evaluated by flow cytometry (FACScan). BaF3 cells selected in puromycin were incubated with rat anti-HA (Roche Molecular Biochemicals) as primary antibody and anti-rat IgG coupled to Cy5 (Dianova, Hamburg, Germany) as secondary antibody and analyzed for green and red fluorescence by flow cytometry. BaF3 cells expressing the wild type EpoR or the EpoR-GFP chimera were starved for 3 h in RPMI with 1 mg/ml bovine serum albumin and then stimulated for 5 min at 37 °C with 50 units/ml Epo. Detergent lysates equivalent to 1 × 107 cells were prepared using Nonidet P-40 buffer as described (8Klingmüller U. Lorenz U. Cantley L.C. Neel B.G. Lodish H.F. Cell. 1995; 80: 729-738Abstract Full Text PDF PubMed Scopus (846) Google Scholar) and subjected to immunoprecipitation using anti-EpoR (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-JAK2 (Upstate Biotechnology, Inc., Lake Placid, NY), anti-STAT5b (Santa Cruz Biotechnology), anti-p85 subunit of phosphatidylinositol 3-kinase (kindly provided by Dr. Lewis Cantley, Harvard Medical School, Boston, MA), and anti-SHP1 (Santa Cruz Biotechnology) antiserum. The immunoprecipitates were eluted, resolved by 15% SDS-PAGE, and transferred to a nitrocellulose membrane. Detection by immunoblotting was performed with an anti-phosphotyrosine monoclonal antibody (4G10, Upstate Biotechnology, Inc., Lake Placid, NY) followed by enhanced chemoluminescence (Amersham Biosciences). The blots were stripped and reprobed with anti-EpoR, anti-STAT5b, anti-JAK2, anti-SHP-1 (all purchased from Santa Cruz Biotechnology), and the anti-p85 subunit of phosphatidylinositol 3-kinase antiserum (kindly provided by Dr. Lewis Cantley). BaF3 cells expressing the wild type EpoR or the EpoR-GFP chimera were washed three times with RPMI and plated at a density of 5 × 104 cells/well in 24-well plates in the presence of Epo concentrations ranging from 0.1 to 10 units/ml or 10% WEHI conditioned medium as a source for interleukin-3. After 3 days, cell numbers were determined using a Coulter counter and expressed as the percentage of growth obtained in a parallel well containing 10% WEHI conditioned medium instead of Epo. The localization of GFP-EpoR fusion proteins and HA-tagged EpoR was assessed in 293T cells transiently transfected with retroviral expression vector constructs. The cells were grown on coverslips in six-well plates and either directly analyzed by immunofluorescence or fixed with 3%para-formaldehyde for 15 min at room temperature prior to immunostaining. For co-staining of HA-tagged receptors, the cells were permeabilized with 0.2% Triton X-100 in phosphate-buffered saline. After three washes in phosphate-buffered saline, the cells were incubated with an antibody raised against HA (Roche Molecular Biochemicals). After three washes, the cells were incubated with an anti-rat IgG coupled to Alexa594 (Molecular Probes, Inc., Eugene, OR). All incubations with antibodies were performed at 4 °C in phosphate-buffered saline supplemented with 0.3% bovine serum albumin. The antibodies were used as 1:100 dilutions. Fetal liver cells from day 13.5 Balb/c were grown on coverslips precoated with 0.2% gelatin (Sigma) for 20 h in Iscove's modified Eagle's medium, 30% fetal calf serum supplemented with 0.5 unit/ml Epo. The cells were washed and analyzed with a Leica DM IRE2 confocal microscope. To visualize the EpoR and to check whether a visible EpoR is capable of activation of signal transduction in response to ligand binding, we inserted the GFP at four positions of the EpoR (Fig.1). In the resulting chimeric proteins, GFP is either located at the junction between the extracellular and TM domains (EpoR-GFP1) or at various positions within the cytoplasmic domain. In EpoR-GFP2, the insertion of GFP directly after the TM domain alters the spacing between the hydrophobic juxtamembrane motif and the JAK2 binding sites, whereas in EpoR-GFP3 the JAK2-activating domain is separated from the eight cytosolic tyrosine residues that mediate the recruitment of signaling molecules. The least invasive chimeric receptor is EpoR-GFP4, where GFP is fused to the C terminus of the EpoR. To test the functionality of the EpoR-GFP receptors, wild type EpoR and chimeric receptors were stable expressed in the interleukin-3-dependent pro-B cell line BaF3. Analysis of total cell lysates by immunoblotting with anti-EpoR antiserum revealed that EpoR-GFP1, EpoR-GFP2, and EpoR-GFP3 were expressed at levels comparable with wild type EpoR, whereas EpoR-GFP4 reproducibly showed reduced expression levels (Fig.2 A). To evaluate whether GFP insertion affected surface transport of the chimeric receptors, we measured 125I-Epo binding to BaF3 cells stable expressing the EpoR derivatives. As shown in Fig. 2 B, chimeric receptors harboring the GFP insertion in the cytoplasmic domain bound the ligand to a similar degree as wild type EpoR. It should be noted that Epo binding to EpoR-GFP2 was reproducibly enhanced. However, EpoR-GFP1 that contains GFP in the extracellular domain did not show significant Epo binding. To distinguish whether the lack of Epo binding was caused by the inability to engage the ligand or by impaired cell surface expression, we introduced an HA tag in the extracellular domain of EpoR-GFP1, EpoR-GFP2, and wild type EpoR. Flow cytometry analysis of BaF3 cells stable expressing the HA-tagged receptors showed that whereas wild type EpoR and EpoR-GFP2 were detected on the cell surface, EpoR-GFP1 was below the detection limit (Fig. 2 C). This suggests that GFP insertion in the extracellular domain of the EpoR blocks transport to the cell surface, whereas insertion at various positions of the cytoplasmic domain does not impair cell surface prevalence. To elucidate whether GFP insertion in the cytoplasmic domain of the EpoR affected the activation of signal transduction, Epo-induced signaling was studied in BaF3 cells expressing wild type EpoR or the EpoR-GFP chimera. Tyrosine-phosphorylated JAK2 and EpoR were measured as indicators for Epo-mediated signal transduction (Fig.3 A). Detergent lysates of cells left untreated or stimulated with Epo were subjected to immunoprecipitation with anti-EpoR or anti-JAK2 antiserum and subsequently analyzed by immunoblotting with an anti-phosphotyrosine monoclonal antibody. As expected, ligand addition to cells expressing wild type receptor resulted in efficient tyrosine phosphorylation of the EpoR and JAK2. A receptor chimera that is not transported to the cell surface (EpoR-GFP1) was unable to trigger tyrosine phosphorylation of the receptor or JAK2. However, despite its presence on the cell surface, EpoR-GFP2 was not able to activate signal transduction, suggesting that structural continuity of the hydrophobic juxtamembrane domain motif and the JAK2 binding sites is required for efficient signal conversion. The chimeric receptor EpoR-GFP4 was tyrosine-phosphorylated upon Epo addition, albeit to a lower extent than wild type EpoR. This may be due to the reduced expression of this receptor variant. Surprisingly, a receptor chimera containing the GFP insertion in the middle of the cytoplasmic domain (EpoR-GFP3) mediated JAK2 and EpoR tyrosine phosphorylation, indicating that the cytosolic domain of the EpoR is capable of coordinating JAK2 activation and receptor tyrosine phosphorylation despite physical separation by GFP insertion. Whereas the unphosphorylated forms of EpoR-GFP3 and EpoR-GFP4 showed comparable mobility, indicating that GFP insertion had no major effect, the tyrosine-phosphorylated form of EpoR-GFP3 showed higher mobility. Therefore, we asked whether this is caused by partial tyrosine phosphorylation of EpoR-GFP3. The phosphorylation of critical tyrosine residues in EpoR-GFP3 compared with EpoR-GFP4 was determined by their capacity to bind the Src homology 2 domain-containing signaling molecules STAT5, SHP-1, and p85 (Fig. 3 B). As evidenced by immunoprecipitation experiments from detergent lysates of cells that were left either unstimulated or treated with Epo, both the tyrosine-phosphorylated forms of EpoR-GFP3 and EpoR-GFP4 were able to associate with STAT5, SHP1, and p85 comparable with wild type EpoR. Therefore, both receptor chimera are indistinguishable regarding their capacity to recruit signaling molecules. It is possible that underphosphorylation of one of the tyrosine residues to which binding partners have not yet been identified accounts for the difference in electrophoretic mobility. To test whether the initiation of signal transduction mediated by the chimeric receptors resulted in efficient biological responses, we first tested the capacity of the EpoR-GFP receptors to support the growth of BaF3 cells in the presence of Epo. BaF3 cells expressing either wild type EpoR or various EpoR-GFP chimeras were cultured in the presence of increasing concentrations of Epo ranging from 0.1 to 10 Epo units/ml for 3 days. The cell numbers shown in Fig.4 A indicate that EpoR-GFP3 supported cell proliferation to a similar extent as wild type receptor, in particular at low Epo concentration, whereas EpoR-GFP4 showed reduced capacity in promoting proliferation. Confirming the biochemical analysis, EpoR-GFP chimera that did not activate signal transduction was unable to support proliferation of BaF3 cells in the presence of Epo. To further test the biological function of the EpoR-GFP chimera, the receptors were introduced into fetal liver cells of EpoR−/− mice by retroviral transduction and tested for their ability to support the formation of CFU-E colonies in the presence of Epo. In agreement with the cell proliferation experiments, EpoR-GFP3 supported similar numbers of CFU-E colonies compared with wild type EpoR, whereas EpoR-GFP4 reproducibly resulted in a lower number of CFU-E colonies. Again, EpoR-GFP1 and EpoR-GFP2 were unable to promote proliferation and terminal differentiation of erythroid progenitor cells. Thus, unexpectedly, an EpoR-GFP receptor containing GFP inserted in the middle of the cytoplasmic domain was functionally indistinguishable from the wild type EpoR, whereas direct fusion of GFP to the C terminus of the EpoR resulted in a receptor with reduced activity. To test whether the EpoR-GFP chimeras are detectable by fluorescence microscopy and whether the fluorescence intensity is sufficient to monitor EpoR trafficking in living cells, we analyzed the chimeric receptors expressed in transiently transfected 293T cells by confocal microscopy (Fig.5 A). The signaling-competent receptors EpoR-GFP3 and EpoR-GFP4 were detectable in intracellular structures resembling the ER, the Golgi, and punctated endosome-like structures. We performed overlay analysis of EpoR-GFP3 and a transiently expressed HA-tagged EpoR detected by anti-HA immunostaining. The HA-tagged EpoR is functionally indistinguishable from wild type EpoR (21Constantinescu S.N. Keren T. Socolovsky M. Nam H. Henis Y.I. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4379-4384Crossref PubMed Scopus (220) Google Scholar) and showed similar subcellular localization (data not shown), thus confirming that the enrichment in punctated structures is not caused by GFP insertion. In erythroid progenitor cells, accumulation of EpoR-GFP3 in similar punctated structures was observed (Fig. 5 B). Expression in other intracellular compartments was observed but was much dimmer compared with the bright endosome-like structures. The EpoR-GFP1 chimera that is unable to reach the cell surface predominantly resides within the ER network, and cells transfected with EpoR-GFP2, a receptor that is transported to the cell surface yet unable to trigger the activation of signaling, show an intermediate phenotype. The EpoR-GFP2 receptor predominantly remains in the ER, and only a minor portion is enriched in punctated structures. Our analysis shows that the EpoR-GFP chimeric receptors facilitate the detection of EpoR and trafficking in living cells and therefore provide the possibility to visualize dynamic processes in vivo. Signal conversion through cytokine receptors relies on intricate communication between the extracellular ligand binding domain and the cytosolic domain that mediates recruitment of signaling molecules. Here, we demonstrate that signal transduction via the EpoR, a member of the cytokine receptor superfamily, can occur despite physical separation of the JAK2 binding site from the cytosolic tyrosine residues, which are phosphorylated upon stimulation with Epo. Insertion of GFP at the junction between the extracellular and TM domains results in a chimeric receptor not transported to the cell surface and unable to bind ligand. This phenotype is reminiscent of mutations in the WSXWS motif in the EpoR extracellular domain, since deletion or alterations in the WSXWS motif resulted in receptors that were retained in the ER and unable to interact with the ligand (22Yoshimura A. Longmore G. Lodish H.F. Nature. 1990; 348: 647-649Crossref PubMed Scopus (271) Google Scholar). The WSXWS motif is conserved in the extracellular domain of cytokine receptors and was initially believed to be involved in ligand binding. However, the crystal structures of the extracellular domain of the growth hormone receptor (23de Vos A.M. Ultsch M. Kossiakoff A.A. Science. 1992; 255: 306-312Crossref PubMed Scopus (2042) Google Scholar) and the EpoR (24Syed R.S. Reid S.W., Li, C. Cheetham J.C. Aoki K.H. Liu B. Zhan H. Osslund T.D. Chirino A.J. Zhang J. Finer-Moore J. Elliott S. Sitney K. Katz B.A. Matthews D.J. Wendoloski J.J. Egrie J. Stroud R.M. Nature. 1998; 395: 511-515Crossref PubMed Scopus (491) Google Scholar) showed that the WSXWS motif is located away from the interfaces that bind the respective ligand. The phenotype of the WSXWS mutants rather suggested that the intact motif is necessary for correct trafficking of the receptor. Our results indicate that not only the amino acid sequence of the motif but also the spatial localization in close proximity to the cell membrane could be critical for successful transport of the EpoR to the cell surface. Recent evidence suggests that JAK2 recruitment to the EpoR mediated by a continuous block of residues in the membrane-proximal segment of the cytoplasmic domain is required for EpoR cell surface expression (6Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Our analysis of EpoR-GFP2 shows that increasing the distance between the JAK2 binding motif in the cytoplasmic part of the EpoR and the cell membrane does not disturb the surface prevalence of the EpoR. However, physical separation of the JAK2 binding sites from the precisely oriented hydrophobic motif in the juxtamembrane segment (5Constantinescu S.N. Huang L.J. Nam H. Lodish H.F. Mol. Cell. 2001; 7: 377-385Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) abrogates the activation of signal transduction. This suggests that the ligand binding domain, the TM domain, the membrane-proximal hydrophobic patch, and the JAK2 binding sites are organized in a structurally rigid entity that requires precise spatial alignment to activate signal transduction. The major part of the cytosolic domain encompassing box 2 and the eight tyrosine residues is contained in exon 8 of the EpoR genomic locus, suggesting a conserved functional entity. Yet we show that insertion of GFP in the middle of the cytoplasmic domain results in a chimeric receptor (EpoR-GFP3) capable of initiating signal transduction and biological responses comparable with wild type EpoR. The cytoplasmic domain of the EpoR is partially unfolded in the absence of JAK2 (25Remy I. Wilson I.A. Michnick S.W. Science. 1999; 283: 990-993Crossref PubMed Scopus (541) Google Scholar), indicating that JAK2 acts as a molecular chaperone (6Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) and is required for structural organization of the cytoplasmic domain. Our results demonstrate that tyrosine phosphorylation of the cytoplasmic domain is maintained despite physical separation of the JAK2-activating domain from the segment harboring the tyrosine residues. This suggests that in the activated state, JAK2 possesses flexibility in accessing substrate tyrosine residues and/or that additional JAK2 coordination sites exist in the membrane-distal segment of the EpoR cytoplasmic domain (6Huang L.J. Constantinescu S.N. Lodish H.F. Mol. Cell. 2001; 8: 1327-1338Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The possibility that another kinase can compensate for JAK2 is rather unlikely, since JAK2 null mice show a dramatic phenotype with fetal anemia and embryonic lethality at day 12.5 comparable with the EpoR null mice (26Neubauer H. Cumano A. Muller M., Wu, H. Huffstadt U. Pfeffer K. Cell. 1998; 93: 397-409Abstract Full Text Full Text PDF PubMed Scopus (697) Google Scholar, 27Parganas E. Wang D. Stravopodis D. Topham D.J. Marine J.C. Teglund S. Vanin E.F. Bodner S. Colamonici O.R. van Deursen J.M. Grosveld G. Ihle J.N. Cell. 1998; 93: 385-395Abstract Full Text Full Text PDF PubMed Scopus (924) Google Scholar). Previous studies in other receptor systems have been limited to the analysis of C-terminally GFP-tagged receptors (28Silverman M.A. Kaech S. Jareb M. Burack M.A. Vogt L. Sonderegger P. Banker G. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7051-7057Crossref PubMed Scopus (77) Google Scholar, 29Wouters F.S. Bastiaens P.I. Curr. Biol. 1999; 9: 1127-1130Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). However, the EpoR that contains GFP fused to the C terminus (EpoR-GFP4) is expressed at reduced levels and has a decreased capacity to promote the formation of CFU-E colonies. In this chimeric protein, GFP is localized in close proximity to Tyr479, a residue that has been shown to be important for the recruitment of the lipid kinase phosphoinositide 3-kinase (12Klingmüller U., Wu, H. Hsiao J.G. Toker A. Duckworth B.C. Cantley L.C. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3016-3021Crossref PubMed Scopus (150) Google Scholar, 13Damen J.E. Cutler R.L. Jiao H., Yi, T. Krystal G. J. Biol. Chem. 1995; 270: 23402-23408Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and sufficient in the absence of other tyrosine residues to promote the biological functions of the EpoR (12Klingmüller U., Wu, H. Hsiao J.G. Toker A. Duckworth B.C. Cantley L.C. Lodish H.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3016-3021Crossref PubMed Scopus (150) Google Scholar, 30Longmore G.D. You Y. Molden J. Liu K.D. Mikami A. Lai S.Y. Pharr P. Goldsmith M.A. Blood. 1998; 91: 870-878Crossref PubMed Google Scholar). Indeed, further separation of Tyr479 and GFP improved signal transmission and the capacity to support the biological functions, although the overall expression levels remained reduced. 2R. Ketteler and U. Klingmüller, manuscript in preparation. In summary, we show by marking a hematopoietic cytokine receptor with a GFP insertion that the extracellular, transmembrane, and membrane-proximal domains form a rigid structure whose specific orientation is essential for initiating signal transduction in response to ligand binding. However, we propose that additional coordinating mechanisms exist, since long range activation of the membrane-distal part is possible, providing a novel concept how ligand binding is converted to receptor activation. We thank Susanne Esser and Melanie Wickert for excellent technical assistance. The anti-p85 antiserum was generously provided by Lewis C. Cantley, and Stefan Constantinescu kindly provided the pMX-HA-EpoR-IRES-GFP. We thank Stephan Kuppig for help with the confocal microscope. We thank Dr. Hong Hu for providing EpoR knockout mice.

Hippo signalling is an evolutionarily conserved pathway that controls organ size by regulating apoptosis, cell proliferation, and stem cell self-renewal. Recently, the pathway has been shown to exert powerful growth regulatory activity in cardiomyocytes. However, the functional role of this stress-related and cell death-related pathway in the human heart and cardiomyocytes is not known. In this study, we investigated the role of the transcriptional co-activators of Hippo signalling, YAP and TAZ, in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in response to cardiotoxic agents and investigated the effects of modulating the pathway on cardiomyocyte function and survival.RNA-sequencing analysis of human heart samples with doxorubicin-induced end-stage heart failure and healthy controls showed that YAP and ERBB2 (HER2) as upstream regulators of differentially expressed genes correlated with doxorubicin treatment. Thus, we tested the effects of doxorubicin on hiPSC-CMs in vitro. Using an automated high-content screen of 96 clinically relevant antineoplastic and cardiotherapeutic drugs, we showed that doxorubicin induced the highest activation of YAP/TAZ nuclear translocation in both hiPSC-CMs and control MCF7 breast cancer cells. The overexpression of YAP rescued doxorubicin-induced cell loss in hiPSC-CMs by inhibiting apoptosis and inducing proliferation. In contrast, silencing of YAP and TAZ by siRNAs resulted in elevated mitochondrial membrane potential loss in response to doxorubicin. hiPSC-CM calcium transients did not change in response to YAP/TAZ silencing.Our results suggest that Hippo signalling is involved in clinical anthracycline-induced cardiomyopathy. Modelling with hiPSC-CMs in vitro showed similar responses to doxorubicin as adult cardiomyocytes and revealed a potential cardioprotective effect of YAP in doxorubicin-induced cardiotoxicity.

The formation of signal-promoting dimeric or oligomeric receptor complexes at the cell surface is modulated by self-interaction of their transmembrane (TM) domains. To address the importance of TM domain packing density for receptor functionality, we examined a set of asparagine mutants in the TM domain of the erythropoietin receptor (EpoR). We identified EpoR-T242N as a receptor variant that is present at the cell surface similar to wild-type EpoR but lacks visible localization in vesicle-like structures and is impaired in efficient activation of specific signaling cascades. Analysis by a molecular modeling approach indicated an increased interhelical distance for the EpoR-T242N TM dimer. By employing the model, we designed additional mutants with increased or decreased packing volume and confirmed a correlation between packing volume and biological responsiveness. These results propose that the packing density of the TM domain provides a novel layer for fine-tuned regulation of signal transduction and cellular decisions.

High-content screening of kinase inhibitors is important in order to identify biogenesis and function mechanisms of subcellular organelles. Here, we present a human kinome siRNA high-content screen on primary human umbilical vein endothelial cells, that were transfected by electroporation. The data descriptor contains a confocal fluorescence, microscopic image dataset. We also describe an open source, automated image analysis workflow that can be reused to perform high-content analysis of other organelles. This dataset is suitable for analysis of morphological parameters that are linked to human umbilical vein endothelial cell (HUVEC) biology.

Cell-based screening assays are frequently used in drug discovery and academic research to identify cellular phenotypes in response to a genetic or chemical perturbation. When investigating cell biology processes, the use of high-content imaging facilitates the analysis of a large number of samples within a reasonably short time frame and at moderate costs. Here, we will provide an overview of the high-content screening process, from experimental design to available platform technologies and bioinformatics analysis.

Primary neurons in culture are considered to be a highly relevant model in the study of neuronal development and activity. They can be cultivated and differentiated in vitro but are difficult to transfect using conventional methods. To address this problem, a capillary electroporation system called Cellaxess Elektra was developed for efficient and reproducible transfection of primary cortical and hippocampal neurons without significant impact on cell morphology and viability. The cells are transfected in any stage of differentiation and development, directly in cell culture plates. Genetic material is delivered in situ to as many as 384 samples at a time, which enables both high-throughput and high-quality screening for hard-to-transfect primary cells, meaning that gene function can be studied on a genome-wide scale in cells previously inaccessible to genetic manipulation.

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways(1-3). Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus(4-6). Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

Scientific Data 4:170022 doi:http://dx.doi.org/10.1038/sdata.2017.22 (2017); Published 1 Mar 2017; Updated 27 Jun 2017 The authors regret that Nicola Stevenson was omitted in error from the author list of the original version of this Data Descriptor. This omission has now been corrected in the HTML and PDF versions of this Data Descriptor, as well as the accompanying Corrigendum.

Mitochondrial dysfunction is linked to many neurological diseases; therefore, the ability to measure mitochondrial function is of great use for researching disease and testing potential therapeutics. Here we describe a high-content assay to simultaneously measure mitochondrial membrane potential, morphology and cell viability in iPSC-derived neurons. Neurons are seeded into plates suitable for fluorescent microscopy, stained with the mitochondrial membrane potential-dependent dye TMRM, cytoplasmic dye Calcein AM, and nuclear stain Hoechst 33342. Images are acquired in live cells and analyzed using automated image analysis software.

Scientific Reports 6: Article number: 32473; published online: 31 August 2016; updated: 26 September 2016. The original version of this Article contained a typographical error in the spelling of the author Mafalda Lopes da Silva which was incorrectly given as Silva Mafalda Lopes da. This has now been corrected in the PDF and HTML versions of the Article.

The quality control of mitochondria is critical for the survival of cells, and defects in the pathways required for this quality control can lead to severe disease. A key quality control mechanism in cells is mitophagy, which functions to remove damaged mitochondria under conditions of various stresses. Defective mitophagy can lead to a number of diseases including neurodegeneration. It has been proposed that an enhancement of mitophagy can improve cell survival, enhance neuronal function in neurodegeneration and extend health and lifespans. In this review, we highlight the role of deubiquitinating enzymes (DUBs) in the regulation of mitophagy. We summarise the current knowledge on DUBs that regulate mitophagy as drug targets and provide a list of small molecule inhibitors that are valuable tools for the further development of therapeutic strategies targeting the mitophagy pathway in neurodegeneration.

Induced pluripotent stem cell (iPSC) derived neurons are an excellent in vitro model of neurological diseases that are often used in early stage drug discovery projects. Thus far, the use of iPSC-derived cells in small molecule drug screening has been limited, and one of the reasons for this has been the challenge of miniaturization of iPSC culture and differentiation in low volume microwell plate formats. Here we describe a method of seeding iPSC-derived neurons into 384-well plates towards the end of the differentiation procedure. This method covers coating the plates with substrates to aid attachment, dissociation of the cells into a single cell suspension, and seeding onto 384-well plates to give an even distribution of neurons. This method facilitates the use of iPSC-derived neurons for high-content imaging, whole-well assays, and small-molecule drug screening.

Oral presentations Session 1: Entry & uncoating O1 Host cell polo-like kinases (PLKs) promote early prototype foamy virus (PFV) replication Irena Zurnic, Sylvia Hütter, Ute Lehmann, Nicole Stanke, Juliane Reh, Tobias Kern, Fabian Lindel, Gesche Gerresheim, Martin Hamann, Erik Müllers, Paul Lesbats, Peter Cherepanov, Erik Serrao, Alan Engelman, Dirk Lindemann O2 A novel entry/uncoating assay reveals the presence of at least two species of viral capsids during synchronized HIV-1 infection Claire Da Silva Santos, Kevin Tartour, Andrea Cimarelli O3 Dynamics of nuclear envelope association and nuclear import of HIV-1 complexes Rya Burdick, Jianbo Chen, Jaya Sastri, Wei-Shau Hu, Vinay Pathak O4 Human papillomavirus protein E4 potently enhances the susceptibility to HIV infection Oliver T. Keppler Session 2: Reverse transcription & integration O5 Structure and function of HIV-1 integrase post translational modifications Karine Pradeau, Sylvia Eiler, Nicolas Levy, Sarah Lennon, Sarah Cianferani, Stéphane Emiliani, Marc Ruff O6 Regulation of retroviral integration by RNA polymerase II associated factors and chromatin structure Vincent Parissi Session 3: Transcription and latency O7 A novel single-cell analysis pipeline to identify specific biomarkers of HIV permissiveness Sylvie Rato, Antonio Rausell, Miguel Munoz, Amalio Telenti, Angela Ciuffi O8 A capsid-dependent integration program linking T cell activation to HIV-1 gene expression Alexander Zhyvoloup, Anat Melamed, Ian Anderson, Delphine Planas, Janos Kriston-Vizi, Robin Ketteler, Chen-Hsuin Lee, Andy Merritt, Petronela Ancuta, Charles Bangham, Ariberto Fassati O9 Characterisation of new RNA polymerase III and RNA polymerase II transcriptional promoters in the Bovine Leukemia Virus genome Anthony Rodari, Benoit Van Driessche, Mathilde Galais, Nadége Delacourt, Sylvain Fauquenoy, Caroline Vanhulle, Anna Kula, Arsène Burny, Olivier Rohr, Carine Van Lint O10 Tissue-specific dendritic cells differentially modulate latent HIV-1 reservoirs Thijs van Montfort, Renee van der Sluis, Dave Speijer, Ben Berkhout Session 4: RNA trafficking & packaging O11 A novel cis-acting element affecting HIV replication Bo Meng, Andrzej Rutkowski, Neil Berry, Lars Dölken, Andrew Lever O12 Tolerance of HIV’s late gene expression towards stepwise codon adaptation Thomas Schuster, Benedikt Asbach, Ralf Wagner Session 5: Assembly & release O13 Importance of the tax-inducible actin-bundling protein fascin for transmission of human T cell leukemia virus Type 1 (HTLV-1) Christine Gross, Veit Wiesmann, Martina Kalmer, Thomas Wittenberg, Jan Gettemans, Andrea K. Thoma-Kress O14 Lentiviral nef proteins antagonize TIM-mediated inhibition of viral release Minghua Li, Eric O. Freed, Shan-Lu Liu Session 6: Pathogenesis & evolution O15 SEVI and semen prolong the half-life of HIV-1 Janis Müller, Jan Münch O16 CD169+ macrophages mediate retrovirus trans-infection of permissive lymphocytes to establish infection in vivo Xaver Sewald, Pradeep Uchil, Mark Ladinsky, Jagadish Beloor, Ruoxi Pi, Christin Herrmann, Nasim Motamedi, Thomas Murooka, Michael Brehm, Dale Greiner, Thorsten Mempel, Pamela Bjorkman, Priti Kumar, Walther Mothes O17 Efficient replication of a vpu containing SIVagm construct in African Green Monkeys requires an HIV-1 nef gene Simone Joas, Erica Parrish, Clement Wesley Gnanadurai, Edina Lump, Christina M. Stürzel, Nicholas F. Parrish, Ulrike Sauermann, Katharina Töpfer, Tina Schultheiss, Steven Bosinger, Guido Silvestri, Cristian Apetrei, Nicholas Huot, Michaela Müller-Trutwin, Daniel Sauter, Beatrice H. Hahn, Christiane Stahl-Hennig, Frank Kirchhoff O18 Reprogramming initiates mobilization of endogenous mutagenic LINE-1, Alu and SVA retrotransposons in human induced pluripotent stem cells with consequences for host gene expression Gerald Schumann, Sabine Jung-Klawitter, Nina V. Fuchs, Kyle R. Upton, Martin Muñoz-Lopez, Ruchi Shukla, Jichang Wang, Marta Garcia-Canadas, Cesar Lopez-Ruiz, Daniel J. Gerhardt, Attila Sebe, Ivana Grabundzija, Patricia Gerdes, Sylvia Merkert, Andres Pulgarin, Anja Bock, Ulrike Held, Anett Witthuhn, Alexandra Haase, Ernst J. Wolvetang, Ulrich Martin, Zoltán Ivics, Zsuzsanna Izsvák, J. Garcia-Perez, Geoffrey J. Faulkner O19 NF-κB activation induces expression of human endogenous retrovirus and particle production Tara Hurst, Aris Katzourakis, Gkikas Magiorkinis Session 7a and b: Innate sensing & intrinsic immunity O20 Identification of the phosphatase acting on T592 in SAMHD1 during M/G1 transition Kerstin Schott, Rita Derua, Janna Seifried, Andreas Reuter, Heike Schmitz, Christiane Tondera, Alberto Brandariz-Nuñez, Felipe Diaz-Griffero, Veerle Janssens, Renate König O21 Vpx overcomes a SAMHD1-independent block to HIV reverse transcription that is specific to resting CD4 T cells Hanna-Mari Baldauf, Lena Stegmann, Sarah-Marie Schwarz, Maud Trotard, Margarethe Martin, Gina Lenzi, Manja Burggraf, Xiaoyu Pan, Oliver I. Fregoso, Efrem S. Lim, Libin Abraham, Elina Erikson, Laura Nguyen, Ina Ambiel, Frank Rutsch, Renate König, Baek Kim, Michael Emerman, Oliver T. Fackler, Oliver T. Keppler O22 The role of SAMHD1 in antiviral restriction and immune sensing in the mouse Sabine Wittmann, Rayk Behrendt, Bianca Volkmann, Kristin Eissmann, Thomas Gramberg O23 T cells expressing reduced restriction factors are preferentially infected in therapy naïve HIV-1 patients Sebastian Bolduan, Herwig Koppensteiner, Stefanie Regensburg, Ruth Brack-Werner, Rika Draenert, Michael Schindler O24 cGAS-mediated innate immunity spreads through HIV-1 env-induced membrane fusion sites from infected to uninfected primary HIV-1 target cells Aurélie Ducroux, Shuting Xu, Aparna Ponnurangam, Sergej Franz, Angelina Malassa, Ellen Ewald, Christine Goffinet O25 Perturbation of innate RNA and DNA sensing by human T cell leukemia virus type 1 oncoproteins Sin-Yee Fung, Ching-Ping Chan, Chun-Kit Yuen, Kin-Hang Kok, Chin-Ping Chan, Dong-Yan Jin O26 Induction and anti-viral activity of Interferon α subtypes in HIV-1 infection Ulf Dittmer O27 Vpu-mediated counteraction of tetherin is a major determinant of HIV-1 interferon resistance Dorota Kmiec, Shilpa Iyer, Christina Stürzel, Daniel Sauter, Beatrice Hahn, Frank Kirchhoff O28 DNA repair protein Rad18 restricts HIV-1 and LINE-1 life cycle Yasuo Ariumi, Mariko Yasuda-Inoue, Koudai Kawano, Satoshi Tateishi, Priscilla Turelli O29 Natural mutations in IFITM3 allow escape from post-translational regulation and toggle antiviral specificity Alex Compton, Nicolas Roy, Françoise Porrot, Anne Billet, Nicoletta Casartelli, Jacob Yount, Chen Liang, Oliver Schwartz Session 8: Adaptive immunity & immune evasion O30 Observing evolution in HIV-1 infection: phylogenetics and mutant selection windows to infer the influence of the autologous antibody response on the viral quasispecies Carsten Magnus, Lucia Reh, Penny Moore, Therese Uhr, Jacqueline Weber, Lynn Morris, Alexandra Trkola O31 Dose and subtype specific analyses of the anti-HIV effects of IFN-alpha family members Rashel V. Grindberg, Erika Schlaepfer, Gideon Schreiber, Viviana Simon, Roberto F. Speck Session 9: Novel antiviral strategies O32 LEDGIN-mediated inhibition of the integrase-LEDGF/p75 interaction reduces reactivation of residual latent HIV Zeger Debyser, Lenard Vranckx, Jonas Demeulemeester, Suha Saleh, Eric Verdin, Anna Cereseto, Frauke Christ, Rik Gijsbers O33 NKG2D-mediated clearance of reactivated viral reservoirs by natural killer cells O34 Inhibition of HIV reactivation in brain cells by AAV-mediated delivery of CRISPR/Cas9 O35 CRISPR-Cas9 as antiviral: potent HIV-1 inhibition, but rapid virus escape and the subsequent design of escape-proof antiviral strategies Ben Berkhout, Gang Wang, Na Zhao, Atze T. Das Session 10: Recent advances in HIV vaccine development O36 Priming with a potent HIV-1 DNA vaccine frames the quality of T cell and antibody responses prior to a poxvirus and protein boost Benedikt Asbach, Josef Köstler, Beatriz Perdiguero, Mariano Esteban, Bertram L. Jacobs, David C. Montefiori, Celia C. LaBranche, Nicole L. Yates, Georgia D. Tomaras, Guido Ferrari, Kathryn E. Foulds, Mario Roederer, Gary Landucci, Donald N. Forthal, Michael S. Seaman, Natalie Hawkins, Steven G. Self, Sanjay Phogat, James Tartaglia, Susan W. Barnett, Brian Burke, Anthony D. Cristillo, Song Ding, Jonathan L. Heeney, Giuseppe Pantaleo, Ralf Wagner O37 Passive immunisation with a neutralising antibody against HIV-1 Env prevents infection of the first cells in a mucosal challenge rhesus monkey model Christiane Stahl-Hennig, Viktoria Stab, Armin Ensser, Ulrike Sauermann, Bettina Tippler, Dennis Burton, Matthias Tenbusch, Klaus Überla O38 HIV antibody Fc-glycoforms drive B cell affinity maturation Galit Alter, Giuseppe Lofano, Anne-Sophie Dugast, Viraj Kulkarni, Todd Suscovich Poster presentations Topic 1: Entry & uncoating P1 Dynein light chain is required for murine leukemia virus infection Tatiana Opazo, Felipe Barraza, Diego Herrera, Andrea Garces, Tomas Schwenke, Diego Tapia, Jorge Cancino, Gloria Arriagada P2 Peptide paratope mimics of the broadly neutralising HIV-1 antibody b12 Christina Haußner, Dominik Damm, Anette Rohrhofer, Barbara Schmidt, Jutta Eichler P3 Investigating cellular pathways involved in the transmission of HIV-1 between dendritic cells and T cells using RNAi screening techniques Rebecca Midgley, James Wheeldon, Vincent Piguet P4 Co-receptor tropism in HIV-1, HIV-2 monotypic and dual infections Priyanka Khopkar, Megha Rohamare, Smita Kulkarni P5 Characterisation of the role of CIB1 and CIB2 as HIV-1 helper factors Ana Godinho-Santos, Allan Hance, Joao Goncalves, Fabrizio Mammano P6 Buffering deleterious polymorphisms in the highly constrained C2 region of HIV-1 envelope by the flexible V3 domain Romain Gasser, Meriem Hamoudi, Martina Pellicciotta, Zhicheng Zhou, Clara Visdeloup, Philippe Colin, Martine Braibant, Bernard Lagane, Matteo Negroni P7 Entry inhibition of HERV-K(HML-2) by an Env-IgG fusion protein Jula Wamara, Norbert Bannert Topic 2: Reverse transcription & integration P8 The R263K/H51Y resistance substitutions in HIV integrase decreases levels of integrated HIV DNA over time Thibault Mesplede, Nathan Osman, Kaitlin Anstett, Jiaming Calvin Liang, Hanh Thi Pham, Mark Wainberg P9 The Retrovirus Integration Database (RID) Wei Shao, Jigui Shan, Mary Kearney, Xiaolin Wu, Frank Maldarelli, John Mellors, Brian Luke, John Coffin, Stephen Hughes P10 The small molecule 3G11 inhibits HIV-1 reverse transcription Thomas Fricke, Silvana Opp, Caitlin Shepard, Dmitri Ivanov, Baek Kim, Jose Valle-Casuso, Felipe Diaz-Griffero P11 Dual and opposite regulation of HIV-1 integration by hRAD51: impact on therapeutical approaches using homologous DNA repair modulators Vincent Parissi P12 A flexible motif essential for integration by HIV-1 integrase Marine Kanja, Pierre Cappy, Matteo Negroni, Daniela Lener P13 Interaction between HIV-1 integrase and the host protein Ku70: identification of the binding site and study of the influence on integrase-proteasome interplay Ekaterina Knyazhanskaya, Andrey Anisenko, Timofey Zatsepin, Marina Gottikh P14 Normalisation based method for deep sequencing of somatic retroelement integrations in human genome Alexander Komkov, Anastasia Minervina, Gaiaz Nugmanov, Vadim Nazarov, Konstantin Khodosevich, Ilgar Mamedov, Yuri Lebedev Topic 3: Transcription and latency P15 BCA2/RABRING7 restricts HIV-1 transcription by preventing the nuclear translocation of NF-κB Marta Colomer-Lluch, Ruth Serra-Moreno P16 MATR3 post-transcriptional regulation of HIV-1 transcription during latency Ambra Sarracino, Anna Kula, Lavina Gharu, Alexander Pasternak, Carine Van Lint, Alessandro Marcello P17 HIV-1 tat intersects the SUMO pathway to regulate HIV-1 promoter activity Ann Marie McCartin, Anurag Kulkarni, Valentin Le Douce, Virginie Gautier P18 Conservation in HIV-1 Vpr guides tertiary gRNA folding and alternative splicing Ann Baeyens, Evelien Naessens, Anouk Van Nuffel, Karin Weening, Anne-Marie Reilly, Eva Claeys, Wim Trypsteen, Linos Vandekerckhove, Sven Eyckerman, Kris Gevaert, Bruno Verhasselt P19 The majority of reactivatable latent HIV are genetically distinct Hoi Ping Mok, Nicholas Norton, Axel Fun, Jack Hirst, Mark Wills, Andrew Lever P20 Do mutations in the tat exonic splice enhancer contribute to HIV-1 latency? Nicholas Norton, Hoi Ping Mok, Jack Hirst, Andrew Lever P21 Culture-to-Ct: A fast and direct RT-qPCR HIV gene reactivation screening method using primary T cell culture Valentin Le Douce, Ann Marie McCartin, Virginie Gautier P22 A novel approach to define populations of early silenced proviruses Dalibor Miklik, Filip Senigl, Jiri Hejnar Topic 4: RNA trafficking & packaging P23 Functional analysis of the structure and conformation of HIV-1 genome RNA DIS Jun-ichi Sakuragi, Sayuri Sakuragi, Masaru Yokoyama, Tatsuo Shioda, Hironori Sato P24 Regulation of foamy viral env splicing controls gag and pol expression Jochen Bodem, Rebecca Moschall, Sarah Denk, Steffen Erkelenz, Christian Schenk, Heiner Schaal Topic 5: Assembly & release P25 Transfer of HTLV-1 p8 to target T cells depends on VASP: a novel interaction partner of p8 Norbert Donhauser, Ellen Socher, Sebastian Millen, Heinrich Sticht, Andrea K. Thoma-Kress P26 COL4A1 and COL4A2 are novel HTLV-1 tax targets with a putative role in virus transmission Christine Gross, Sebastian Millen, Melanie Mann, Klaus Überla, Andrea K. Thoma-Kress P27 The C terminus of foamy virus gag protein is required for particle formation, and virus budding: starting assembly at the C terminus? Guochao Wei, Matthew J. Betts, Yang Liu, Timo Kehl, Robert B. Russell, Martin Löchelt P28 Generation of an antigen-capture ELISA and analysis of Rec and Staufen-1 effects on HERV-K(HML-2) virus particle production Oliver Hohn, Saeed Mostafa, Kirsten Hanke, Stephen Norley, Norbert Bannert P29 Antagonism of BST-2/tetherin is a conserved function of primary HIV-2 Env glycoproteins Chia-Yen Chen, Masashi Shingai, Pedro Borrego, Nuno Taveira, Klaus Strebel P30 Mutations in the packaging signal region of the HIV-1 genome cause a late domain mutant phenotype Chris Hellmund, Bo Meng, Andrew Lever P31 p6 regulates membrane association of HIV-1 gag Melanie Friedrich, Friedrich Hahn, Christian Setz, Pia Rauch, Kirsten Fraedrich, Alina Matthaei, Petra Henklein, Maximilian Traxdorf, Torgils Fossen, Ulrich Schubert Topic 6: Pathogenesis & evolution P32 Molecular and structural basis of protein evolution during viral adaptation Aya Khwaja, Meytal Galilee, Akram Alian P33 HIV-1 enhancement and neutralisation by soluble gp120 and its role for the selection of the R5-tropic “best fit” Birco Schwalbe, Heiko Hauser, Michael Schreiber P34 An insertion of seven amino acids in the Env cytoplasmic tail of Human Immunodeficiency Virus type 2 (HIV-2) selected during disease progression enhances viral replication François Dufrasne, Mara Lucchetti, Patrick Goubau, Jean Ruelle P35 Cell-associated HIV-1 unspliced to multiply spliced RNA ratio at 12 weeks ART correlates with markers of immune activation and apoptosis and predicts the CD4 T-cell count at 96 weeks ART Mirte Scherpenisse, Ben Berkhout, Alexander Pasternak P36 Faster progression in non-B subtype HIV-1-infected patients than Korean subclade of subtype B is accompanied by higher variation and no induction of gross deletion in non-B nef gene by Korean red ginseng treatment Young-Keol Cho, Jungeun Kim, Daeun Jeong P37 Aberrant expression of ERVWE1 endogenous retrovirus and overexpression of TET dioxygenases are characteristic features of seminoma Katerina Trejbalova, Martina Benesova, Dana Kucerova, Zdenka Vernerova, Rachel Amouroux, Petra Hajkova, Jiri Hejnar P38 Life history of the oldest lentivirus: characterisation of ELVgv integrations and the TRIM5 selection pattern in dermoptera Daniel Elleder, Tomas Hron, Helena Farkasova, Abinash Padhi, Jan Paces P39 Characterisation of a highly divergent endogenous retrovirus in the equine germ line Henan Zhu, Robert Gifford, Pablo Murcia P40 The emergence of pandemic retroviral infection in small ruminants Maria Luisa Carrozza, Anna-Maria Niewiadomska, Maurizio Mazzei, Mounir Abi-Said, Joseph Hughes, Stéphane Hué, Robert Gifford P41 Near full-length genome (NFLG) Characterisation of HIV-1 subtype B identified in South Africa Adetayo Obasa, Graeme Jacobs, Susan Engelbrecht P42 Acquisition of Vpu-mediated tetherin antagonism by an HIV-1 group O strain Katharina Mack, Kathrin Starz, Daniel Sauter, Matthias Geyer, Frederic Bibollet-Ruche, Christina Stürzel, Marie Leoz, Jean Christophe Plantier, Beatrice H. Hahn, Frank Kirchhoff P43 The human endogenous retrovirus type K is involved in cancer stem cell markers expression and in human melanoma malignancy Ayele Argaw-Denboba, Emanuela Balestrieri, Annalucia Serafino, Ilaria Bucci, Chiara Cipriani, Corrado Spadafora, Paolo Sinibaldi-Vallebona, Claudia Matteucci P44 Natural infection of Indian non-human primates by unique lentiviruses S. Nandi Jayashree, Ujjwal Neogi, Anil K. Chhangani, Shravan Sing Rathore, Bajrang R. J. Mathur P45 Free cervical cancer screening among HIV-positive women receiving antiretroviral treatment in Nigeria Adeyemi Abati P46 Molecular evolutionary status of feline immunodeficiency virus in Turkey B. Taylan Koç, Tuba Çiğdem Oğuzoğlu Topic 7: Innate sensing & intrinsic immunity P47 Cell-to-cell contact with HTLV-1-infected T cells reduces dendritic cell immune functions and contributes to infection in trans. Takatoshi Shimauchi, Stephan Caucheteux, Jocelyn Turpin, Katja Finsterbusch, Charles Bangham, Yoshiki Tokura, Vincent Piguet P48 Deciphering the mechanisms of HIV-1 exacerbation induced by Mycobacterium tuberculosis in monocytes/macrophages Shanti Souriant, Luciana Balboa, Karine Pingris, Denise Kviatcowsky, Brigitte Raynaud-Messina, Céline Cougoule, Ingrid Mercier, Marcelo Kuroda, Pablo González-Montaner, Sandra Inwentarz, Eduardo Jose Moraña, Maria del Carmen Sasiain, Olivier Neyrolles, Isabelle Maridonneau-Parini, Geanncarlo Lugo-Villarino, Christel Vérollet P49 The SAMHD1-mediated inhibition of LINE-1 retroelements is regulated by phosphorylation Alexandra Herrmann, Sabine Wittmann, Caitlin Shepard, Dominique Thomas, Nerea Ferreirós Bouzas, Baek Kim, Thomas Gramberg P50 Activities of nuclear envelope protein SUN2 in HIV infection Xavier Lahaye, Anvita Bhargava, Takeshi Satoh, Matteo Gentili, Silvia Cerboni, Aymeric Silvin, Cécile Conrad, Hakim Ahmed-Belkacem, Elisa C. Rodriguez, Jean-François Guichou, Nathalie Bosquet, Matthieu Piel, Roger Le Grand, Megan King, Jean-Michel Pawlotsky, Nicolas Manel P51 Activation of TLR7/8 with a small molecule agonist induces a novel restriction to HIV-1 infection of monocytes Henning Hofmann, Benedicte Vanwalscappel, Nicolin Bloch, Nathaniel Landau P52 Steady state between the DNA polymerase and Rnase H domain activities of reverse transcriptases determines the sensitivity of retroviruses to inhibition by APOBEC3 proteins Stanislav Indik, Benedikt Hagen P53 HIV restriction in mature dendritic cells is related to p21 induction and p21-mediated control of the dNTP pool and SAMHD1 activity. José Carlos Valle-Casuso, Awatef Allouch, Annie David, Françoise Barré-Sinoussi, Michaela Müller-Trutwin, Monsef Benkirane, Gianfranco Pancino, Asier Saez-Cirion P54 IFITM protens restrict HIV-1 protein synthesis Wing-Yiu Lee, Chen Liang, Richard Sloan P55 Characterisation and functional analysis of the novel restriction factor Serinc5 Bianca Schulte, Silvana Opp, Felipe Diaz-Griffero P56 piRNA sequences are common in Human Endogenous Retroviral Sequences (HERVs): An antiretroviral restriction mechanism? Jonas Blomberg, Luana Vargiu, Patricia Rodriguez-Tomé, Enzo Tramontano, Göran Sperber P57 Ferroportin restricts HIV-1 infection in sickle cell disease Namita Kumari, Tatiana Ammosova, Sharmeen Diaz, Patricia Oneal, Sergei Nekhai P58 APOBEC3G modulates the response to antiretroviral drugs in humanized mice Audrey Fahrny, Gustavo Gers-Huber, Annette Audigé, Roberto F. Speck, Anitha Jayaprakash, Ravi Sachidanandam, Matt Hernandez, Marsha Dillon-White, Viviana Simon P59 High-throughput epigenetic analysis of evolutionarily young endogenous retrovirus presents in the mule deer (Odocoileus hemionus) genome Tomas Hron, Helena Farkasova, Daniel Elleder P60 Characterisation of the expression of novel endogenous retroviruses and immune interactions in a macaque model Neil Berry, Emmanuel Maze, Claire Ham, Neil Almond, Greg Towers, Robert Belshaw P61 HIV-1 restriction by orthologs of SERINC3 and SERINC5 Patrícia de Sousa-Pereira, Joana Abrantes, Massimo Pizzato, Pedro J. Esteves, Oliver T. Fackler, Oliver T. Keppler, Hanna-Mari Baldauf P62 TRIM19/PML restricts HIV infection in a cell type-dependent manner Bianca Volkmann, Tanja Kahle, Kristin Eissmann, Alexandra Herrmann, Sven Schmitt, Sabine Wittmann, Laura Merkel, Nina Reuter, Thomas Stamminger, Thomas Gramberg P63 Recent invasion of the mule deer genome by a retrovirus Helena Farkasova, Tomas Hron, Daniel Elleder P64 Does the antiviral protein SAMHD1 influence mitochondrial function? Ilaria Dalla Rosa, Kate Bishop, Antonella Spinazzola, Harriet Groom P65 cGAMP transfers intercellularly via HIV-1 Env-mediated cell–cell fusion sites and triggers an innate immune response in primary target cells Shuting Xu, Aurélie Ducroux, Aparna Ponnurangam, Sergej Franz, Gabrielle Vieyres, Mathias Müsken, Thomas Zillinger, Angelina Malassa, Ellen Ewald, Veit Hornung, Winfried Barchet, Susanne Häussler, Thomas Pietschmann, Christine Goffinet P66 Pre-infection transcript levels of FAM26F in PBMCS inform about overall plasma viral load in acute and postacute phase after SIV-infection Ulrike Sauermann, Aneela Javed, Nicole Leuchte, Gabriela Salinas, Lennart Opitz, Christiane Stahl-Hennig, Sieghart Sopper P67 Sequence-function analysis of three T cell receptors targeting the HIV-1 p17 epitope SLYNTVATL Christiane Mummert, Christian Hofmann, Angela G. Hückelhoven, Silke Bergmann, Sandra M. Müller-Schmucker, Ellen G. Harrer, Jan Dörrie, Niels Schaft, Thomas Harrer P68 An immunodominant region of the envelope glycoprotein of small ruminant lentiviruses may function as decoy antigen Laure Cardinaux, M.-L. Zahno, H.-R. Vogt, R. Zanoni, G. Bertoni P69 Impact of immune activation, immune exhaustion, broadly neutralising antibodies and viral reservoirs on disease progression in HIV-infected children Maximilian Muenchhoff, Philip Goulder, Oliver Keppler Topic 9: Novel antiviral strategies P70 Identification of natural compounds as new antiviral products by bioassay-guided fractionation Alexandra Herrmann, Stephanie Rebensburg, Markus Helfer, Michael Schindler, Ruth Brack-Werner P71 The PPARG antagonism disconnects the HIV replication and effector functions in Th17 cells Yuwei Zhang, Huicheng Chen, Delphine Planas, Annie Bernier, Annie Gosselin, Jean-Pierre Routy, Petronela Ancuta P72 Characterisation of a multiresistant subtype AG reverse transcriptase: AZT resistance, sensitivity to RNase H inhibitors and inhibitor binding Birgitta Wöhrl, Anna Schneider, Angela Corona, Imke Spöring, Mareike Jordan, Bernd Buchholz, Elias Maccioni, Roberto Di Santo, Jochen Bodem, Enzo Tramontano, Kristian Schweimer P73 Insigths into the acetylation pattern of HDAC inhibitors and their potential role in HIV therapy Christian Schölz, Brian Weinert, Sebastian Wagner, Petra Beli, Yasuyuki Miyake, Jun Qi, Lars Jensen, Werner Streicher, Anna McCarthy, Nicholas Westwood, Sonia Lain, Jürgen Cox, Patrick Matthias, Matthias Mann, James Bradner, Chunaram Choudhary P74 HPV-derived and seminal amyloid peptides enhance HIV-1 infection and impair the efficacy of broadly neutralising antibodies and antiretroviral drugs Marcel Stern, Oliver T. Keppler P75 D(−)lentiginosine inhibits both proliferation and virus expression in cells infected by HTLV-1 in vitro Elena Valletta, Caterina Frezza, Claudia Matteucci, Francesca Marino-Merlo, Sandro Grelli, Anna Lucia Serafino, Antonio Mastino, Beatrice Macchi P76 HIV-1 resistance analyses of the Cape Winelands districts, South Africa Sello Mikasi, Graeme Jacobs, Susan Engelbrecht Topic 10: Recent advances in HIV vaccine development P77 Induction of complex retrovirus antigen-specific immune responses by adenovirus-based vectors depends on the order of vector administration Meike Kaulfuß, Sonja Windmann, Wibke Bayer P78 Direct impact of structural properties of HIV-1 Env on the regulation of the humoral immune response Rebecca Heß, Michael Storcksdieck gen. Bonsmann, Viktoria Stab, Carsten Kirschning, Bernd Lepenies, Matthias Tenbusch, Klaus Überla P79 Lentiviral virus-like particles mediate gerenration of T-follicular helper cells in vitro Anne Kolenbrander, Klaus Überla, Vladimir Temchura P80 Recruitment of HIV-1 Vpr to DNA damage sites and protection of proviral DNA from nuclease activity Kenta Iijima, Junya Kobayashi, Yukihito Ishizaka

Richard Feynman postulated in 1948 that the path of an electron can be best described by the sum or functional integral of all possible trajectories rather than by the notion of a single, unique trajectory. As a consequence, the position of an electron does not harbor any information about the paths that contributed to this position. This observation constitutes a classical endpoint observation. The endpoint assay is the desired type of experiment for high-throughput screening applications, mainly because of limitations in data acquisition and handling. Quite contrary to electrons, it is possible to extract information about the path of a protein using endpoint assays, and these types of applications are reviewed in this article.

Abstract Development of therapeutic approaches for rare respiratory diseases is hampered by the lack of systems that allow medium-to-high-throughput screening of fully differentiated respiratory epithelium from affected patients. This is a particular problem for primary ciliary dyskinesia (PCD), a rare genetic disease caused by mutations in genes that adversely affect ciliary movement and consequently mucociliary transport. Primary cell culture of basal epithelial cells from nasal brush biopsies, followed by ciliated differentiation at air-liquid interface (ALI) has proven to be a useful tool in PCD diagnostics but the technique’s broader utility, including in pre-clinical PCD research, has been limited by the number of basal cells that it is possible to expand from such biopsies. Here, we describe a high-content, imaging-based screening method, enabled by extensive expansion of PCD patient basal cells and their culture into differentiated human respiratory epithelium in miniaturised 96-well transwell format ALI cultures. Analyses of ciliary beat pattern, beat frequency and ultrastructure indicate that a range of different PCD defects are retained in these cultures. We perform a proof-of-principle personalized investigation in reduced generation of motile cilia (RGMC), a rare and very severe form of PCD, in this case caused by a homozygous nonsense mutation (c.441C&gt;A; p.Cys147*) in the MCIDAS gene. The screening system allowed multiple drugs inducing translational readthrough to be evaluated alone or in combination with inhibitors of nonsense-mediated decay. Restoration of basal body formation in the patient’s nasal epithelial cells was seen in vitro , suggesting a novel avenue for drug evaluation and development in PCD. Summary We describe primary cell culture of nasal epithelial cells from patients with primary ciliary dyskinesia including differentiatiation of these to a ciliary phenotype and high-content screening in miniaturised air-liquid interface cultures.

Cell-based screening assays are frequently used in drug discovery and academic research to identify cellular phenotypes in response to a genetic or chemical perturbation. When investigating cell biology processes, the use of high-content imaging facilitates the analysis of a large number of samples within a reasonably short time frame and at moderate costs. Here, we will provide an overview of the high-content screening process, from experimental design to available platform technologies and bioinformatics analysis.

Growing evidence has shown that high autophagic flux is related to tumor progression and cancer therapy resistance. Assaying individual autophagy proteins is a prerequisite for therapeutic strategies targeting this pathway. Inhibition of the autophagy protease ATG4B has been shown to increase overall survival, suggesting that ATG4B could be a potential drug target for cancer therapy. Our laboratory has developed a selective luciferase-based assay for monitoring ATG4B activity in cells. For this assay, the substrate of ATG4B, LC3B, is tagged at the C-terminus with a secretable luciferase from the marine copepod Gaussia princeps (GLUC). This reporter is linked to the actin cytoskeleton, thus keeping it in the cytoplasm of cells when uncleaved. ATG4B-mediated cleavage results in the release of GLUC by non-conventional secretion, which then can be monitored by harvesting supernatants from cell culture as a correlate of cellular ATG4B activity. This paper presents the adaptation of this luciferase-based assay to automated high-throughput screening. We describe the workflow and optimization for exemplary high-throughput analysis of cellular ATG4B activity.

Beta-Propeller Protein-Associated Neurodegeneration (BPAN) is one of the commonest forms of Neurodegeneration with Brain Iron Accumulation, caused by mutations in the gene encoding the autophagy-related protein, WDR45. The mechanisms linking autophagy, iron overload and neurodegeneration in BPAN are poorly understood and, as a result, there are currently no disease-modifying treatments for this progressive disorder. We have developed a patient-derived, induced pluripotent stem cell (iPSC)-based midbrain dopaminergic neuronal cell model of BPAN (3 patient, 2 age-matched controls and 2 isogenic control lines) which shows defective autophagy and aberrant gene expression in key neurodegenerative, neurodevelopmental and collagen pathways. A high content imaging-based medium-throughput blinded drug screen using the FDA-approved Prestwick library identified 5 cardiac glycosides that both corrected disease-related defective autophagosome formation and restored BPAN-specific gene expression profiles. Our findings have clear translational potential and emphasise the utility of iPSC-based modelling in elucidating disease pathophysiology and identifying targeted therapeutics for early-onset monogenic disorders.

Autophagy is the process by which cellular proteins and organelles are degraded and recycled and is essential to the survival of cells. Defective autophagic degradation has been linked to many neurodegenerative diseases and in particular lysosomal storage diseases. Here we describe a high-content assay to detect defects in the autophagy pathway in induced pluripotent stem cell-derived neurons. This assay utilizes immunofluorescence to stain autophagosomes and uses automated image analysis to measure changes in autophagosome levels in response to modulators of autophagy.

The identification of host cell factors for virus replication holds great promise for the development of new antiviral therapies. Recently, high-throughput screening methods have emerged as powerful tools to identify candidate host factors for therapeutic intervention. The development of assay systems suitable for large-scale automated screening is of particular importance for novel viruses with high pathogenic potential for which limited biological information can be developed in a short period of time. This report presents a general enzymatic reporter system for the detection and characterization of multiple enveloped viruses that does not rely on engineering of the virus. Instead, reporter enzymes are incorporated into virus particles by targeting to lipid microdomains in producer cells. The approach allows a variety of human pathogenic enveloped viruses to be detected by sensitive, inexpensive and automatable enzymatic assays. Tagged viruses can be purified quickly and efficiently by a magnetic bead-based capture method. The method allows general detection of enveloped viruses without prior reference to their sequence.

High-content screening is a useful tool to understand complex cellular processes and to identify genes, proteins or small molecule compounds that modulate such pathways. High-content assays monitor the function of a protein or pathway by visualizing a change in an image-based readout, such as a change in the localization of a reporter protein. Examples of this can be the translocation of a fluorescently tagged protein from the cytoplasm to the nucleus or to the plasma membrane. One protein that is known to undergo such translocation is the Growth Factor Receptor-bound protein 2 (GRB2) that is recruited to the plasma membrane upon stimulation of a growth factor receptor and subsequently undergoes internalization. We have used GFP-tagged Grb2 previously to identify genes that are involved in EGFR signaling (Petschnigg et al., 2017). Ultimately, the assay can be adapted to cDNA expression cloning (Freeman et al., 2012) and can be used in early stage drug discovery to identify compounds that modulate or inhibit EGFR signaling and internalization (Antczak and Djaballah, 2016).

Abstract It is long-established that Von Willebrand Factor (VWF) is central to haemostasis and thrombosis. Endothelial VWF is stored in cell-specific secretory granules, Weibel-Palade bodies (WPBs), uniquely rod-like exocytic organelles generated in a wide range of lengths (0.5 to 5.0 µm). It has been shown that WPB size responds to physiological cues and pharmacological treatment and that, under flow, VWF secretion from shortened WPBs produces a dramatic reduction of platelet and plasma VWF adhesion to an endothelial surface. WPB-shortening therefore represents a novel target for antithrombotic therapy acting via modulation of VWF adhesive activity. To this aim, we screened a library of licenced drugs and identified several that prompt WPB size reduction. These compounds therefore constitute a novel set of potentially antithrombotic compounds. Summary The size of the endothelial secretory granules that store Von Willebrand Factor correlates with its activity, central to haemostasis and thrombosis. Here, human-licenced drugs that reduce the size of these secretory granules are identified, providing a set of novel potential anti-thrombotic compounds.

Summary Mitochondrial dysfunction is implicated in Parkinson disease (PD). Mutations in Parkin, an E3 ubiquitin ligase, can cause juvenile-onset Parkinsonism probably through impairment of mitophagy. Inhibition of the de-ubiquitinating enzyme USP30 may counter this effect to enhance mitophagy. Using different tools and cellular approaches, we wanted to independently confirm this claimed role for USP30. Pharmacological characterization of additional tool compounds that selectively inhibit USP30 are reported. The consequence of USP30 inhibition by these compounds, siRNA knockdown and overexpression of dominant-negative USP30 in the mitophagy pathway in different disease-relevant cellular models was explored. Knockdown and inhibition of USP30 showed increased p-Ser65-ubiquitin levels and mitophagy in neuronal cell models. Furthermore, patient-derived fibroblasts carrying pathogenic mutations in Parkin showed reduced p-Ser65-ubiquitin levels compared to wild-type cells, levels that could be restored using either USP30 inhibitor or dominant-negative USP30 expression. Our data provide additional support for USP30 inhibition as a regulator of the mitophagy pathway.

The ATG4 proteases are key regulators of autophagy. Until recently it was thought that their main function was to mediate the processing of ATG8 family members. A new study by Nguyen et al. reveals a role for ATG4s, independent of their catalytic activity, and proposes novel functions in mediating lipid transfer and mitophagy.

<h3>Background</h3> Mitochondrial dysfunction is a known component of HD pathogenesis, but the precise mechanisms and temporal order of events linked to mHTT inclusion formation and neurodegeneration are unclear. For example, observations from clinical samples and <i>in vitro</i> models implicate respiratory impairment and enhanced glycolysis in HD cells. <h3>Aim</h3> To identify early signs of mitochondrial dysfunction and perturbed bioenergetics in a human HD neuronal model, and better understand the interaction between the proteotoxic environment of mHTT-expressing cells and their metabolic capacity. <h3>Methods</h3> We are using a human neural stem cell (NSC) line (ReNcell VM) that over-expresses huntingtin (HTT) exon 1 fragments with either normal (29Q) or pathogenic polyglutamine tracts (71Q and 129Q), and can be rapidly differentiated into mixed populations of neurons and glia in various culture formats. The pathogenic HTT exon 1 lines develop both intranuclear and cytoplasmic mHTT-containing inclusions, as well as accumulating a diffuse aggregated form of mHTT in a time and poly Q-dependent manner. We have correlated with this measures of oxygen consumption rate and extracellular acidification rates using an XF96 Seahorse Bioanalyser, mitochondrial volume and basal ΔΨm by live confocal imaging and potential impairment individual respiratory chain complexes using<i> in vitro</i> assays. <h3>Results</h3> Despite no overt reduction in cell viability in the mHTT exon 1 expressing lines we have uncovered sub-pathological alterations to mitochondrial function. We report differences in key respiratory parameters (e.g. basal and maximal respiration) in line with a significant decrease in basal mitochondrial ΔΨm in mHTT exon 1 cells. Here, we will present our findings correlating the inclusion phenotypes and mitochondrial dysfunction in the presence of mHTT exon 1. <h3>Conclusions</h3> We have established a robust and disease-relevant <i>in vitro</i> cellular model of HD that has revealed potentially important early changes to neuronal bioenergetics and mitochondrial function. Using this system we aim to not only identify novel pathways in disease pathogenesis that may be amenable to therapeutic targeting, but also to simultaneously develop a novel <i>in vitro</i> cellular platform for high-throughput compound screening.

<h3>Background</h3> Neuronal inclusion formation is a pathognomonic feature of Huntington’s disease (HD). Recent evidence has suggested that these inclusion bodies (IBs) may comprise a heterogeneous population of huntingtin (HTT) protein species, a subset of which may lead to impaired cell viability and ultimately cell death. <h3>Aim</h3> To investigate the formation of inclusions in human neurons derived from neural stem cells (NSCs) (ReNcell VM) stably transduced to over-express HTT exon 1 fragments with either normal (29Q) or pathogenic polyglutamine tracts (71 Q and 129Q). <h3>Methods</h3> High-content screening of neurons stained with anti-HTT antibody S830 was carried out using a Perkin Elmer Opera LX confocal microscope. Subsequent quantitative image analysis was carried out using ImageJ and R. Individual cells were analysed in more detail using super resolution imaging (N-SIM Super Resolution System). The effect of mHTT exon 1 on neuronal viability was assessed using a variety of biochemical techniques (LDH assay, MTT and Alamar Blue). <h3>Results</h3> Inclusion bodies form in a small proportion of (1%) of neurons, and are predominantly intra-nuclear, though a lower proportion of cells display smaller perinuclear inclusions. The formation of nuclear inclusions increases in a polyQ- length dependent manner and over time. Super resolution microscopy of individual cells has demonstrated this to be an all-or-nothing phenomenon. A diffuse cytosolic form of mHTT also accumulates in the cells over time. There is no overt effect on basal viability in these mHTT exon 1 expressing neurons. <h3>Conclusions</h3> We have characterised the spatio-temporal profile of mHTT inclusion formation in mHTT expressing NSCs and neurons, and have demonstrated poly-Q and time-dependent accumulation of IBs. A deeper understanding of relationship between the different forms of mHTT and neuronal vulnerability is essential for the design of targeted therapeutics.

Methods in Molecular Biology, Volume 716: Drug Design and Discovery: Methods and Protocols . Editor Seetharama D. Satyanarayanajois . Published by Humana Press , New York, NY, USA , 2011 . 295 pp, price $119.00, ISBN: 978-1-61779-011-9 Over the past few years, small molecule screening has undergone a dramatic shift from simple ‘cheap and fast’ high-throughput assay systems to more complex screening setups where one aims to use more physiologically relevant systems. The new edition of ‘Methods in Molecular Biology: Drug Design and Discovery’ aims to cover aspects of this new approach with a comprehensive volume about various strategies in drug discovery. This volume is an easily accessible collection of protocols covering a broad range of different screening methodologies. The book is divided into 16 chapters that cover topics such as the design of large therapeutic molecules (DNA or peptides), revised protocols for the use of conventional assays in primary screening, computational methods and animal models for pre-clinical evaluation of drug candidates. One particular emphasis is the use of computational methods in drug discovery, which is covered in the first four chapters of the book. Chapter 4 (‘Methods for evaluation of structural and biological properties of anti-invasive natural products’) deserves particular mention in this regard, and the authors provide protocols for isolation and purification of compounds, the use of 3D model systems in cell culture and 3D QSAR modelling applications. Another major emphasis of this book is the use of animal models in primary screening (such as chapter 12, ‘Chemical screening with zebrafish embryos’ and chapter 10, ‘Imaging NFkB signaling in mice for screening anticancer drugs’) or in pre-clinical testing (chapter 15, ‘Evaluation of antibacterial activity of proteins and peptides using a specific animal model for wound healing’). Other noteworthy areas covered are the use of mass spectroscopy, in vivo imaging in drug discovery, and the use of in vitro phage display for selection of peptide ligands in human placental transcytosis systems (chapter 8; clearly a gem in this book). The strong academic background of the authors makes this book highly recommendable to everyone involved in drug screening and has a particular appeal to an academic audience. Written in the highly successful format of the Methods in Molecular Biology series, each chapter includes a background on the topic, a list of materials required, a detailed step-by-step description of the methods and a notes section that draws attention to potential pitfalls and highlights some of the key steps in the procedure. All protocols provide very detailed instructions, a characteristic of the Methods in Molecular Biology series. The only minor drawback of this volume is the slightly misleading title. This collection of protocols touches very briefly on drug design. The book rather focuses on novel approaches to drug screening and physiological validation. It is highly recommended for all those who are performing drug screening or who aim to be doing so in the near future. There are no competing interests to declare.

The potential of stem cell-based disease modelling is enhanced by the realisation that cardiomyocytes from human embryonic stem cells (hESC-CM) and induced pluripotent stem cells (hiPSC-CM) can be obtained with disease-specificity. Hypertrophy is a high priority target because of its central role in the transition to heart failure. Strikingly, here we found that hiPSC-CM are relatively unresponsive to major hypertrophic signals compared to hESC-CM. We show that the normal alpha-adrenergic receptor 1A subtype (ADRA1A) is not expressed robustly in either cell type. ADRA1A is reversibly silenced during differentiation, accompanied by up-regulation of ADRA1B, resulting in a distinct gene profile from that in adult human cardiomyocytes. Loss of ADRA1A is more pronounced in hiPSC-CM, due to greater epigenetic silencing and more marked up-regulation of HIF-1α, but ultimately both cell types differ from adult in their reliance on active ADRA1B rather than ADRA1A. ADRA1B up-regulation is sufficient in hESC-CM for hypertrophic changes such as cell size, cell volume and ANF. However, in hiPSC-CM, additional decreased G-protein signalling and tonically inhibitory pathway networks suppress the effect of alpha-adrenoceptor stimulation on growth. Superficial similarities between hESC-CM, hiPSC-CM and adult cardiomyocytes may mask complex differences in signalling. These data raise serious questions regarding the hiPSC-CM as a valid model system for certain aspects of cardiac disease.

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3. Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

Purpose : Autophagy is thought to be a response to increased oxidative stress in age related macular degeneration (AMD) and it has been suggested that modulation of autophagy flux could become a treatment strategy for the disease. The ARED study showed that zinc supplementation can reduce the progression of AMD, raising the possibility, that zinc might be directly involved in regulating cellular processes during disease progression. In this study we tested the hypothesis that zinc supplementation can directly affect autophagy. Methods : Primary human fetal RPE cells (ScienceCell) were cultured on polyester transwells (Corning), coated with Geltrex for 4 and 31 days in the presence or absence of 125 μM externally added ZnSO₄. Autophagosome number was induced by Chloroquine or Bafilomycin, visualized using CytoID kit (Enzo) and the Opera high content screening system (PerkinElmer) and quantified by ImageJ. Distribution of autophagosomes were examined by confocal (Zeiss LSM 700) and transmission electron microscopy (Jeol 1010). Results : There was a small, but significant decrease in autophagosome numbers with increasing differentiation in non-treated cells (1.77±0.31 vs. 0.65±0.15; p<0.05). Treatment with Chloroquine or Bafilomycin increased autophagosome numbers to 7.92±0.45 and 11.25±2.34 respectively, at day 4. The treatment with Bafilomycin did not affect the cells at day 31, while Chloroquine had a small, but significant effect (0.65±0.15 vs 1.17±0.21; p<0.05). Treatment with zinc significantly attenuated the effects of both Chloroquine and Bafilomycin (p<0.05). Examination of the autophagosomes with confocal and transmission electron microscopy showed that autophagosomes were not only present in the cells but also in the sub-RPE space. Conclusions : Our results support the hypothesis, that zinc supplementation can directly affect autophagy. The observation that autophagosomes are present in the sub-RPE space suggest that they may contribute to deposit formation in AMD. Therefore, the effects observed in the ARED study, at least in part, might be due to a direct influence on autophagy and this could indeed be a target for future treatment strategies for AMD. Aknowledgement: This work was supported by COST TD 1304 ‘The Network for the Biology of Zinc (Zinc-Net)’and EYE-RISK European Union’s Horizon 2020 Research and Innovation Programme under Grant Agreement No 634479. This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

Abstract Deregulation of the cyclin-dependent kinases 4 and 6 (CDK4/6) is highly prevalent in cancer yet inhibitors against these kinases currently show use in restricted tumour contexts. The extent to which cancers depend on CDK4/6 and what may undermine such dependency is poorly understood. Here we report that signalling engaging the MET proto-oncogene receptor tyrosine kinase/focal adhesion kinase (FAK) axis leads to CDK4/6-independent CDK2-activation, involving as a critical mechanistic events loss of the CDK inhibitor p21 CIP1 and gain of its regulator, the ubiquitin ligase subunit SKP2. Combined inhibition of MET/FAK and CDK4/6 eliminates proliferation capacity of cancer cells in culture, and enhances tumour growth inhibition in vivo . Activation of the MET/FAK axis is known to arise through cancer extrinsic and intrinsic cues. Our work predicts that such cues support cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases and identifies MET/FAK as a tractable route to broaden the utility of CDK4/6 inhibitor-based therapies in the clinic.

The original version of this Article contained an error in the spelling of the author Harry Hemingway, which was incorrectly given as Harry Hemmingway. This has been corrected in both the PDF and HTML versions of the Article.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mitochondrial quality control is essential for maintaining a healthy population of mitochondria. Two proteins associated with Parkinson disease, the kinase PINK1 and the E3 ubiquitin ligase PRKN, play a central role in the selective degradation of heavily damaged mitochondria (mitophagy), thus avoiding their toxic accumulation. Most of the knowledge on PINK1-PRKN mitophagy comes from <i>in vitro</i> experiments involving the treatment of mammalian cells with high concentrations of mitochondrial uncouplers, such as CCCP. These chemicals have been shown to mediate off target effects, other than mitochondrial depolarization. A matter of controversy between mitochondrial physiologists and cell biologists is the discrepancy between concentrations of CCCP needed to activate mitophagy (usually &gt;10 μM), when compared to the much lower concentrations used to depolarize mitochondria (&lt;1 μM). Thus, there is an urgent need for optimizing the current methods to assess PINK1-PRKN mitophagy <i>in vitro</i>. In this study, we address the utilization of high CCCP concentrations commonly used to activate mitophagy. Combining live fluorescence microscopy and biochemistry, we show that the FBS/BSA in the cell culture medium reduces the ability of CCCP to induce PINK1 accumulation at depolarized mitochondria, subsequent PRKN recruitment and ubiquitin phosphorylation, and ultimately mitochondrial clearance. As a result, high concentrations of CCCP are required to induce mitophagy in FBS/BSA containing media. These data unite mitochondrial physiology and mitophagy studies and are a first step toward a consensus on optimal experimental conditions for PINK1-PRKN mitophagy and mitochondrial physiology investigations to be carried out in parallel. <b>Abbreviations:</b> BSA: bovine serum albumin; CCCP: carbonyl cyanide m-chlorophenylhydrazone; DMEM: dulbecco’s Modified Eagle’s Medium; DNP: 2,4-dinitrophenol; FBS: fetal bovine serum; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GSH: glutathione; HBSS: Hanks‘ balanced salt solution; mtKeima: mitochondria-targeted monomeric keima-red; PBS: phosphate buffered saline; PD: Parkinson disease; PINK1: PTEN induced kinase 1; POE SHSY5Ys: FLAG-PRKN over-expressing SHSY5Y cells; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; TMRM: tetramethylrhodamine methyl ester; WB: western blot; WT: wild-type; ΔΨm: mitochondrial membrane potential

Mitochondrial quality control is essential for maintaining a healthy population of mitochondria. Two proteins associated with Parkinson disease, the kinase PINK1 and the E3 ubiquitin ligase PRKN, play a central role in the selective degradation of heavily damaged mitochondria (mitophagy), thus avoiding their toxic accumulation. Most of the knowledge on PINK1-PRKN mitophagy comes from <i>in vitro</i> experiments involving the treatment of mammalian cells with high concentrations of mitochondrial uncouplers, such as CCCP. These chemicals have been shown to mediate off target effects, other than mitochondrial depolarization. A matter of controversy between mitochondrial physiologists and cell biologists is the discrepancy between concentrations of CCCP needed to activate mitophagy (usually &gt;10 μM), when compared to the much lower concentrations used to depolarize mitochondria (&lt;1 μM). Thus, there is an urgent need for optimizing the current methods to assess PINK1-PRKN mitophagy <i>in vitro</i>. In this study, we address the utilization of high CCCP concentrations commonly used to activate mitophagy. Combining live fluorescence microscopy and biochemistry, we show that the FBS/BSA in the cell culture medium reduces the ability of CCCP to induce PINK1 accumulation at depolarized mitochondria, subsequent PRKN recruitment and ubiquitin phosphorylation, and ultimately mitochondrial clearance. As a result, high concentrations of CCCP are required to induce mitophagy in FBS/BSA containing media. These data unite mitochondrial physiology and mitophagy studies and are a first step toward a consensus on optimal experimental conditions for PINK1-PRKN mitophagy and mitochondrial physiology investigations to be carried out in parallel. <b>Abbreviations:</b> BSA: bovine serum albumin; CCCP: carbonyl cyanide m-chlorophenylhydrazone; DMEM: dulbecco’s Modified Eagle’s Medium; DNP: 2,4-dinitrophenol; FBS: fetal bovine serum; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; GSH: glutathione; HBSS: Hanks‘ balanced salt solution; mtKeima: mitochondria-targeted monomeric keima-red; PBS: phosphate buffered saline; PD: Parkinson disease; PINK1: PTEN induced kinase 1; POE SHSY5Ys: FLAG-PRKN over-expressing SHSY5Y cells; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; TMRM: tetramethylrhodamine methyl ester; WB: western blot; WT: wild-type; ΔΨm: mitochondrial membrane potential

Abstract LC3 (Light Chain 3) is a key player of autophagy, a major stress-responsive proteolysis pathway promoting cellular homeostasis. It coordinates the formation and maturation of autophagosomes and recruits cargo to be further degraded upon autophagosome-lysosome fusion. To orchestrate its functions, LC3 binds to multiple proteins from the autophagosomes’ inner and outer membranes, but the full extent of these interactions is not known. Moreover, LC3 has been increasingly reported in other cellular locations than the autophagosome, with cellular outcome not fully understood and not all related to autophagy. Furthermore, novel functions of LC3 as well as autophagy can occur in cells growing in a normal medium thus in non-stressed conditions. A better knowledge of the molecule in proximity to LC3 in normal growth conditions will improve the understanding of LC3 function in autophagy and in other cell biology function. Using an APEX2 based proteomic approach, we have detected 407 proteins in proximity to the well-characterised LC3B isoform in non-stress conditions. These include known and novel LC3B proximity proteins, associated with various cell localisation and biological functions. Sixty-nine of these proteins contain a putative LIR (LC3 Interacting Region) including 41 not reported associated to autophagy. Several APEX2 hits were validated by co-immunoprecipitation and co-immunofluorescence. This study uncovers the LC3B global interactome and reveals novel LC3B interactors, irrespective of LC3B localisation and function. This knowledge could be exploited to better understand the role of LC3B in autophagy and non-autophagy cellular processes.