Biology subjects

Winkler, J.

Publications and source records attributed to Winkler, J..

33 records · Page 2Linked to original sources

bioRxiv2022

Dissecting the contributions of tumor heterogeneity on metastasis at single-cell resolution

Metastasis is the leading cause of cancer-related deaths, but metastasis research is challenged by limited access to patient material and a lack of experimental models that appropriately recapitulate tumor heterogeneity. Here, we analyzed single-cell transcriptomes of matched primary tumor and metastasis from patient-derived xenograft models of breast cancer, demonstrating that primary tumor and metastatic cells show profound transcriptional differences across heterogeneous tumors. While primary tumor cells upregulated several metabolic genes, metastatic cells displayed a motility phenotype in micrometastatic lesions and increased stress response signaling during metastatic progression. Additionally, we identified gene signatures that are associated with the metastatic potential and correlated with patient outcomes. Poorly metastatic primary tumors showed increased immune-regulatory control that may prevent metastasis, whereas highly metastatic primary tumors upregulated markers of epithelial-mesenchymal transition (EMT). We found that intra-tumor heterogeneity is dominated by epithelial-mesenchymal plasticity (EMP) which presented as a dynamic continuum with intermediate cell states that were characterized by novel, specific markers. These intermediate EMP markers correlated with worse patient outcomes and could serve as potential new therapeutic targets to block metastatic development.

cancer biology↗

bioRxiv2022

Systematic functional interrogation of SARS-CoV-2 host factors using Perturb-seq

Genomic and proteomic screens have identified numerous host factors of SARS-CoV-2, but efficient delineation of their molecular roles during infection remains a challenge. Here we use Perturb-seq, combining genetic perturbations with a single-cell readout, to investigate how inactivation of host factors changes the course of SARS-CoV-2 infection and the host response in human lung epithelial cells. Our high-dimensional data resolve complex phenotypes such as shifts in the stages of infection and modulations of the interferon response. However, only a small percentage of host factors showed such phenotypes upon perturbation. We further identified the NF-{kappa}B inhibitor I{kappa}B (NFKBIA), as well as the translation factors EIF4E2 and EIF4H as strong host dependency factors acting early in infection. Overall, our study provides massively parallel functional characterization of host factors of SARS-CoV-2 and quantitatively defines their roles both in virus-infected and bystander cells.

systems biology↗

bioRxiv2022

Targeting acetyl-CoA metabolism attenuates the formation of fear memories through reduced activity-dependent histone acetylation

Histone acetylation is a key component in the consolidation of long-term fear memories. Epigenetic enzymes involved in histone acetylation, including histone acetyltransferases and deacetylases, have been put forward as potential pharmacological targets in the treatment of pathological fear memories, such as those that underlie post-traumatic stress disorder (PTSD). However, these enzymes typically play a ubiquitous role in gene regulation, which precludes the clinical use of systemic manipulations. Recently, we have found that a nuclear-localized metabolic enzyme, Acetyl-coA synthetase 2 (Acss2), modulates histone acetylation during learning and memory. Loss of Acss2 is well-tolerated in mice, with no impact on general health or baseline behavior. Here, we show that an Acss2 null mouse model shows reduced acquisition of long-term fear memories in assays of contextual and cued fear conditioning. We find that loss of Acss2 leads to consolidation-specific reductions in both histone acetylation and the expression of critical learning and memory-related genes in the dorsal hippocampus. Further, we show that systemic administration of blood-brain-barrier (BBB)-permeable Acss2 inhibitors during the consolidation window reduces fear memory formation in mice and rats, and also reduces anxiety in a predator-scent-stress (PSS) paradigm. Our findings suggest that Acss2 plays a critical role in the formation of fear memories, and represents a potential pharmacological target in the treatment of PTSD.

neuroscience↗

bioRxiv2022

Reduced synaptic activity and dysregulated extracellular matrix pathways are common phenotypes in midbrain neurons derived from sporadic and mutation-associated Parkinson's disease patients

Several mutations that cause Parkinsons disease (PD) have been identified over the past decade. These account for 15-25% of PD cases; the rest of the cases are considered sporadic. Currently, it is accepted that PD is not a single monolithic disease but rather a constellation of diseases with some common phenotypes. While rodent models exist for some of the PD-causing mutations, research on the sporadic forms of PD is lagging due to a lack of cellular models. In our study, we differentiated PD patient-derived dopaminergic (DA) neurons from induced pluripotent stem cells (iPSCs) of several PD-causing mutations as well as from sporadic PD patients. Strikingly, we observed a common neurophysiological phenotype: Neurons derived from PD patients had a severe reduction in the rate of synaptic currents compared to those derived from healthy controls. While the relationship between mutations in genes such as the SNCA and LRRK2 and a reduction in synaptic transmission has been investigated before, here we show evidence that the pathogenesis of the synapses in neurons is a general phenotype in PD. Analysis of RNA sequencing results displayed changes in gene expression in different synaptic mechanisms as well as other affected pathways such as extracellular matrix-related pathways. Some of these dysregulated pathways are common to all PD patients (monogenic or idiopathic). Our data, therefore, shows changes that are central and convergent to PD and suggests a strong involvement of the tetra-partite synapse in PD pathophysiology.

neuroscience↗

bioRxiv2021

An mRNA processing pathway suppresses metastasis by governing translational control from the nucleus

Cancer cells often co-opt post-transcriptional regulatory mechanisms to achieve pathologic expression of gene networks that drive metastasis. Translational control is a major regulatory hub in oncogenesis, however its effects on cancer progression remain poorly understood. To address this, we used ribosome profiling to compare genome-wide translation efficiencies of poorly and highly metastatic breast cancer cells and patient-derived xenografts. We developed novel regression-based methods to analyze ribosome profiling and alternative polyadenylation data, and identified HNRNPC as a translational controller of a specific mRNA regulon. Mechanistically, HNRNPC, in concert with PABPC4, binds near to poly(A) signals, thereby governing the alternative polyadenylation of a set of mRNAs. We found that HNRNPC and PABPC4 are downregulated in highly metastatic cells, which causes HNRNPC-bound mRNAs to undergo 3 UTR lengthening and subsequently, translational repression. We showed that modulating HNRNPC expression impacts the metastatic capacity of breast cancer cells in xenograft mouse models. We also found that a small molecule, previously shown to induce a distal-to-proximal poly(A) site switching, counteracts the HNRNPC-PABPC4 driven deregulation of alternative polyadenylation and decreases the metastatic lung colonization by breast cancer cells in vivo.

cancer biology↗

bioRxiv2021

Combinatorial immunotherapies overcome MYC-driven immune evasion

For many human cancers, including triple negative breast cancer (TNBC), a modest number of patients benefit from immune checkpoint inhibitors, and few experience cancer remission1. Expression of programed death-ligand 1 (PD-L1), tumor immune infiltration, or tumor mutation burden have been widely investigated for predicting cancer immunotherapy response1-5. Whether specific oncogenes diminish response to immunotherapy6-10 and whether these effects are reversible remains poorly understood. We predicted that MYC, an oncogene that is frequently overexpressed11,12 and is associated with worse prognosis12, may predict immunotherapy response in patients with TNBC. Here, we report that MYC-elevated TNBCs are resistant to immune checkpoint inhibitors. Using mouse models of TNBC and patient data we report that MYC signaling is associated with low tumor cell PD-L1, low overall immune cell infiltration, and low tumor cell MHC-I expression. Restoring interferon signaling in the tumor reduces MYC expression and increases MHC-I expression. By combining a TLR9 agonist and an agonistic antibody against OX40 with anti-PD-L1, most mice experience complete tumor regression and are protected from new TNBC tumor outgrowth. Our findings demonstrate that MYC-dependent immune evasion is reversible and druggable, and if strategically targeted, may improve outcomes for patients treated with immune checkpoint inhibitors.

cancer biology↗

bioRxiv2020

The TPLATE subunit is essential for structural assembly of the endocytic TSET complex

All eukaryotic cells rely on endocytosis to regulate the plasma membrane proteome and lipidome. Most eukaryotic groups, with the exception of fungi and animals, have retained the evolutionary ancient TSET complex as a regulator of endocytosis. Despite the presence of similar building blocks in TSET, compared to other coatomer complexes, structural insight into this adaptor complex is lacking. Here, we elucidate the molecular architecture of the octameric plant TSET complex (TPLATE complex/TPC) using an integrative structural approach. This allowed us to describe a plant-specific connection between the TML subunit and the AtEH/Pan1 proteins and show a direct interaction between the complex and the plasma membrane without the need for any additional protein factors. Furthermore, we identify the appendage of TPLATE as crucial for complex assembly. Structural elucidation of this ancient adaptor complex vastly advances our functional as well as evolutionary insight into the process of endocytosis. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

cell biology↗

bioRxiv2020

Acute Exercise Following Skill Practice Promotes Motor Memory Consolidation in Parkinson's Disease

Acute cardiovascular exercise has shown to promote neuroplastic processes, and thus to improve the consolidation of newly acquired motor skills in healthy adults. First results suggest that this concept may be transferred to populations with motor and cognitive dysfunctions. In this context, Parkinsons disease (PD) is highly relevant since patients demonstrate deficits in motor learning. Hence, in the present study we sought to explore the effect of a single post-practice exercise bout on motor memory consolidation in PD patients. For this purpose, 17 PD patients (Hoehn and Yahr: 1 - 2.5, age: 60.1 {+/-} 7.9 y) practiced a whole-body task followed by either (i) a moderate-intense bout of cycling, or (ii) seated rest for a total of 30 minutes. The motor task required the participants to balance on a tiltable platform (stabilometer) for 30 seconds. During skill practice, patients performed 15 trials followed by a retention test 1 day and 7 days later. We calculated time in balance (platform within {+/-} 5{degrees} from horizontal) for each trial and within- and between-group differences in memory consolidation (i.e. offline learning = skill change from last acquisition block to retention tests) were analyzed. Groups revealed similar improvements during skill practice (F4,60 = .316, p = .866), but showed differences in offline learning, which was only evident after 7 days (F1,14 = 5.602, p = .033). Our results suggest that a single post-practice exercise bout is effective in enhancing long-term motor memory consolidation in a population with motor learning impairments. This may point at unique promoting effects of exercise on dopamine neurotransmission involved in memory formation. Future studies should investigate the potential role of exercise-induced effects on the dopaminergic system. HighlightsO_LIAcute exercise enhanced motor memory consolidation in PD C_LIO_LIEffects were evident only at 7-day retention C_LIO_LIResults may indicate unique exercise-effects on the dopaminergic system C_LIO_LIFindings show promising potential of exercise for motor rehabilitation C_LI

neuroscience↗

bioRxiv2020

Rapamycin-dependent delocalization as a novel tool to reveal protein-protein interactions in plants

Identifying protein-protein interactions (PPI) is crucial for understanding biological processes. Many PPI tools are available, yet only some function within the context of a plant cell. Narrowing down even further, only a few tools allow complex multi-protein interactions to be visualized. Here, we present a conditional in vivo PPI tool for plant research that meets these criteria. Knocksideways in plants (KSP) is based on the ability of rapamycin to alter the localization of a bait protein and its interactors via the heterodimerization of FKBP and FRB domains. KSP is inherently free from many limitations of other PPI systems. This in vivo tool does not require spatial proximity of the bait and prey fluorophores and it is compatible with a broad range of fluorophores. KSP is also a conditional tool and therefore the visualization of the proteins in the absence of rapamycin acts as an internal control. We used KSP to confirm previously identified interactions in Nicotiana benthamiana leaf epidermal cells. Furthermore, the scripts that we generated allow the interactions to be quantified at high throughput. Finally, we demonstrate that KSP can easily be used to visualize complex multi-protein interactions. KSP is therefore a versatile tool with unique characteristics and applications that complements other plant PPI methods.

cell biology↗

bioRxiv2020

Nanobody-dependent delocalization of endocytic machinery in Arabidopsis root cells dampens their internalization capacity

Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein complexes and the scaffolding molecule clathrin. Two adaptor protein complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric Adaptor Protein 2 (AP-2) complex and the octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promotor, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants.

cell biology↗

bioRxiv2019

Protein Quality Control is a Risk Factor and Therapeutic Target in Toxin-Induced Biliary Atresia

BACKGROUND and AIMS Extra-hepatic biliary atresia (BA) is a pediatric liver disease with no approved medical therapy. Recent studies using human samples and experimental modeling suggest that glutathione redox metabolism and heterogeneity play a role in disease pathogenesis. We sought to dissect the mechanistic basis of liver redox variation and explore how other stress responses affect cholangiocyte injury in BA.METHODS We performed quantitative in situ hepatic glutathione redox mapping in zebrafish larvae carrying targeted mutations in glutathione metabolism genes and correlated these findings with sensitivity to the plant-derived BA-linked toxin biliatresone. We also determined whether genetic disruption of HSP90 protein quality control pathway genes implicated in human BA altered biliatresone toxicity in zebrafish and human cholangiocytes. An in vivo screen of a known drug library was performed to identify novel modifiers of cholangiocyte injury in the zebrafish experimental BA model with subsequent validation.RESULTS Glutathione metabolism gene mutations caused regionally distinct changes in the redox potential of cholangiocytes that differentially sensitized them to biliatresone. Disruption of human BA-implicated HSP90 pathway genes sensitized zebrafish and human cholangiocytes to biliatresone-induced injury independent of glutathione. Phosphodiesterase-5 inhibitors (PDE5i) and other cGMP signaling activators worked synergistically with the glutathione precursor N- acetylcysteine (NAC) in preventing biliatresone-induced injury in zebrafish and human cholangiocytes. PDE5i enhanced proteasomal degradation and required intact HSP90 chaperone.CONCLUSION Regional variation in glutathione metabolism underlies sensitivity to the biliary toxin biliatresone, and mirrors recently reported BA risk stratification linked to glutathione metabolism gene expression. Human BA can be causatively linked to genetic modulation of protein quality control. Combined treatment with NAC and cGMP signaling enhancers warrants further investigation as therapy for BA.Background and Context Biliary atresia (BA) is an obstructive fibrosing cholangiopathy that is the leading indication for liver transplantation in the pediatric population. There are no known treatments to prevent progressive liver injury after surgical restoration of bile flow.New Findings The authors identify factors that affect susceptibility of cholangiocytes to oxidative injury using a toxin-induced BA model. This information is used to validate genetic risk factors for human BA and identified PDE5i as a potential treatment for biliary atresia, either on its own or in combination with the anti-oxidant N-acetyl-cysteine.Limitations The work done in animal and cell culture models needs further study in human tissue-derived models and a larger cohort of BA patients.Impact The findings from this study provide a rationale for identifying new genetic risk factors that predispose to BA and for an interventional study to prevent progressive liver injury in this enigmatic disease.Short Summary This study uses zebrafish and human cell culture models to identify novel injury mechanisms, genetic risk factors and new therapies for the pediatric liver disease biliary atresia.Competing Interest StatementThe authors have declared no competing interest.Abbreviations17-AAG7-N-allylamino-17-demethoxygeldanamycin;BAbiliary atresia;bilbiliatresone;8-Br-cGMP8-Bromoguanosine 3’,5’-cyclic monophosphate;EHCextrahepatic cholangiocytes;Gclglutamate cysteine ligase;GGAgeranylgeranyl acetate;gsrglutathione reductase;GSHreduced glutathione;GSSGoxidized glutathione;IHCintrahepatic cholangiocytes;NACN-acetylcysteine;PDE5iPhosphodiesterase-5 inhibitors;PQCprotein quality control;WTwild-typeView Full Text

pathology↗

bioRxiv2018

The Resistome: updating a standardized resource for analyzing resistance phenotypes

Advances in genome engineering have enabled routine engineering and interrogation of microbial resistance on a scale previously impossible, but developing an integrated understanding of resistance from these data remains challenging. As part of our continued efforts to address this challenge, we present a significant update of our previously released Resistome database of standardized genotype-resistance phenotype relationships, along with a new web interface to enable facile searches of genomic, transcriptomic, and phenotypic data within the database. Revisiting our previous analysis of resistance, we again find distinct mutational biases associated with random selection versus genome-scale libraries, along with pervasive pleiotropy among resistant mutants. Attempts to predict mutant phenotypes using machine learning identified the lack of comprehensive phenotype screening and small size of the Resistome corpus as challenges for effective model training. Overall, the Resistome represents a unique platform for understanding the interconnections between both current and future resistant mutants, and is available for use at https://resistome-web-interface.herokuapp.com.

bioinformatics↗

bioRxiv2018

MULTI-seq: Scalable sample multiplexing for single-cell RNA sequencing using lipid-tagged indices

We describe MULTI-seq: A rapid, modular, and universal scRNA-seq sample multiplexing strategy using lipid-tagged indices. MULTI-seq reagents can barcode any cell type from any species with an accessible plasma membrane. The method is compatible with enzymatic tissue dissociation, and also preserves viability and endogenous gene expression patterns. We leverage these features to multiplex the analysis of multiple solid tissues comprising human and mouse cells isolated from patient-derived xenograft mouse models. We also utilize MULTI-seqs modular design to perform a 96-plex perturbation experiment with human mammary epithelial cells. MULTI-seq also enables robust doublet identification, which improves data quality and increases scRNA-seq cell throughput by minimizing the negative effects of Poisson loading. We anticipate that the sample throughput and reagent savings enabled by MULTI-seq will expand the purview of scRNA-seq and democratize the application of these technologies within the scientific community.

genomics↗

bioRxiv2018

Need for high-resolution Genetic Analysis in iPSC: Results and Lessons from the ForIPS Consortium

Genetic integrity of induced pluripotent stem cells (iPSCs) is essential for their validity as disease models and for potential therapeutic use. We describe the comprehensive analysis in the ForIPS consortium: an iPSC collection from donors with neurological diseases and healthy controls. Characterization included pluripotency confirmation, fingerprinting, conventional and molecular karyotyping in all lines. In the majority, somatic copy number variants (CNVs) were identified. A subset with available matched donor DNA was selected for comparative exome sequencing. We identified single nucleotide variants (SNVs) at different allelic frequencies in each clone with high variability in mutational load. Low frequencies of variants in parental fibroblasts highlight the importance of germline samples. Somatic variant number was independent from reprogramming, cell type and passage. Comparison with disease genes and prediction scores suggest biological relevance for some variants. We show that high-throughput sequencing has value beyond SNV detection and the requirement to individually evaluate each clone.

genomics↗

bioRxiv2018

Tumor cell-adipocyte gap junctions activate lipolysis and are essential for breast tumorigenesis

A pro-tumorigenic role for adipocytes has been identified in breast cancer, and reliance on fatty acid catabolism found in aggressive tumors. The molecular mechanisms by which tumor cells coopt neighboring adipocytes, however, remain elusive. Here, we describe a direct interaction linking tumorigenesis to adjacent adipocytes. We examine breast tumors and their normal adjacent tissue from several patient cohorts, patient-derived xenografts and mouse models, and find that lipolysis and lipolytic signaling are activated in neighboring adipose tissue. We find that functional gap junctions form between breast cancer cells and adipocytes. As a result, cAMP is transferred from breast cancer cells to adipocytes and activates lipolysis in a gap junction-dependent manner. We identify connexin 31 (GJB3), which promotes receptor triple negative breast cancer growth and activation of lipolysis in vivo. Thus, direct tumor cell-adipocyte interaction contributes to tumorigenesis and may serve as a new therapeutic target in breast cancer. One sentence summaryGap junctions between breast cancer cells and adipocytes transfer cAMP and activate lipolysis in the breast tumor microenvironment to support growth.

cancer biology↗