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Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2017

The Ndc80 complex targets Bod1 to human mitotic kinetochores

Regulation of protein phosphatase activity by endogenous protein inhibitors is an important mechanism to control protein phosphorylation in cells. We recently identified Biorientation defective 1 (Bod1) as a small protein inhibitor of PP2A-B56. This phosphatase controls the amount of phosphorylation of mitotic kinetochore proteins and thus the establishment of load-bearing chromosome-spindle attachments in time for accurate separation of sister chromatids in mitosis. The PP2A-B56 regulatory protein Bod1 directly localises to mitotic kinetochores and is required for correct segregation of mitotic chromosomes. In this report, we have probed the spatio-temporal regulation of Bod1 during mitotic progression. Kinetochore localisation of Bod1 increases from nuclear envelope breakdown until metaphase. Phosphorylation of Bod1 at threonine 95 (T95), which increases Bod1s binding and inhibition of PP2A-B56, peaks in prometaphase, when PP2A-B56 localisation to kinetochores is highest. Kinetochore targeting of Bod1 depends on the outer kinetochore protein Ndc80 and not PP2A-B56. Additionally, Bod1 depletion leads to a defect in the phosphorylation kinetics of Ndc80 at serine 55 (S55) within its N-terminus. Therefore, Ndc80 recruits a phosphatase inhibitor to kinetochores which directly feeds forward to regulate Ndc80 phosphorylation and the stability of the attachments of microtubules to kinetochores.

cell biology↗

bioRxiv2017

Necroptosis promotes the Aging of the Male Reproductive System in Mice

Necroptosis is a form of programmed necrotic cell death in mammals that is mediated by a pair of kinases, RIP1 and RIP3, as well as the RIP3 substrate MLKL. We report here that male reproductive organs of both RIP3-and MLKL-knockout mice retain \"youthful\" morphology and function into advanced age, while those of age-matched wild type mice deteriorate. The RIP3 phosphorylation of MLKL, the activation marker of necroptosis, is detected in spermatogonial stem cells in the testes of old but not in young wild type mice. When the testes of young wild type mice are given a local necroptotic stimulus, their reproductive organs showed accelerated aging. Feeding of wild type mice with an RIP1 inhibitor prior to the normal onset of age-related changes in their reproductive organs blocked the appearance of signs of aging. Thus, necroptosis in testes promotes the aging-associated deterioration of the male reproductive system in mice.

cell biology↗

bioRxiv2017

Real-Time Observation Of Light-Controlled Transcription In Living Cells

Gene expression is a tightly controlled process that is coordinated in space and time. To dissect its dynamic regulation with high temporal resolution, we introduce an optogenetic tool termed BLInCR (Blue Light-Induced Chromatin Recruitment) that combines rapid and reversible light-dependent recruitment of effector proteins with a real-time readout for transcription. We used BLInCR to control the activity of a reporter gene cluster in the human osteosarcoma cell line U2OS by reversibly recruiting the viral transactivator VP16. RNA production was detectable [~]2 minutes after VP16 recruitment and readily decreased when VP16 dissociated from the cluster in the absence of light. Quantitative assessment of the activation process revealed biphasic activation kinetics with a pronounced early phase in cells treated with the histone deacetylase inhibitor SAHA. Comparison with kinetic models for transcription activation suggests that the gene cluster undergoes a maturation process when activated. BLInCR will facilitate the study of transcription dynamics in living cells.

cell biology↗

bioRxiv2017

Rapid DNA Re-Identification for Cell Line Authentication and Forensics

DNA re-identification is used for a broad range of applications, ranging from cell line authentication to crime scene sample identification. However, current re-identification schemes suffer from high latency. Here, we describe a rapid, inexpensive, and portable strategy to re-identify human DNA called MinION sketching. Using data from Oxford Nanopore Technologies sequencer, MinION sketching requires only 3min of sequencing and [~]91 random SNPs to identify a sample, enabling near real-time applications of DNA re-identification. This method capitalizes on the vastly growing availability of genomic reference data for individuals and cancer cell lines. Hands-on preparation of the samples can be reduced to <1 hour. This empowers the application of MinION sketching in research settings for routine cell line authentication or in forensics.\n\nSoftware is available at https://github.com/TeamErlich/personal-identification-pipeline

cell biology↗

bioRxiv2017

An Anisotropic Attraction Model For The Diversity And Robustness Of Cell Arrangement In Nematodes

In early embryogenesis stages in animals, cells are arranged into a species-specific pattern in a robust manner. The cell arrangement patterns are diverse, even among close relatives. We evaluated how the diversity and robustness are achieved in developing embryos. We succeeded in reproducing different patterns of cell arrangements observed in various nematode species using Caenorhabditis elegans embryos by changing the eggshell shapes. This implies that the diversity of cell arrangements can be explained by differences in a shape parameter. Additionally, we found that the cell arrangement was robust against eggshell deformation. Computational modeling revealed that, in addition to repulsion forces, attraction forces are sufficient for this robustness. Genetic perturbation experiments demonstrated that attraction forces derived from cell adhesion are necessary for the robustness. The proposed model accounts for both diversity and robustness of cell arrangements and contributes to our understanding of how diversity and robustness are achieved in developing embryos.

cell biology↗

bioRxiv2017

Deletion Of EP4 In S100a4-Lineage Cells Reduces Scar Tissue Formation During Early But Not Later Stages Of Tendon Healing

Tendon injuries heal via scar tissue rather than regeneration. This healing response forms adhesions between the flexor tendons in the hand and surrounding tissues, resulting in impaired range of motion and hand function. Mechanistically, inflammation has been strongly linked to adhesion formation, and Prostaglandin E2 (PGE2) is associated with both adhesion formation and tendinopathy. In the present study we tested the hypothesis that deletion of the PGE2 receptor EP4 in S100a4-lineage cells would decrease adhesion formation. S100a4-Cre; EP4flox/flox (EP4cKOS100a4) repairs healed with improved gliding function at day 14, followed by impaired gliding at day 28, relative to wild type. Interestingly, EP4cKOS100a4 resulted in only transient deletion of EP4, suggesting up-regulation of EP4 in an alternative cell population in these mice. Loss of EP4 in Scleraxis-lineage cells did not alter gliding function, suggesting that Scx-lineage cells are not the predominant EP4 expressing population. In contrast, a dramatic increase in -SMA+, EP4+ double-positive cells were observed in EP4cKOS100a4 suggesting that EP4cKOS100a4 repairs heal with increased infiltration of EP4 expressing -SMA myofibroblasts, identifying a potential mechanism of late up-regulation of EP4 and impaired gliding function in EP4cKOS100a4 tendon repairs.

cell biology↗

bioRxiv2017

Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay

When marked for degradation, surface receptor and transporter proteins are internalized and delivered to endosomes where they are packaged into intralumenal vesicles (ILVs). Many rounds of ILV formation create multivesicular bodies (MVBs) that fuse with lysosomes exposing ILVs to hydrolases for catabolism. Despite being critical for protein degradation, the molecular underpinnings of MVB-lysosome fusion remain unclear, although machinery underlying other lysosome fusion events is implicated. But how then is specificity conferred? And how is MVB maturation and fusion coordinated for efficient protein degradation? To address these questions, we developed a cell-free MVB-lysosome fusion assay using S. cerevisiae as a model. After confirming that the Rab7 ortholog Ypt7 and the multisubunit tethering complex HOPS are required, we found that the Qa-SNARE Pep12 distinguishes this event from homotypic lysosome fusion. Mutations that impair MVB maturation block fusion by preventing Ypt7 activation, confirming that a Rab-cascade mechanism harmonizes MVB maturation with lysosome fusion.\n\nIMPACT STATEMENTEndocytosis culminates with multivesicular bodies (MVBs) fusing with lysosomes. But the molecular underpinnings of this event remain unclear. Here, using S. cerevisiae as a model, Karim et al. employ a new in vitro assay to show that MVB-lysosome fusion is driven by ESCRT-dependent Rab-GTPase activation and the syntaxin ortholog Pep12, distinguishing it from other lysosome membrane fusion events.

cell biology↗

bioRxiv2017

Mitochondria Are Physiologically Maintained At Close To 50 C

In endothermic species, heat released as a product of metabolism ensures stable internal temperature throughout the organism, despite varying environmental conditions. Mitochondria are major actors in this thermogenic process. Part of the energy released by the oxidation of respiratory substrates drives ATP synthesis and metabolite transport, while a noticeable proportion is released as heat. Using a temperature-sensitive fluorescent probe targeted to mitochondria, we measured mitochondrial temperature in situ under different physiological conditions. At a constant external temperature of 38 {degrees}C, mitochondria were more than 10 {degrees}C warmer when the respiratory chain was fully functional, both in HEK293cells and primary skin fibroblasts. This differential was abolished in cells lacking mitochondrial DNA or by respiratory inhibitors, but preserved or enhanced by expressing thermogenic enzymes such as the alternative oxidase or the uncoupling protein 1. The activity of various RC enzymes was maximal at, or slightly above, 50 {degrees}C. Our study prompts a re-examination of the literature on mitochondria, taking account of the inferred high temperature.

cell biology↗

bioRxiv2017

A Guide To Analysis And Reconstruction Of Serial Block Face Scanning Electron Microscopy Data

Serial block face scanning electron microscopy (SBF-SEM) is a relatively new technique that allows the acquisition of serially sectioned, imaged and digitally aligned ultrastructural data. There is a wealth of information that can be obtained from the resulting image stacks but this presents a new challenge for researchers - how to computationally analyse and make best use of the large data sets produced. One approach is to reconstruct structures and features of interest in 3D. However the software programs can appear overwhelming, time consuming and not intuitive for those new to image analysis. There are a limited number of published articles that provide sufficient detail on how to do this type of reconstruction. Therefore the aim of this paper is to provide a detailed step-by-step protocol, videos and explanation on the types of analysis and programs that can be used. To showcase the programs, skeletal muscle from fetal and adult guinea pigs were used. The tissue was processed using the heavy metal protocol developed specifically for SBFSEM. Trimmed resin blocks were placed into a Zeiss Sigma SEM incorporating the Gatan 3View and the resulting image stacks were analysed in 3 different programs, Fiji, Amira and MIB, using a range of tools available for segmentation. The results from the image analysis comparison show that the analysis tools are often more suited to a type of structure. For example larger structures, such as nuclei and cells, can be segmented using interpolation, which speeds up analysis; single contrast structures, such as the nucleolus, can be segmented using the contrast-based thresholding tools. Knowing the nature of the tissue and its specific structures (complexity, contrast, if there are distinct membranes, size) will help to determine the best method for reconstruction and thus maximising output from valuable tissue.

cell biology↗

bioRxiv2017

COPI Mediates Recycling Of An Exocytic SNARE From Endosomes By Recognition Of A Ubiquitin Sorting Signal

ABSTRACTThe COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI mediates delivery of a budding yeast SNARE (Snc1) from early endosomes to the Golgi complex through recognition of a polyubiquitin sorting signal. Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through early endosomes back to the Golgi for reuse. Removal of ubiquitin from Snc1, or deletion of a {beta}-COP subunit propeller domain that binds K63-linked polyubiquitin, causes aberrant accumulation of Snc1 in early endosomes. Moreover, replacement of the {beta}-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles at early endosomes for Golgi delivery.

cell biology↗

bioRxiv2017

ATR Is Required To Complete Meiotic Recombination In Mice

Precise execution of recombination during meiosis is essential for forming chromosomally balanced gametes. Meiotic recombination initiates with the formation and resection of DNA double-strand breaks (DSBs). Binding of replication protein A (RPA) at resected DSBs fosters association of RAD51 and DMC1, the primary effectors of homology search. It is well appreciated that cellular responses to meiotic DSBs are critical for efficient repair and quality control, but molecular features of these responses remain poorly understood, particularly in mammals. Here we provide evidence that the DNA damage response protein kinase ATR is crucial for meiotic recombination and completion of meiotic prophase in mice. Using a hypomorphic Atr mutation and pharmacological inhibition of ATR in vivo and in cultured spermatocytes, we show that ATR, through its effector kinase CHK1, promotes efficient RAD51 and DMC1 assembly at RPA-coated DSB sites and establishment of interhomolog connections during meiosis. Furthermore, our findings suggest that ATR promotes local accumulation of recombination markers on unsynapsed axes during meiotic prophase to favor homologous chromosome synapsis. These data reveal that ATR plays multiple roles in mammalian meiotic recombination.

cell biology↗

bioRxiv2017

ADAM17 Is The Main Sheddase For The Generation Of Human Triggering Receptor Expressed In Myeloid Cells (hTREM2) Ectodomain And Cleaves TREM2 After Histidine 157

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Using both pharmacological and genetic approaches we report here that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNF converting enzyme) while ADAM10 plays a minor role. Using mutational analysis, we demonstrate that the main cleavage site of the sheddases is located within the stalk region of TREM2 proximal to the plasma membrane. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimers disease. O-glycosylation is detected within the stalk region, but distant to the cleavage site. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered.

cell biology↗

bioRxiv2017

Identification and dynamics of the DHHC16-DHHC6 palmitoylation cascade

S-Palmitoylation is the only reversible post-translational lipid modification. Knowledge about the DHHC family of palmitoyltransferases is very limited. Here we show that mammalian DHHC6, which modifies key proteins of the endoplasmic reticulum, is controlled by an upstream palmitoyltransferase, DHHC16, revealing the first palmitoylation cascade. Combination of site specific mutagenesis of the three DHHC6 palmitoylation sites, experimental determination of kinetic parameters and data-driven mathematical modelling allowed us to obtain detailed information on the 8 differentially palmitoylated DHHC6 species. We found that species rapidly interconvert through the action of DHHC16 and the Acyl Protein Thioesterase APT2, that each species varies in terms of turnover rate and activity, altogether allowing the cell to robustly tune its DHHC6 activity.

cell biology↗

bioRxiv2017

Role of Membrane-tension Gated Ca Flux in Cell Mechanosensation

Eukaryotic cells are sensitive to mechanical forces that they experience from the environment. The process of mechanosensation is complex, and involves elements such as the cytoskeleton and active contraction from myosin motors. Ultimately, mechanosensation is connected to changes in gene expression in the cell, or mechanotransduction. While the involvement of the cytoskeleton in mechanosensation is known, processes upstream to cytoskeletal changes is unclear. In this paper, using a microfluidic device that mechanically compresses live cells, we demonstrate that calcium currents and membrane tension-sensitive ion channels directly signals to the Rho GTPase and myosin contraction. In response to membrane tension changes, cell actively regulates cortical myosin contraction to balance external forces. The process is captured by a mechanochemical model where membrane tension, myosin contraction and the osmotic pressure difference between the cytoplasm and extracellular environment are connected by mechanical force-balance. Finally, to complete the picture of mechanotransduction, we find that the tension-sensitive transcription factor YAP translocates from the nucleus to the cytoplasm in response to mechanical compression.

cell biology↗

bioRxiv2017

A Deep Learning And Novelty Detection Framework For Rapid Phenotyping In High-Content Screening

Supervised machine learning is a powerful and widely used method to analyze high-content screening data. Despite its accuracy, efficiency, and versatility, supervised machine learning has drawbacks, most notably its dependence on a priori knowledge of expected phenotypes and time-consuming classifier training. We provide a solution to these limitations with CellCognition Explorer, a generic novelty detection and deep learning framework. Application to several large-scale screening data sets on nuclear and mitotic cell morphologies demonstrates that CellCognition Explorer enables discovery of rare phenotypes without user training, which has broad implications for improved assay development in high-content screening.

cell biology↗

bioRxiv2017

The unfolded protein response turns from pro-survival to pro-death to cap the extent of endoplasmic reticulum expansion

Insufficient folding capacity of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to restore homeostasis. Yet, how the UPR achieves and evaluates ER homeostatic readjustment is poorly understood. In a HeLa cell model we show that, upon a severe proteostatic insult that eclipses the ER chaperone BiP, the UPR transitions from acute full-gear activation to chronic submaximal activation, when BiP is in excess again. As such, the UPR-driven ER expansion is pro-survival, primarily via the UPR transducer ATF6. Simultaneous abrogation of ER-associated degradation (ERAD) leads to chronic full-gear UPR activation and further ER expansion, but the UPR transducers IRE1 and PERK turn pro-apoptotic. Pro-survival XBP1 mRNA processing by IRE1 is then trumped since unspliced XBP1 mRNA is depleted and IRE1s endonuclease activity is unleashed against other RNAs. Thus, the IRE1/XBP1 relay serves as a capacitor of ER expansion that heralds cell death if the expansion is deemed excessive.

cell biology↗

bioRxiv2017

Spindle assembly checkpoint strength is governed by cell size and PAR-mediated cell fate determination in C. elegans

The spindle assembly checkpoint (SAC) is a conserved mitotic regulator that preserves genome stability. Despite its central role in preserving the fidelity of mitosis, the strength of the SAC varies widely between cell types. How the SAC is adapted to different cellular contexts remains largely unknown. Here we show that both cell size and cell fate impact SAC strength. While smaller cells have a stronger SAC, cells with a germline fate show increased SAC activity relative to their somatic counterparts across all cell sizes. We find that enhanced SAC activity in the germline blastomere P1 requires proper specification of cell fate downstream of the conserved PAR polarity proteins, supporting a model in which checkpoint factors are distributed asymmetrically during early germ cell divisions. Our results indicate that size scaling of SAC activity is modulated by cell fate and reveal a novel interaction between asymmetric cell division and the SAC.

cell biology↗

bioRxiv2017

Targeting xCT-Mediated Glutamate Release Normalizes Tumor Angiogenesis In The Brain

Brain tumors are among the most malignant primary tumors, hallmarked by angiogenesis, neuronal destruction and brain swelling. Inhibition of the glutamate-cystein antiporter xCT (system xc-/SLC7A11) alleviates seizures, neuronal cell death and tumor-associated brain edema. Here we show enhanced tumor vessel growth and increased brain edema in xCT-expressing brain tumors. Furthermore, xCT-mediated glutamate impacts directly on endothelial cells in an N-methyl-D-aspartate receptor (NMDAR) dependent manner with intracellular Ca2+ release. Cerebral intravital microscopy revealed that xCT-driven tumor vessels are functional and display increased permeability. Endothelial-cell-specific NMDAR1 knockout mice (GRINi{Delta}EC) show suppressed endothelial sprouting and vascular density compared to control littermates. In addition, implanted gliomas in GRINi{Delta}EC mice display reduced tumor vessels in contrast to gliomas in wildtype animals. Moreover, therapeutic targeting of xCT in gliomas alleviates tumor angiogenesis to normalized levels comparable to controls. Our data reveal that xCT and its substrate glutamate specifically operate on endothelial cells and promote neoangiogenesis. Thus, targeting xCT expression and glutamate secretion in gliomas provides a novel therapeutic roadmap for normalizing tumor angiogenesis.

cell biology↗