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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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Mitochondrial heteroplasmy is responsible for Atovaquone drug resistance in Plasmodium falciparum

Malaria is the most significant parasitic disease affecting humans, with 212 million cases and 429,000 deaths in 2 0151, and resistance to existing drugs endangers the global malaria elimination campaign. Atovaquone (ATO) is a safe and potent antimalarial drug that acts on cytochrome b (cyt. b) of the mitochondrial electron transport chain (mtETC) in Plasmodium falciparum, yet treatment failures result in resistance-conferring SNPs in cyt. b. Herein we report that rather than the expected de novo selection of resistance, previously unknown mitochondrial diversity is the genetic mechanism responsible for resistance to ATO, and potentially other cyt. b targeted drugs. We found that P. falciparum harbors cryptic cyt. b. Y268S alleles in the multicopy ([~]22 copies) mitochondrial genome prior to drug treatment, a phenomenon known as mitochondrial heteroplasmy. Parasites with cryptic Y268S alleles readily evolve into highly resistant parasites with >95% Y268S copies under in vitro ATO selection. Further we uncovered high mitochondrial diversity in a global collection of 1279 genomes in which heteroplasmic polymorphisms were >3-fold more prevalent than homoplasmic SNPs. Moreover, significantly higher mitochondrial genome copy number was found in Asia (e.g., Cambodia) versus Africa (e.g., Ghana). Similarly, ATO drug selections in vitro induced >3-fold mitochondrial copy number increases in ATO resistant lines. Hidden mitochondrial diversity is a previously unknown mechanism of antimalarial drug resistance and characterization of mitochondrial heteroplasmy will be of paramount importance in combatting resistance to antimalarials targeting the electron transport chain.

cell biology↗

bioRxiv2017

mTORC1 activates PASK-Wdr5 signaling to epigenetically connect the nutrient status with myogenesis.

In the tissue microenvironment, stem cell functions are modulated by extrinsic signaling cues such as peptide hormones and dietary nutrients. These signaling cues maintain the balance between self-renewal and differentiation of its resident stem cells. The mechanistic Target of Rapamycin Complex 1 (mTORC1) is implicated to play an important role in regulating this balance, although its downstream effectors in stem cells have been elusive. We have recently shown that the PASK protein kinase phosphorylates Wdr5 to stimulate muscle stem cell differentiation by epigenetically activating the Myogenin promoter. Here, we show that the PASK-Wdr5 signaling pathway is a nutrient-sensitive downstream target of mTORC1 in muscle stem cells. We show that phosphorylation of PASK, and in turn of Wdr5, by mTORC1 is required for the activation of Myogenin transcription, exit from the self-renewal and induction of the myogenesis program. Thus, mTOR connects the diverse extrinsic signaling cues to a central epigenetic process to regulate the muscle stem cell fate between self-renewal and differentiation.

cell biology↗

bioRxiv2017

Ultrastructural analysis of intracellular membrane and microtubule behavior during mitosis of Drosophila S2 cells

S2 cells are one of the most widely used Drosophila melanogaster cell lines for molecular dissection of mitosis using RNA interference (RNAi). However, a detailed and complete description of S2 cell mitosis at the ultrastructural level is still missing. Here, we analyzed by transmission electron microscopy (TEM) a random sample of 144 cells undergoing mitosis, focusing on intracellular membrane and microtubule (MT) behavior. This unbiased approach allowed us to discover that S2 cells exhibit a characteristic behavior of intracellular membranes, involving the formation of a quadruple nuclear membrane in early prometaphase and its disassembly during late prometaphase. After nuclear envelope disassembly, the mitotic apparatus becomes encased by a discontinuous network of ER membranes that associate with mitochondria preventing their diffusion into the spindle area. We also observed a peculiar metaphase spindle organization. We found that kinetochores with attached k-fibers are almost invariably associated with lateral MT bundles that can be either interpolar bundles or k-fibers connected to a different kinetochore. This spindle organization is likely to favor chromosome alignment at metaphase and subsequent segregation during anaphase. In summary, we describe several previously unknown features of membrane and microtubule organization during S2 cell mitosis. The genetic determinants of these mitotic features of can now be investigated using an RNAi-based approach, which is particularly easy and efficient in S2 cells

cell biology↗

bioRxiv2017

Acto-myosin driven functional nanoclusters of GPI-anchored proteins are generated by integrin receptor signaling

GPI-anchored protein (GPI-AP) nanoclusters are generated by cortical acto-myosin activity. While our understanding of the physical principles behind this process is emerging, the molecular machinery required for the generation of these nanoclusters is unknown. Here, we show that ligand-mediated membrane receptor signaling triggers nanocluster formation. Both soluble and surface-tethered RGD ligands bind the {beta}1-integrin receptor and activate focal adhesion and src-kinases, resulting in RhoA signaling. This cascade ultimately triggers actin-nucleation via specific formins, driving nanoclustering of both GPI-APs and a model transmembrane protein with an actin-binding domain. Integrin signaling concurrently results in talin mediated activation of vinculin. This is necessary for the coupling of the dynamic actin machinery to the inner leaflet driving GPI-AP nanoclustering. Disruption of GPI-AP nanoclustering in either GPI-anchor remodeling mutants or in cells that express vinculin mutants, provide evidence that these nanoclusters are necessary for activating cell spreading, a hallmark of integrin function.

cell biology↗

bioRxiv2017

GORAB, a Golgi protein required for centriole structure and duplication

Genome wide screens are widely believed to have identified the great majority of genes required for centriole duplication. However, seeking to clarify the partners of the Drosophila cartwheel protein Sas6, we identified Gorab, a known trans-Golgi associated protein that is mutated in the human wrinkly skin disease, gerodermia osteodysplastica. We now report that Gorab is present not only in the trans-Golgi but also in association with Sas6 at the core of the centriole. Flies lacking Gorab show defects in centriole duplication in many tissues and are also uncoordinated due to basal body defects in sensory cilia, which lose their 9-fold symmetry. We demonstrate the separation of centriole and Golgi functions of Drosophila Gorab in two ways: First, we have created Gorab variants that are unable to localize to trans-Golgi but can still rescue the centriole and cilia defects of gorab null flies. Secondly, we show that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype overcome by a second mutation preventing Golgi targeting. We discuss the tissue specific requirement of Gorab for centriole duplication in the context of its split functions.

cell biology↗

bioRxiv2017

Mir-132 controls beta cell proliferation and survival in mouse model through the PTEN/AKT/FOXO3 signaling

Aim and hypothesismicroRNAs (miRNAs) play an integral role in maintaining beta cell function and identity. Deciphering their targets and precise role, however, remains a challenge. In this study we aimed to identify miRNAs and their downstream targets involved in regeneration of islet beta cells following partial pancreatectomy in mice.\n\nMethodsRNA from laser capture microdissected (LCM) islets of partially pancreatectomized and sham-operated mice were profiled with microarrays to identify putative miRNAs implicated in control of beta cell regeneration. Altered expression of selected miRNAs, including miR-132, was verified by RT-PCR. Potential targets of miR-132 were seleced through bioinformatic data mining. Predicted miR-132 targets were validated for their changed RNA and protein expression levels and signaling upon miR-132 knockdown or overexpression in MIN6 cells. The ability of miR-132 to foster beta cell proliferation in vivo was further assessed in pancreatectomized miR-132-/- and control mice.\n\nResultsPartial pancreatectomy significantly increased the number of BrdU+/insulin+ positive islet cells. Microarray profiling revealed 14 miRNAs, including miR-132 and -141, to be significantly upregulated in LCM islets of partially pancreatectomized compared to LCM islets of control mice. In the same comparison miR-760 was the only miRNA found to be downregulated. Changed expression of these miRNAs in islets of partially pancreatectomized mice was confirmed by RT-PCR only in the case of miR-132 and -141. Based on previous knowledge of its function, we chose to focus our attention on miR-132. Downregulation of miR-132 in MIN6 cells reduced proliferation while enhancing the expression of proapoptic genes, which was instead reduced in miR-132 overexpression MIN6 cells. Microarray profiling, RT-PCR and immunoblotting of miR-132 overexpressing MIN6 cells revealed their downregulated expression of Pten, with concomitant increased levels of pro-proliferative factors phospho-Akt and phospho-Creb as well as inactivation of pro-apoptotic Foxo3 via its phosphorylation. Finally, we show that regeneration of beta cells following partial pancreatectomy was reduced in miR-132-/- mice compared to control mice.\n\nConclusions/InterpretationsOur study provides compelling evidence for upregulation of miR-132 being critical for regeneration of mouse islet beta cells in vivo through downregulation of its target Pten. Hence, the miR-132/Pten/Akt/Foxo3 signaling pathway may represent a suitable target to enhance beta cell mass.\n\nResearch in ContextWhat is already known?\n\nO_LISeveral miRNAs, including miR-132, are known to regulate beta cell function and mass in several mouse models of diabetes db/db, ob/ob and high fat-diet.\nC_LI\n\nWhat is the key question?\n\nO_LIWhich are miRNAs implicated in control of beta cell regeneration upon partial pancreatectomy and how?\nC_LI\n\nWhat are the new findings?\n\nO_LImiR-132 is critical to promote regeneration of mouse beta cells in vivo following partial pancreatectomy\nC_LIO_LIIn vitro studies in mouse MIN6 cells indicate that miR-132 fosters beta cell proliferation by down-regulating the expression of phosphatase Pten, thereby tilting the balance between anti-apoptotic factor Akt and pro-apoptotic factor Foxo3 activities towards proliferation through regulation of their phosphorylation.\nC_LI\n\nHow might this impact on clinical practice in the foreseeable future?\n\nO_LIThese findings strengthen the rationale for targeting the expression of miR-132 to increase beta cell mass in vivo (type 2 diabetes) or ex-vivo (islet transplantation in type 1 diabetes) for the treatment of diabetes.\nC_LI

cell biology↗

bioRxiv2017

ZMYND10 functions in a chaperone relay during axonemal dynein assembly

Molecular chaperones promote the folding and macromolecular assembly of a diverse set of substrate client proteins. How the ubiquitous chaperone machinery directs its activities towards a specific set of substrates and whether this selectivity could be targeted for therapeutic intervention is of intense research. Through the use of mouse genetics, imaging and quantitative proteomics we uncover that ZMYND10 is a novel co-chaperone for the FKBP8-HSP90 chaperone complex during the biosynthesis of axonemal dynein heavy chains required for cilia motility. In the absence of ZMYND10, defects in dynein heavy chains trigger broader dynein motor degradation. We show that FKBP8 inhibition phenocopies dynein motor instability in airway cells, and human disease-causing variants of ZMYND10 disrupt its ability to act as FKBP8-HSP90 co-chaperone. Our study indicates that the motile ciliopathy Primary Ciliary Dyskinesia (PCD) should be considered a cell-type specific protein-misfolding disease and opens the potential for rational drug design that could restore specificity to the ubiquitous chaperone apparatus towards dynein subunits.

cell biology↗

bioRxiv2017

mRNA structure determines specificity of a polyQ-driven phase separation

RNA promotes liquid-liquid phase separation (LLPS) to build membrane-less compartments in cells. How distinct molecular compositions are established and maintained in these liquid compartments is unknown. Here we report that secondary structure allows mRNAs to self-associate and determines if an mRNA is recruited to or excluded from liquid compartments. The polyQ-protein Whi3 induces conformational changes in RNA structure and generates distinct molecular fluctuations depending on the RNA sequence. These data support a model in which structure-based, RNA-RNA interactions promote assembly of distinct droplets and protein-driven, conformational dynamics of the RNA maintain this identity. Thus, the shape of RNA can promote the formation and coexistence of the diverse array of RNA-rich liquid compartments found in a single cell.\n\nOne Sentence SummaryIdentity in cellular, phase-separated compartments arises from RNA-RNA complexes encoded by mRNA secondary structures.

cell biology↗

bioRxiv2017

Neural network control of focal position during time-lapse microscopy of cells

Live-cell microscopy is quickly becoming an indispensable technique for studying the dynamics of cellular processes. Maintaining the specimen in focus during image acquisition is crucial for high-throughput applications, especially for long experiments or when a large sample is being continuously scanned. Automated focus control methods are often expensive, imperfect, or ill-adapted to a specific application and are a bottleneck for widespread adoption of high-throughput, live-cell imaging. Here, we demonstrate a neural network approach for automatically maintaining focus during bright-field microscopy. Z-stacks of yeast cells growing in a microfluidic device were collected and used to train a convolutional neural network to classify images according to their z-position. We studied the effect on prediction accuracy of the various hyperparameters of the neural network, including downsampling, batch size, and z-bin resolution. The network was able to predict the z-position of an image with {+/-}1 m accuracy, outperforming human annotators. Finally, we used our neural network to control microscope focus in real-time during a 24 hour growth experiment. The method robustly maintained the correct focal position compensating for 40 m of focal drift and was insensitive to changes in the field of view. Only ~100 annotated z-stacks were required to train the network making our method quite practical for custom autofocus applications.

cell biology↗

bioRxiv2017

Transcription establishes microenvironments that organize euchromatin

Chromatin is organized into heterochromatin, which is transcriptionally inactive, and euchromatin, which can switch between transcriptionally active and inactive states. This switch in euchromatin activity is accompanied by changes in its spatial distribution. How euchromatin rearrangements are established is unknown. Here we use super-resolution and live-cell microscopy to show that transcriptionally inactive euchromatin moves away from transcriptionally active euchromatin. This movement is driven by the formation of RNA-enriched microenvironments that exclude inactive euchromatin. Using theory, we show that the segregation into RNA-enriched microenvironments and euchromatin domains can be considered an active microemulsion. The tethering of transcripts to chromatin via RNA polymerase II forms effective amphiphiles that intersperse the two segregated phases. Taken together with previous experiments, our data suggest that chromatin is organized in the following way: heterochromatin segregates from euchromatin by phase separation, while transcription organizes euchromatin similar to an active microemulsion.

cell biology↗

bioRxiv2017

Reticular adhesions: A new class of adhesion complex that mediates cell-matrix attachment during mitosis

Adhesion to the extracellular matrix (ECM) persists during mitosis in most cell types. Yet, classical adhesion complexes (ACs), such as focal adhesions and focal complexes, do and must disassemble to enable cytoskeletal rearrangements associated with mitotic rounding. Given this paradox, mechanisms of mitotic cell-ECM adhesion remain undefined. Here, we identify reticular adhesions, a new class of AC that is mediated by integrin v{beta}5, formed during interphase and preserved at cell-ECM attachment sites throughout cell division. Consistent with this role, integrin {beta}5 depletion perturbs mitosis and disrupts spatial memory transmission between cell generations. Quantitative imaging reveals reticular adhesions to be both morphologically and dynamically distinct from classic focal adhesions, while mass spectrometry defines their unique composition; lacking virtually all consensus adhesome components. Indeed, remarkably, reticular adhesions are functionally independent of both talin and F-actin, yet are promoted by phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). Overall, the distinct characteristics of reticular adhesions provide a unique solution to the problem of maintaining cell-ECM attachment during mitotic rounding and division.

cell biology↗

bioRxiv2017

A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions

A variety of self-interacting domains, defined at different levels of resolution, have been described in mammalian genomes. These include Chromatin Compartments (A and B)1, Topologically Associated Domains (TADs)2,3, contact domains4,5, sub-TADs6, insulated neighbourhoods7 and frequently interacting regions (FIREs)8. Whereas many studies have found the organisation of self-interacting domains to be conserved across cell types389, some do form in a lineage-specific manner6710. However, it is not clear to what degree such tissue-specific structures result from processes related to gene activity such as enhancer-promoter interactions or whether they form earlier during lineage commitment and are therefore likely to be prerequisite for promoting gene expression. To examine these models of genome organisation in detail, we used a combination of high-resolution chromosome conformation capture, a newly-developed form of quantitative fluorescence in-situ hybridisation and super-resolution imaging to study a 70 kb self-interacting domain containing the mouse -globin locus. To understand how this self-interacting domain is established, we studied the region when the genes are inactive and during erythroid differentiation when the genes are progressively switched on. In contrast to many current models of long-range gene regulation, we show that an erythroid-specific, decompacted self-interacting domain, delimited by convergent CTCF/cohesin binding sites, forms prior to the onset of robust gene expression. Using previously established mouse models we show that formation of the self-interacting domain does not rely on interactions between the -globin genes and their enhancers. As there are also no tissue-specific changes in CTCF binding, then formation of the domain may simply depend on the presence of activated lineage-specific cis-elements driving a transcription-independent mechanism for opening chromatin throughout the 70 kb region to create a permissive environment for gene expression. These findings are consistent with a model of loop-extrusion in which all segments of chromatin, within a region delimited by CTCF boundary elements, can contact each other. Our findings suggest that activation of tissue-specific element(s)within such a self-interacting region is sufficient to influence all chromatin within the domain.

cell biology↗

bioRxiv2017

The IRE1a-XBP1 pathway promotes T helper cell differentiation by resolving secretory stress and accelerating proliferation

The IRE1a-XBP1 pathway, a conserved adaptive mediator of the unfolded protein response, is indispensable for the development of secretory cells. It maintains endoplasmic reticulum homeostasis by facilitating protein folding and enhancing secretory capacity of the cells. Its role in immune cells is emerging. It is involved in dendritic cell, plasma cell and eosinophil development and differentiation. Using genome-wide approaches, integrating ChIPmentation and mRNA-sequencing data, we have elucidated the regulatory circuitry governed by the IRE1a-XBP1 pathway in type-2 T helper cells (Th2). We show that the XBP1 transcription factor is activated by splicing in vivo in T helper cell lineages. We report a comprehensive repertoire of XBP1 target genes in Th2 lymphocytes. We found that the pathway is conserved across cell types in terms of resolving secretory stress, and has T helper cell-specific functions in controlling activation-dependent Th2 cell proliferation and regulating cytokine expression in addition to secretion. These results provide a detailed picture of the regulatory map governed by the XBP1 transcription factor during Th2 lymphocyte activation.

cell biology↗

bioRxiv2018

Basal mitophagy is widespread in Drosophila but minimally affected by loss of Pink1 or parkin

Parkinsons disease factors, PINK1 and parkin, are strongly implicated in stress-induced mitophagy in vitro, but little is known about their impact on basal mitophagy in vivo. We generated transgenic Drosophila expressing fluorescent mitophagy reporters to evaluate the impact of Pink1/parkin mutations on basal mitophagy under physiological conditions. We find that mitophagy is readily detectable and abundant in many tissues including Parkinsons disease relevant dopaminergic neurons. However, we did not detect mitolysosomes in flight muscle. Surprisingly, in Pink1 or parkin null flies we did not observe any substantial impact on basal mitophagy. As these flies exhibit locomotor defects and dopaminergic neuron loss, our findings raise questions about current assumptions of the pathogenic mechanism associated with the PINK1/Parkin pathway. Our findings provide evidence that Pink1 and parkin are not essential for bulk basal mitophagy in Drosophila. They also emphasize that mechanisms underpinning basal mitophagy remain largely obscure.\n\nSummaryPINK1/parkin are key mediators of stress-induced mitophagy in vitro but their impact on basal mitophagy in vivo is unclear. Novel Drosophila reporters lines reveal abundant mitophagy in many tissues including dopaminergic neurons but is unaffected by loss of PINK1/parkin.

cell biology↗

bioRxiv2017

AP-4 vesicles unmasked by organellar proteomics to reveal their cargo and machinery

Adaptor protein 4 (AP-4) is an ancient membrane trafficking complex, whose function has largely remained elusive. In humans, AP-4 deficiency causes a severe neurological disorder of unknown aetiology. We apply unbiased proteomic methods, including Dynamic Organellar Maps, to find proteins whose subcellular localisation depends on AP-4. We identify three transmembrane cargo proteins, ATG9A, SERINC1 and SERINC3, and two AP-4 accessory proteins, RUSC1 and RUSC2. We demonstrate that AP-4 deficiency causes missorting of ATG9A in diverse cell types, including patient-derived cells, as well as dysregulation of autophagy. RUSC2 facilitates the transport of AP-4-derived, ATG9A-positive vesicles from the TGN to the cell periphery. These vesicles cluster in close association with autophagosomes, suggesting they are the \"ATG9A reservoir\" required for autophagosome biogenesis. Our study uncovers ATG9A trafficking as a ubiquitous function of the AP-4 pathway. Furthermore, it provides a potential molecular pathomechanism of AP-4 deficiency, through dysregulated spatial control of autophagy.

cell biology↗

bioRxiv2017

Muscle specific stress fibers give rise to sarcomeres and are mechanistically distinct from stress fibers in non-muscle cells

The sarcomere is the basic contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating thin (i.e., actin) and thick (i.e., myosin) filament assembly during sarcomere formation. Thus, we developed an assay using human cardiomyocytes to test de novo sarcomere assembly. Using this assay, we report a population of muscle-specific stress fibers are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from these muscle stress fibers. This process requires formin-mediated but not Arp2/3-mediated actin polymerization and nonmuscle myosin IIB but not non-muscle myosin IIA. Furthermore, we show a short species of {beta} cardiac myosin II filaments grows to form ~1.5 long filaments that then \"stitch\" together to form the stack of filaments at the core of the sarcomere (i.e., A-band). Interestingly, these are different from mechanisms that have previously been reported during stress fiber assembly in non-muscle cells. Thus, we provide a new model of cardiac sarcomere assembly based on distinct mechanisms of stress fiber regulation between non-muscle and muscle cells.

cell biology↗

bioRxiv2017

Regulation of mitochondrial dynamics by Aurora A kinase

Aurora A kinase (AURKA) is a major regulator of mitosis and an important driver of cancer progression. The roles of AURKA outside of mitosis, and how these might contribute to cancer progression, are not well understood. Here we show that a fraction of cytoplasmic AURKA is associated with mitochondria, co-fractionating in cell extracts and interacting with mitochondrial proteins by reciprocal co-immunoprecipitation. We have also found that the dynamics of the mitochondrial network are sensitive to AURKA inhibition, depletion or overexpression. This can account for the different mitochondrial morphologies observed in RPE1 and U2OS cell lines, which show very different levels of expression of AURKA. We identify the mitochondrial fraction of AURKA as influencing mitochondrial morphology, since an N-terminally truncated version of the kinase that does not localize to mitochondria does not affect the mitochondrial network. We identify a cryptic mitochondrial targeting sequence in the AURKA N-terminus and discuss how alternative conformations of the protein may influence its cytoplasmic fate.

cell biology↗

bioRxiv2017

Deficient autophagy drives aging in Hydra

Hydra exhibits a negligible senescence as its epithelial and interstitial stem cell populations continuously divide. Here we identified two H. oligactis strains that respond differently to interstitial stem cell loss. Cold-resistant (Ho_CR) animals adapt and remain healthy while cold-sensitive (Ho_CS) ones die within three months, after their epithelial stem cells lose their selfrenewal potential. In Ho_CS but not in Ho_CR animals, the autophagy flux is deficient, characterized by a low induction upon starvation, proteasome inhibition or Rapamycin treatment, and a constitutively repressed Ulk activity. In the non-aging Hydra vulgaris, WIPI2 silencing suffices to induce aging. Rapamycin can delay aging by sustaining epithelial self-renewal and regeneration, although without enhancing the autophagy flux. Instead Rapamycin promotes engulfment in epithelial cells where p62/SQSTM1-positive phagocytic vacuoles accumulate. This study uncovers the importance of autophagy in the longevity of early-branched eumetazoans by maintaining stem cell renewal, and a novel anti-aging effect of Rapamycin via phagocytosis.

cell biology↗