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 Read source abstracts and check manuscript versions; preprints are not peer reviewed.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

Rational cell culture optimization enhances experimental reproducibility in cancer cells.

Optimization of experimental conditions is critical in ensuring robust experimental reproducibility. Through detailed metabolomic analysis we found that cell culture conditions significantly impacted on glutaminase (GLS1) sensitivity resulting in variable sensitivity and irreproducibility in data.\n\nBaseline metabolite profiling highlighted that untreated cells underwent significant changes in metabolic status. Both the extracellular levels of glutamine and lactate and the intracellular levels of multiple metabolites changed drastically during the assay. We show that these changes compromise the robustness of the assay and make it difficult to reproduce.\n\nWe then devised metabolically rationalized standard assay conditions, in which glutaminase-1 inhibition reduced glutamine metabolism differently in both cell lines assayed, and decreased the proliferation of one of them. The adoption of optimized conditions such as the ones described here should lead to an improvement in reproducibility and help eliminate false negatives as well as false positives in these assays.

cell biology↗

bioRxiv2017

ESCRT-Independent Surface Receptor And Transporter Protein Degradation By The ILF Pathway

Surface receptor and transporter protein down-regulation drives cell signaling, quality control and metabolism underlying diverse physiology. After endocytosis, proteins are delivered to endosomes where ESCRTs package them into intralumenal vesicles, which are degraded by acid hydrolases upon fusion with lysosomes. However, reports of ESCRT-independent surface protein degradation are emerging suggesting that alternative, non-canonical pathways exist. Using Saccharomyces cerevisiae as a model, here we show that in response to substrates, protein misfolding or TOR signaling, some internalized surface transporters (Hxt3, Itr1, Aqr1) bypass ESCRTs en route to the lysosome membrane where they are sorted into an area that is internalized as an intralumenal fragment (ILF) and degraded upon organelle fusion. This ILF pathway also degrades typical ESCRT client proteins (Mup1, Can1, Ste3) when ESCRT function is impaired. As the underlying machinery is conserved, we speculate that the ILF pathway is an important contributor to receptor and transporter down-regulation in all eukaryotes.

cell biology↗

bioRxiv2017

The mitotic spindle is chiral due to torques generated by motor proteins

Mitosis relies on forces generated in the spindle, a micro-machine composed of microtubules and associated proteins1,2. Forces are required for the congression of chromosomes to the metaphase plate and separation of chromatids in anaphase3-6. However, torques may also exist in the spindle, yet they have not been investigated. Here we show that the spindle is chiral. Chirality is evident from the finding that microtubule bundles follow a left-handed helical path, which cannot be explained by forces but rather by torques acting in the bundles. STED super-resolution microscopy, as well as confocal microscopy, of human spindles shows that the bundles have complex curved shapes. The average helicity of the bundles with respect to the spindle axis is 1.2{degrees}/m. Inactivation of kinesin-5 (Eg5/Kif11) abolished the chirality of the spindle, suggesting that this motor generates the helical shape of microtubule bundles. To explain the observed shapes, we introduce a theoretical model for the balance of forces and torques acting in the spindle, and show that torque is required to generate the helical shapes. We conclude that torques generated by motor proteins, in addition to forces, exist in the spindle and determine its architecture.

cell biology↗

bioRxiv2017

The cytoplasmic DNA sensor cGAS promotes mitotic cell death

The cyclic GMP-AMP (cGAMP) synthase cGAS counteracts infections by detecting and binding foreign cytoplasmic DNA1. DNA-induced synthesis of cGAMP activates innate immune signalling and apoptosis through the cGAMP receptor STING and the downstream effector IRF31-7. During interphase the nuclear envelope protects chromosomal self-DNA from cGAS, but the consequences of exposing chromosomes to cGAS following mitotic nuclear envelope disassembly are unknown. Here we demonstrate that cGAS associates with chromosomes during mitosis and binds nucleosomes with even higher affinity than naked DNA in vitro. Nucleosomes nevertheless competitively inhibit the DNA-dependent stimulation of cGAS, and accordingly, chromosomal cGAS does not affect mitotic progression under normal conditions. This suggests that nucleosomes prevent the inappropriate activation of cGAS during mitosis by acting as a signature of self-DNA. During prolonged mitotic arrest, however, cGAS becomes activated to promote cell death, limiting the fraction of cells that can survive and escape mitotic arrest induced by the chemotherapeutic drug taxol. Induction of mitotic cell death involves cGAMP synthesis by cGAS, as well as signal transduction to IRF3 by STING. We thus propose that cGAS plays a previously unappreciated role in guarding against mitotic errors, promoting cell death during prolonged mitotic arrest. Our data also indicate that the cGAS pathway, whose activity differs widely among cell lines, impacts cell fate determination upon treatment with taxol and other anti-mitotic drugs. Thus, we propose the innate immune system may be harnessed to selectively target cells with mitotic abnormalities.

cell biology↗

bioRxiv2017

AKT isoforms have distinct hippocampal expression and roles in synaptic plasticity

AKT is a kinase that regulates numerous cellular processes in the brain and mutations in AKT are known to affect brain function. AKT is indirectly implicated in synaptic plasticity, but its direct role has not been studied. Moreover, three highly related AKT isoforms are expressed in the brain, but their individual roles are poorly understood. We find that each AKT isoform has a unique expression pattern in the hippocampus, with AKT1 and AKT3 primarily in neurons but displaying local differences, while AKT2 is in astrocytes. We also find isoform-specific roles for AKT in multiple paradigms of hippocampal synaptic plasticity. AKT1, but not AKT2 or AKT3, is required for L-LTP through regulating activity-induced protein synthesis. Interestingly, AKT activity inhibits mGluR-LTD, with overlapping functions for AKT1 and AKT3. In summary, our studies identify distinct expression patterns and roles in synaptic plasticity for AKT isoforms in the hippocampus.

cell biology↗

bioRxiv2017

Remodeling of ER-exit sites initiates a membrane supply pathway forautophagosome biogenesis

Autophagosomes are double-membrane vesicles generated during autophagy. Biogenesis of the autophagosome requires membrane acquisition from intracellular compartments, the mechanisms of which are unclear. We previously found that a relocation of COPII machinery to the ER-Golgi intermediate compartment (ERGIC) generates ERGIC-derived COPII vesicles which serve as a membrane precursor for the lipidation of LC3, a key membrane component of the autophagosome. Here we employed super-resolution microscopy to show that starvation induces the enlargement of ER-exit sites (ERES) positive for the COPII activator, SEC12, and the remodeled ERES patches along the ERGIC. A SEC12 binding protein, CTAGE5, is required for the enlargement of ERES, SEC12 relocation to the ERGIC, and modulates autophagosome biogenesis. Moreover, FIP200, a subunit of the ULK protein kinase complex, facilitates the starvation-induced enlargement of ERES independent of the other subunits of this complex and associates via its C-terminal domain with SEC12. Our data indicate a pathway wherein FIP200 and CTAGE5 facilitate starvation-induced remodeling of the ERES, a prerequisite for the production of COPII vesicles budded from the ERGIC that contribute to autophagosome formation.

cell biology↗

bioRxiv2017

Inferring cell state by quantitative motility analysis reveals a dynamic state system and broken detailed balance

Cell populations display heterogeneous phenotypic states at multiple scales. Similar to molecular features commonly used to explore cell heterogeneity, cell behavior is a rich phenotypic space that may allow for identification of relevant cell states. Inference of cell state from cell behavior across a time course may enable the investigation of dynamics of transitions between heterogeneous cell states, a task difficult to perform with destructive molecular observations. Cell motility is one such easily observed cell behavior with known biomedical relevance. To investigate cell heterogeneity through the lens of cell behavior, we developed Heteromotility, a software tool to extract quantitative motility features from timelapse cell images. In mouse embryonic fibroblasts (MEFs), myoblasts, and muscle stem cells (MuSCs), Heteromotility analysis identifies multiple motility phenotypes within the population. In all three systems, the motility state identity of individual cells is dynamic. Quantification of state transitions reveals that MuSCs undergoing activation transition through progressive motility states toward the myoblast phenotype. By probability flux analysis, we find that this MuSC motility state system breaks detailed balance, while the MEF and myoblast systems do not. Our data indicate that the system regulating cell behavior can be decomposed into a set of attractor states which depend on the identity of the cell, together with a set of transitions between states governed by inputs from signaling pathways such as oncogenes and growth factors. Within one state, equilibrium formalisms can capture variation in behavior, while switching between states violates equilibrium conditions and would require an external driving force. These results support a conceptual view of the cell as a non-deterministic state automaton, responding to inputs from signaling pathways and generating outputs in the form of observable motile behaviors.

cell biology↗

bioRxiv2017

A compartmentalized, self-extinguishing signaling network mediates crossover control in meiosis

Meiotic recombination between homologous chromosomes is tightly regulated to ensure proper chromosome segregation. Each chromosome pair typically undergoes at least one crossover event (crossover assurance) but these exchanges are also strictly limited in number and widely spaced along chromosomes (crossover interference). This has implied the existence of chromosome-wide signals that regulate crossovers, but their molecular basis remains mysterious. Here we characterize a family of four related RING finger proteins in C. elegans. These proteins are recruited to the synaptonemal complex between paired homologs, where they act as two heterodimeric complexes, likely as E3 ubiquitin ligases. Genetic and cytological analysis reveals that they act with additional components to create a self-extinguishing circuit that controls crossover designation and maturation. These proteins also act at the top of a hierarchical chromosome remodeling process that enables crossovers to direct stepwise segregation. Work in diverse phyla indicates that related mechanisms mediate crossover control across eukaryotes.

cell biology↗

bioRxiv2017

Alpha-synuclein fibrils induce autophagy in microglial cells as a consequence of lysosomal damage.

Autophagy is a constitutive lysosomal catabolic pathway that degrades damaged organelles and protein aggregates. In Parkinsons disease, the synaptic protein alpha-synuclein (AS) accumulates in neuronal cell bodies and axons. Recent studies indicate that aggregation-prone proteins can spread to other brain cells - such as glia - contributing to progressive deterioration.\n\nAlthough autophagic dysfunction and protein aggregation have been linked to several neurodegenerative disorders, exact mechanisms are not clear and most work was done in neurons and not on microglial cells.\n\nHere we report that AS fibrils but not monomers induce lysosomal damage and autophagy in microglial cells and we extensively characterized the dynamics of this response by both live-cell imaging and correlative light-electron microscopy (CLEM). In addition, we found that autophagy inhibition in these cells impairs mitochondrial quality and leads to microglial cell death. We propose that AS accumulation in lysosomes leads to lysosomal damage, which in turn activates canonical autophagy as a rescue mechanism.\n\nOur results provide novel findings about the interaction between AS and the autophagy pathway in microglial cells, which may be important for targeting protein misfolding-associated neurodegenerative diseases.

cell biology↗

bioRxiv2017

The ABC transporter Snu drives formation of the lipid-based inward and outward barrier in the skin of Drosophila

Lipids in extracellular matrices (ECM) contribute to barrier function and stability of epithelial tissues such as the pulmonary alveoli and the skin. In insects, skin waterproofness depends on the outermost layer of the extracellular cuticle termed envelope that contains cuticulin, an unidentified water-repellent complex molecule composed of proteins, lipids and catecholamines. Based on live-imaging analyses of fruit fly larvae, we find that initially envelope units are assembled within vesicles harbouring the ABC transporter Snu and the extracellular protein Snsl. In a second step, the content of these vesicles is distributed to cuticular lipid-transporting nanotubes named pore canals and to the cuticle surface in dependence of Snu function. Consistently, the surface of snu and snsl mutant larvae is depleted from lipids and cuticulin. By consequence, these animals suffer uncontrolled water loss and penetration of xenobiotics. Our data allude to a two-step model of envelope i.e. barrier formation. The proposed mechanism in principle parallels the events occurring during differentiation of the lipid-based ECM by keratinocytes in the vertebrate skin suggesting establishment of analogous mechanisms of skin barrier formation in vertebrates and invertebrates.

cell biology↗

bioRxiv2017

Tropomyosin and a-actinin cooperation inhibits fimbrin association with actin filament networks in fission yeast

We previously discovered that competition between fission yeast actin binding proteins (ABPs) for association with F-actin helps facilitate their sorting to different F-actin networks. Specifically, competition between actin patch ABPs fimbrin Fim1 and cofilin Adf1 enhances each others activities, and rapidly displaces tropomyosin Cdc8 from the F-actin network. However, these interactions dont explain how Fim1, a robust competitor, is prevented from associating equally well with other F-actin networks. Here, with a combination of fission yeast genetics, live cell fluorescent imaging, and in vitro TIRF microscopy, we identified the contractile ring ABP -actinin Ain1 as a key sorting factor. Fim1 competes with Ain1 for association with F-actin, which is dependent upon their residence time on F-actin. Remarkably, although Fim1 outcompetes both contractile ring ABPs Ain1 and Cdc8 individually, Cdc8 enhances the bundling activity of Ain1 10-fold, allowing the combination of Ain1 and Cdc8 to inhibit Fim1 association with contractile ring F-actin.

cell biology↗

bioRxiv2017

The Na+(K+)/H+ exchanger Nhx1 controls multivesicular body-vacuolar lysosome fusion

Loss-of-function mutations in human endosomal Na+(K+)/H+ Exchangers (NHEs) NHE6 and NHE9 are implicated in neurological disorders including Christianson Syndrome, autism and attention deficit and hyperactivity disorder (ADHD). These mutations disrupt retention of surface receptors within neurons and glial cells by affecting their delivery to lysosomes for degradation. However, the molecular basis of how these endosomal NHEs control endocytic trafficking is unclear. Using Saccharomyces cerevisiae as a model, we conducted cell-free organelle fusion assays to show that transport activity of the orthologous endosomal NHE Nhx1 is important for multivesicular body (MVB)-vacuolar lysosome fusion, the last step of endocytosis required for surface protein degradation. We find that deleting Nhx1 disrupts the fusogenicity of the MVB, not vacuole, by targeting pH-sensitive machinery downstream of the Rab-GTPase Ypt7 needed for SNARE-mediated lipid bilayer merger. All contributing mechanisms are evolutionarily conserved offering new insight into the etiology of human disorders linked to loss of endosomal NHE function.

cell biology↗

bioRxiv2017

Small GTPases and BAR domain proteins regulate branched actin to make clathrin and dynamin independent endocytic vesicles

Numerous endocytic pathways operate simultaneously at the cell surface. Here we focus on the molecular machinery involved in the generation of endocytic vesicles of the clathrin and dynamin-independent CLIC/GEEC (CG) pathway. This pathway internalises many GPI-anchored proteins and a large fraction of the fluid-phase in different cell types. We developed a real-time TIRF assay using pH-sensitive GFP-GPI to identify nascent CG endocytic sites. The temporal profile of known CG pathway modulators showed that ARF1/GBF1 (GTPase/GEF pair) and CDC42 (RhoGTPase) are recruited sequentially to CG endocytic sites, [~]60s and [~]9s prior to scission. Using a limited RNAi screen, we found several BAR domain proteins affecting CG endocytosis and focused on IRSp53 and PICK1 that have interactions with CDC42 and ARF1 respectively. IRSp53, an I-BAR domain containing protein, was recruited to the plasma membrane at the site of forming CG endocytic vesicles and in its absence, nascent endocytic CLICs, did not form. The requirement for actin polymerization in the CG pathway suggested a role for nucleators of actin polymerization, and ARP2/3 was found enriched at the site of the forming endocytic vesicle. PICK1, a BAR domain containing protein and the ARP2/3 inhibitor is recruited at an early stage along with ARP2/3, but is removed from the endocytic site coincident with CDC42 recruitment and a burst of Factin polymerization. This study provides a spatio-temporal understanding of the molecular machinery necessary to build a CG endocytic vesicle.

cell biology↗

bioRxiv2017

Kif2 microtubule depolymerase is required for unequal cell division and localizes to a subdomain of cortical endoplasmic reticulum

Unequal cell division (UCD) is a fundamental process responsible for creating sibling cell size asymmetry; however, how microtubules are specifically depolymerized on one aster of the mitotic spindle creating a smaller sibling cell during UCD has remained elusive. Using invertebrate chordate embryos (ascidian) that possess a large cortical structure (CAB) that causes UCD, we identified a microtubule depolymerase (Kif2) involved in creating cell size asymmetry. Kif2 localizes to the cortical subdomain of endoplasmic reticulum in the CAB. During three successive UCDs, Kif2 protein accumulates at the CAB during interphase and is delocalized from the CAB in mid mitosis. Rapid imaging of microtubule dynamics at the cortex revealed that microtubules do not penetrate the CAB during interphase. Inhibition of Kif2 function prevents the development of mitotic aster asymmetry and centrosome movement towards the CAB thereby blocking UCD, whereas locally increasing microtubule depolymerization causes exaggerated asymmetric spindle positioning. This study provides insights into the fundamental process of UCD and for the first time shows that a microtubule depolymerase is localized to a cortical site controlling UCD.

cell biology↗

bioRxiv2017

The impact of experimental design choices on parameter inference for models of growing cell colonies

To better understand development, repair and disease progression it is useful to quantify the behaviour of proliferative and motile cell populations as they grow and expand to fill their local environment. Inferring parameters associated with mechanistic models of cell colony growth using quantitative data collected from carefully designed experiments provides a natural means to elucidate the relative contributions of various processes to the growth of the colony. In this work we explore how experimental design impacts our ability to infer parameters for simple models of the growth of proliferative and motile cell populations. We adopt a Bayesian approach, which allows us to characterise the uncertainty associated with estimates of the model parameters. Our results suggest that experimental designs that incorporate initial spatial heterogeneities in cell positions facilitate parameter inference without the requirement of cell tracking, whilst designs that involve uniform initial placement of cells require cell tracking for accurate parameter inference. As cell tracking is an experimental bottleneck in many studies of this type, our recommendations for experimental design provide for significant potential time and cost savings in the analysis of cell colony growth.

cell biology↗

bioRxiv2017

Kinesin-6 KIF20B is required for efficient cytokinetic furrowing and timely abscission in human cells

Cytokinesis requires the cooperation of many cytoskeletal and membrane regulators. Most of the major players required for cytokinesis are known, but the temporal regulation and adaptations for different cell types are less understood. KIF20B (previously called MPHOSPH1 or MPP1) is a member of the Kinesin-6 family, which also includes the better-known members KIF23/MKLP1 and KIF20A/MKLP2. Previously, we showed that mouse Kif20b is involved in cerebral cortex growth and midbody organization of neural stem cells. Here we show that KIF20B has a cell-autonomous role in cytokinesis in isolated human cells. It localizes to microtubules of the central spindle and midbody throughout cytokinesis, at sites distinct from the other Kinesin-6 family members. KIF20B is not required for central spindle or midbody assembly, but affects midbody shape and late maturation steps. KIF20B appears to temporally regulate both furrow ingression and abscission.

cell biology↗

bioRxiv2017

The contributions of the actin machinery to endocytic membrane bending and vesicle formation

Branched and crosslinked actin networks mediate cellular processes that move and shape membranes. To understand how actin contributes during the different stages of endocytic membrane reshaping, we analysed deletion mutants of yeast actin network components using a hybrid imaging approach that combines live imaging with correlative microscopy. We could thereby temporally dissect the effects of different actin network perturbations, revealing distinct stages of actin-based membrane reshaping. Our data show that initiation of membrane bending requires the actin network to be physically linked to the plasma membrane and to be optimally crosslinked. Once initiated, the membrane invagination process is driven by nucleation and polymerization of new actin filaments, independently of the degree of cross-linking and unaffected by a surplus of actin network components. A key transition occurs 2 seconds before scission when the filament nucleation rate drops. From that time point on, invagination growth and vesicle scission are driven by an expansion of the assembled actin network. The expansion is sensitive to the amount of filamentous actin and its crosslinking. Our results suggest that the mechanism by which actin reshapes the membrane adapts to force requirements that vary during the progress of endocytosis.

cell biology↗

bioRxiv2017

ATR is a multifunctional regulator of male mouse meiosis

Meiotic cells undergo genetic exchange between homologous chromosomes through programmed DNA double-strand break (DSB) formation, recombination and synapsis1, 2. In mice, the DNA damage-regulated phosphatidylinositol-3-kinase-like kinase (PIKK) ATM regulates all of these processes3-6. However, the meiotic functions of another major PIKK, ATR, have remained elusive, because germ line-specific depletion of this kinase is challenging. Using an efficient conditional strategy, we uncover roles for ATR in male mouse prophase I progression. Deletion of ATR causes chromosome axis fragmentation and germ cell elimination at mid pachynema. ATR is required for homologous synapsis, in a manner genetically dissociable from DSB formation. In addition, ATR regulates loading of recombinases RAD51 and DMC1 to DSBs and maintenance of recombination foci on synapsed and asynapsed chromosomes. Mid pachytene spermatocyte elimination in ATR deficient mice cannot be rescued by deletion of ATM and the third DNA damage-regulated PIKK, PRKDC, consistent with the existence of a PIKK-independent surveillance mechanism in the mammalian germ line. Our studies identify ATR as a multifunctional regulator of mouse meiosis.

cell biology↗