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bioRxiv2018

Inner lumen proteins stabilize doublet microtubules in cilia/flagella

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although it is known that microtubules in cilia are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dynein and intraflagellar transport. To address this question, we identified a new class of microtubule-associated proteins, named FAP45 and FAP52, in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we established that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These inner protrusions are located near the inner junctions of doublet microtubules and lack of FAP45, FAP52, and FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrated that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

cell biology↗

bioRxiv2018

Spermatoproteasome-deficient mice are proficient in meiotic DNA repair but defective in meiotic exit

Meiotic recombination generates crossovers which are essential to ensure genome haploidization. The ubiquitin proteasome system regulates meiotic recombination through its association to the synaptonemal complex, a zipper-like structure that holds homologs and provides the structural framework for meiotic recombination. Here we show that the testis-specific 4s subunit (PSMA8) of the spermatoproteasome is located at the synaptonemal complex and is essential for the assembly of its activator PA200. Accordingly, synapsis-deficient mice show delocalization of PSMA8 from the synaptonemal complex. Genetic analysis of Psma8-deficient mice shows normal meiotic DNA repair, crossing over formation and an increase of spermatocytes at metaphase I and metaphase II which either enter into apoptosis or slip to give rise to an early spermatid arrest and infertility. Thus, spermatoproteasome-dependent histone degradation is dispensable for meiotic recombination. We show that PSMA8 deficiency alters the proteostasis of several key meiotic players such as acetylated histones, SYCP3, SYCP1, CDK1 and TRIP13 which in turn leads to an aberrant meiotic exit and early spermatid arrest prior to the histone displacement process that take place subsequently.

cell biology↗

bioRxiv2018

Condensin controls mitotic chromosome stiffness and stability without forming a structurally contiguous scaffold

During cell division, chromosomes must be folded into their compact mitotic form to ensure their segregation. This process is thought to be largely controlled by the action of condensin SMC protein complexes on chromatin fibers. However, how condensins organize metaphase chromosomes is not understood. We have combined micromanipulation of single human mitotic chromosomes, sub-nanonewton force measurement, siRNA interference of condensin subunit expression, and fluorescence microscopy, to analyze the role of condensin in large-scale chromosome organization. Condensin depletion leads to a dramatic (~10 fold) reduction in chromosome elastic stiffness relative to the native, non-depleted case. We also find that prolonged metaphase stalling of cells leads to overloading of chromosomes with condensin, with abnormally high chromosome stiffness. These results demonstrate that condensin is a main element controlling the stiffness of mitotic chromosomes. Isolated, slightly stretched chromosomes display a discontinuous condensing staining pattern, suggesting that condensins organize mitotic chromosomes by forming isolated compaction centers that do not form a continuous scaffold.

cell biology↗

bioRxiv2018

Mechanical heterogeneity and roles of parallel microtubule arrays in governing meiotic spindle length

Metaphase spindles are arrays of microtubules whose architecture provides the mechanism for regulated force generation required for proper segregation of chromosomes during cell division. Whereas long-standing models are based on continuous antiparallel microtubule arrays connecting two spindle poles and overlapping at the equator, spindles typically possess a more complex architecture with randomly arranged short filaments. How these heterogeneous multifilament arrays generate and respond to forces has been mysterious, as it has not been possible to directly measure and perturb spindle force while observing relevant filament motility. Here, we combined microneedle-based quantitative micromanipulation with high-resolution microtubule tracking of Xenopus egg extract spindles to simultaneously examine the force and individual filament motility in situ. We found that the microtubule arrays at the middle of the spindle half are considerably weak and fluid-like, being more adaptable to perturbing forces as compared to those near the pole and the equator. We also found that a force altering spindle length induces filament translocation nearer the spindle pole, where parallel microtubules predominate, while maintaining equatorial antiparallel filaments. Molecular perturbations suggested that the distinct mechanical heterogeneity of the spindle emerges from activities of kinesin-5 and dynein, two key spindle motor proteins. Together, our data establish a link between spindle architecture and mechanics, and highlight the importance of parallel microtubule arrays in maintaining its structural and functional stability.

cell biology↗

bioRxiv2018

Efa6 regulates axon growth, branching and maintenance by eliminating off-track microtubules at the cortex

Cortical collapse factors affect microtubule (MT) dynamics at the plasma membrane. They play important roles in neurons, as suggested by inhibition of axon growth and regeneration through the Arf activator Efa6 in C. elegans, and by neurodevelopmental disorders linked to the mammalian kinesin Kif21A. How cortical collapse factors influence axon growth is little understood. Here we studied them, focussing on the function of Drosophila Efa6 in experimentally and genetically amenable fly neurons. First, we show that Drosophila Efa6 can inhibit MTs directly without interacting molecules via an N-terminal 18 amino acid motif (MT elimination domain/MTED) that binds tubulin and inhibits microtubule growth in vitro and cells. If N-terminal MTED-containing fragments are in the cytoplasm they abolish entire microtubule networks of mouse fibroblasts and whole axons of fly neurons. Full-length Efa6 is membrane-attached, hence primarily blocks MTs in the periphery of fibroblasts, and explorative MTs that have left axonal bundles in neurons. Accordingly, loss of Efa6 causes an increase of explorative MTs: in growth cones, they enhance axon growth, in axon shafts, explorative MTs cause excessive branching, as well as atrophy through perturbations of MT bundles. Efa6 over-expression causes the opposite phenotypes. Taken together, our work conceptually links molecular and sub-cellular functions of cortical collapse factors to axon growth regulation and reveals new roles in axon branching and in the prevention of axonal atrophy. Furthermore, the MTED delivers a promising tool that can be used to inhibit MTs in a compartmentalised fashion when fusing it to specifically localising protein domains.\n\nSummary statementThe cortical collapse factor Efa6 inhibits microtubule polymerising outside axonal bundles. Thereby it limits axon growth and branching, but preserves microtubule bundle organisation crucial for axon maintenance.

cell biology↗

bioRxiv2018

Melatonin: The smart molecule that differentially modulates autophagy in tumor and normal placental cells

HighlightsO_LIH/R induces autophagy and Nrf2 in tumoral and primary trophoblast cells.\nC_LIO_LIMelatonin inhibits autophagy and Nrf2 under H/R, inducing BeWo cell death.\nC_LIO_LIMelatonin increases autophagy and Nrf2 under H/R conditions, promoting primary villous trophoblast cells survival.\nC_LI\n\nAbstractHypoxia/reoxygenation (H/R) induces oxidative damage and apoptosis. These consequences activate autophagy, which degrades damaged cellular content, as well as inducing the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor, and thereby the expression of protective genes. Melatonin has protective roles in normal cells and cytotoxic actions in cancer cells, with effects involving autophagy and Nrf2 pathways. The current study shows melatonin to differentially modulate autophagy and Nrf2 pathways in tumor and normal placental cells exposed to H/R. BeWo, a human placental choriocarcinoma cell line, and primary villous cytotrophoblasts isolated from normal term placenta, were maintained in normoxia (8% O2) for 24 h or exposed to hypoxia (0.5% of O2 for 4 h) followed by 20 h of normoxia, creating a H/R, in the presence or absence of 1 mM melatonin. Melatonin induced a 7-fold increase in the activation of 5 adenosine monophosphate-activated protein kinase (AMPK), an upstream modulator of autophagy, rising to a 16-fold increase in cells co-exposed to H/R and melatonin, compared to controls. H/R induced autophagosome formation via the increased expression of Beclin-1 (by 94 %) and ATG7 (by 97%). H/R also induced autophagic activity, indicated by the by the 630% increase in P62, and increased Nrf2 by 314%. In H/R conditions, melatonin reduced autophagy by 74% and Nrf2 expression by 66%, leading to BeWo cell apoptosis. In contrast, in human primary villous cytotrophoblasts, H/R induced autophagy and Nrf2, which melatonin further potentiated, thereby affording protection against H/R. This study demonstrates that melatonin differentially modulates autophagy and the Nrf2 pathway in normal vs. tumor trophoblast cells, being cytoprotective in normal cells whilst increasing apoptosis in tumoral trophoblast cells.

cell biology↗

bioRxiv2018

ELYS coordinates NF-κB pathway dynamics during development in Drosophila

Summary StatementELYS, a nucleoporin spatiotemporally regulates NF-{kappa}B pathway dynamics during development in Drosophila and its misregulation in post-embryonic stages leads to apoptosis mediated abnormalities.\n\nAbstractNuclear pores are the exclusive conduit to facilitate the nucleocytoplasmic transport in a precisely regulated manner. ELYS, a constituent protein of nuclear pores, initiates assembly of nuclear pore complexes (NPCs) into functional nuclear pores towards the end of mitosis. Using cellular, molecular and genetic tools, here, we report that ELYS orthologue (dElys) plays critical roles during Drosophila development. Through in silico analyses, we find all conserved structural features in dElys except for the presence of non-canonical AT-hook motif strongly binding with DNA. dElys localized to nuclear rim in interphase cells, but during mitosis, it was present on chromatin. RNAi mediated depletion of dElys leads to aberrant development and defects in the nuclear lamina and NPCs assembly at the cellular level. Furthermore, we demonstrate that in dElys depletion NF-{kappa}B is activated and accumulates inside the nucleus which results in illimed expression of critical molecules. dElys depletion sustains NF-{kappa}B into the nucleus in post-embryonic stages. Prolonged NF-{kappa}B inside nucleus induces apoptosis in response to hitherto unknown quality check mechanism and highlights on the under-appreciated apoptotic paradigm of NF-{kappa}B pathway.

cell biology↗

bioRxiv2018

The endosomal sorting adaptor HD-PTP is required for ephrin-B:EphB signalling in cell collapse and motor axon guidance

The signalling output of many transmembrane receptors that mediate cell-cell communication is restricted by the endosomal sorting complex required for transport (ESCRT), but the impact of this machinery on Eph tyrosine kinase receptor function is unknown. We identified the ESCRT-associated adaptor protein HD-PTP as part of an EphB2 BioID interactome, and confirmed this association using co-immunoprecipitation. Although HD-PTP loss does not change EphB2 expression, it attenuates the ephrin-B2:EphB2 signalling-induced collapse of cultured cells and axonal growth cones, and results in aberrant guidance of chick spinal motor neuron axons in vivo HD-PTP depletion abrogates ligand-induced EphB2 clustering, and EphB2 and Src family kinase activation. HD-PTP deficiency also accelerates ligand-induced EphB2 degradation, contrasting the phenotypes reported for other cell surface receptors. Our results link Eph signalling to the ESCRT machinery and demonstrate a role for HD-PTP in the earliest steps of ephrin-B:EphB signalling, as well as in obstructing premature receptor depletion.

cell biology↗

bioRxiv2018

TP promotes malignant progression in hepatocellular carcinoma through pentose Warburg effect

Tumor progression is dependent on metabolic reprogramming. Metastasis and vasculogenic mimicry (VM) are typical tumor progression. The relationship of metastasis, VM and metabolic reprogramming is not clear. In this study, we identified the novel role of Twist1, a VM regulator, in the transcriptional regulation of the expression of thymidine phosphorylase (TP). We demonstrated that TP promoted extracellular thymidine metabolization into ATP and amino acids through pentose Warburg effect by coupling the pentose phosphate pathway and glycolysis. Moreover, Twist1 relied on TP-induced metabolic reprogramming to promote hepatocellular carcinoma (HCC) metastasis and VM formation mediated by VE-Cad, VEGFR1, and VEGFR2 in vitro and in vivo. TP inhibitor tipiracil reduced promotion effect of TP enzyme activity on HCC VM formation and metastasis. Our findings demonstrate that TP, transcriptionally activated by Twist1, promotes HCC VM formation and metastasis through pentose Warburg effect, contributing to tumor progression.

cell biology↗

bioRxiv2018

Ena/VASP processive elongation is modulated by avidity on actin filaments bundled by the filopodia crosslinker fascin

Ena/VASP are tetrameric assembly factors that bind F-actin barbed ends continuously while increasing their elongation rate within dynamic bundled networks such as filopodia. We used single-molecule TIRFM and developed a kinetic model to dissect Ena/VASPs processive mechanism on bundled filaments. Notably, Ena/VASPs processive run length increases with the number of both bundled filaments and Ena arms, revealing avidity facilitates enhanced processivity. Moreover, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Finally, enhanced processivity on trailing barbed ends of bundled filaments is an evolutionarily conserved property of Ena/VASP homologs and is specific to fascin-bundled filaments. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, we discovered a novel regulatory mechanism whereby bundle size and bundling protein specificity control activities of a processive assembly factor.

cell biology↗

bioRxiv2018

HSP-4/BiP expression in secretory cells is regulated by a lineage-dependent differentiation program and not by the unfolded protein response

Differentiation of secretory cells leads to sharp increases in protein synthesis, challenging ER proteostasis. Anticipatory activation of the unfolded protein response (UPR) prepares cells for the onset of secretory function by expanding the ER size and folding capacity. How cells ensure that the repertoire of induced chaperones matches their post-differentiation folding needs is not well understood. We find that during differentiation of stem-like seam cells, a typical UPR target, the C. elegans BiP homologue HSP-4, is selectively induced in alae-secreting daughter cells, but is repressed in hypodermal daughter cells. Surprisingly, this lineage-dependent induction bypasses the requirement for UPR signaling, and instead is controlled by a specific developmental program. The repression of HSP-4 in hypodermal-fated cells requires a transcriptional regulator BLMP-1/BLIMP1, involved in differentiation of mammalian secretory cells. The HSP-4 induction is anticipatory, and is required for the integrity of secreted alae. Thus, differentiation programs can directly control a broad-specificity chaperone that is normally stress-dependent, to ensure the integrity of secreted proteins.

cell biology↗

bioRxiv2018

MAP7 family proteins are microtubule-tethered allosteric activators of kinesin-1.

Kinesin-1 is responsible for microtubule-based transport of numerous cellular cargoes. Here, we explored the regulation of kinesin-1 by MAP7 proteins. We found that all four mammalian MAP7 family members bind to kinesin-1. In HeLa cells, MAP7, MAP7D1 and MAP7D3 act redundantly to enable kinesin-1-dependent transport and microtubule recruitment of the truncated kinesin-1 KIF5B-560, which contains the stalk but not the cargo-binding and autoregulatory regions. In vitro, purified MAP7 and MAP7D3 increase microtubule landing rate and processivity of kinesin-1 through transient association with the motor. MAP7 proteins promote binding of kinesin-1 to microtubules both directly, through the N-terminal microtubule-binding domain and unstructured linker region, and indirectly, through an allosteric effect exerted by the kinesin-binding C-terminal domain. Compared to MAP7, MAP7D3 has a higher affinity for kinesin-1 and a lower affinity for microtubules and, unlike MAP7, can be co-transported with the motor. We propose that MAP7 proteins are microtubule-tethered kinesin-1 activators, with which the motor transiently interacts as it moves along microtubules.\n\nSummaryA combination of experiments in cells and in vitro reconstitution assays demonstrated that mammalian MAP7 family proteins act redundantly to activate kinesin-1 and promote its microtubule binding and processivity by transiently associating with the stalk region of the motor.

cell biology↗

bioRxiv2018

Focal cholesterol depletion during Fcγ receptor-mediated phagocytosis contributes to Lyn kinase regulation

Cholesterol-rich nanodomains, historically referred to as lipid rafts, have previously been reported to be critical for proper Fc{gamma} Receptor and Lyn kinase signaling during phagocytosis. Throughout the initial stages of phagocytosis, the nascent phagosome is actively remodeled by localized lipid metabolism and exocytosis. However, to date, little is known about the dynamics of cholesterol during this stage of particle engulfment. Using a genetically-encoded biosensor for cholesterol, we find that cholesterol is depleted from the nascent phagosome prior to sealing. Additionally, protein markers of both cholesterol-rich and cholesterol-poor nanodomains also clear from the site of phagocytosis arguing against the selective depletion of specific membrane domains. Consistent with previous studies we find that exocytosis contributes to the remodeling of the nascent phagosome. The displacement of cholesterol from the forming phagosome was paralleled by Lyn kinase helping to explain the reduction of phosphotyrosine signal in the nascent phagosome. This diminution of cholesterol and Lyn from the base of the cup may aid in the processivity of the phagocytic signal during pseudopod extension, and provide an unappreciated mechanism by which Lyn kinase signaling is regulated during phagocytosis.\n\nSummary Statement: Localized exocytosis dilutes cholesterol from the phagocytic cup leading to the displacement of Lyn kinase and an attenuation of signaling.

cell biology↗

bioRxiv2018

The single mitochondrion of the kinetoplastid parasite Crithidia fasciculata is a dynamic network

Mitochondria are central organelles in cellular metabolism. Their structure is highly dynamic, allowing them to adapt to different energy requirements, to be partitioned during cell division, and to maintain functionality. Mitochondrial dynamics, including membrane fusion and fission reactions, are well studied in yeast and mammals but it is not known if these processes are conserved throughout eukaryotic evolution. Kinetoplastid parasites are some of the earliest-diverging eukaryotes to retain a mitochondrion. Each cell has only a single mitochondrial organelle, making them an interesting model for the role of dynamics in controlling mitochondrial architecture. We have investigated the mitochondrial division cycle in the kinetoplastid Crithidia fasciculata. The majority of mitochondrial biogenesis occurs during the G1 phase of the cell cycle, and the mitochondrion is divided symmetrically in a process coincident with cytokinesis. Mitochondrial division was not inhibited by the putative dynamin inhibitor mdivi-1, although mitochondrial membrane potential and cell size were affected. Live cell imaging revealed that the mitochondrion is highly dynamic, with frequent changes in the topology of the branched network. These remodeling reactions include tubule fission, fusion, and sliding, as well as new tubule formation. We hypothesize that the function of this dynamic remodeling is to homogenize mitochondrial contents and to facilitate rapid transport of mitochondria-encoded gene products from the area containing the mitochondrial nucleoid to other parts of the organelle.

cell biology↗

bioRxiv2018

Very high-density platelets determine reactivity and activity of circulating platelets

BackgroundFor many decades, platelets have been known to display a substantial density heterogeneity. Knowledge about the origins and functions of specific platelet density subpopulations is scarce. This study investigates if very high-density (1.09 kg/L) platelets impact upon the reactivity and activity of all platelets.\n\nMethodsSubjects (n=52) were recruited in conjunction with a nurse-guided blood pressure control. Platelet reactivity in citrate anticoagulated whole blood, i.e. surface-bound P-selectin after provocation, was analysed using a flow cytometry technique. ADP (8.5 mol/L) was employed as an agonist. Subsequently, the entire population was separated according to density into 17 subpopulations, with fraction 1 containing the densest platelets (1.09 kg/L). In each subfraction surface P-selectin expression was determined. Subjects were then divided according to the number of very high-density platelets in 1.09high (n=17) and 1.09low (n=35) demonstrating >8x109/L and [≤]8x109/L 1.09 kg/L fraction 1 platelets, respectively.\n\nResultsSurface-attached P-selectin after provocation reveals that 1.09high associates with increased whole blood reactivity of the entire platelet population. The level of significance was p[≤]0.01 (8.5 mol/L ADP). Furthermore, 1.09high relates to increased spontaneous activity of density populations, as evidenced by membrane-bound P-selectin. For the fractions nos. 2, 4-7, 9, 10 the differences were significant, with p-values ranging from p[≤]0.05 to p[≤]0.01.\n\nConclusionThe number of very high-density (1.09 kg/L) platelets reflects the reactivity of the entire population. It is also closely related to subfraction P-selectin activity. It is unlikely that platelets gain density when circulating. Therefore, evidence suggests that very dense cells are created for this purpose at thrombopoiesis. It is tenable that such platelets regulate the reactivity of the entire population.

cell biology↗

bioRxiv2018

Adaptor protein Bbc1 regulates localization of Wsp1 and Vrp1 during endocytic actin patch assembly

Arp2/3 complex-nucleated branched actin networks provide the force necessary for endocytosis. The Arp2/3 complex is activated by Nucleation Promoting Factors (NPFs) including the Schizosaccharomyces pombe proteins WASp Wsp1 and myosin-1 Myo1. There are >40 known yeast endocytic proteins with distinct spatial and temporal localizations and functions; however, it is still unclear how these proteins work together to drive endocytosis. We used quantitative live cell imaging to determine the function of the uncharacterized S. pombe protein Bbc1. We discovered Myo1 interacts with and recruits Bbc1 to sites of endocytosis. Bbc1 competes with verprolin Vrp1 for Myo1 binding, thus releasing Vrp1 and its binding partner Wsp1 from Myo1. Normally Myo1 remains at the base of the endocytic invagination and Vrp1-Wsp1 internalize with the endocytic vesicle; however, in the absence of Bbc1, a portion of Vrp1-Wsp1 remains with Myo1 at the base of the invagination and endocytic invaginations are twice as long. We propose that Bbc1 disrupts a transient Myo1-Vrp1-Wsp1 interaction and limits Arp2/3 complex-nucleation of actin branches at the plasma membrane.

cell biology↗

bioRxiv2018

PCH-2TRIP13 regulates spindle checkpoint strength

Spindle checkpoint strength is dictated by three criteria: the number of unattached kinetochores, cell volume and cell fate. We show that the conserved AAA-ATPase, PCH-2/TRIP13, which remodels the checkpoint effector Mad2 from an active conformation to an inactive one, controls checkpoint strength in C. elegans. When we manipulate embryos to decrease cell volume, PCH-2 is no longer required for the spindle checkpoint or recruitment of Mad2 at unattached kinetochores. This role in checkpoint strength is not limited to large cells: the stronger checkpoint in germline precursor cells also depends on PCH-2. PCH-2 is enriched in germline precursor cells and this enrichment relies on conserved factors that induce asymmetry in the early embryo. Finally, the stronger checkpoint in germline precursor cells is regulated by CMT-1, the ortholog of p31comet, which is required for both PCH-2s localization to unattached kinetochores and its enrichment in germline precursor cells. Thus, PCH-2, likely by regulating the availability of inactive Mad2 at and near unattached kinetochores, governs checkpoint strength. This role may be specifically relevant in scenarios where maintaining genomic stability is particularly challenging, such as in oocytes and early embryos enlarged for developmental competence and germline cells that maintain immortality.

cell biology↗

bioRxiv2018

A specialized MreB-dependent complex mediates the formation of stalk-specific peptidoglycan in Caulobacter crescentus

Many bacteria have complex cell shapes, but the mechanisms producing their distinctive morphologies are still poorly understood. Caulobacter crescentus, for instance, exhibits a stalk-like extension that carries an adhesive holdfast mediating surface attachment. This structure forms through zonal peptidoglycan biosynthesis at the old cell pole and elongates extensively under phosphate-limiting conditions. We analyzed the composition of cell body and stalk peptidoglycan and identified significant differences in the nature and proportion of peptide crosslinks, indicating that the stalk represents a distinct subcellular domain with specific mechanical properties. To identify factors that participate in stalk formation, we systematically inactivated and localized predicted components of the cell wall biosynthetic machinery of C. crescentus. Our results show that the biosynthesis of stalk peptidoglycan involves a dedicated peptidoglycan biosynthetic complex that combines specific components of the divisome and elongasome, suggesting that the repurposing of pre-existing machinery provides a straightforward means to evolve new morphological traits.

cell biology↗