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bioRxiv2018

Imaging beyond the super-resolution limits using ultrastructure expansion microscopy (UltraExM)

For decades, electron microscopy (EM) was the only method able to reveal the ultrastructure of cellular organelles and molecular complexes because of the diffraction limit of optical microscopy. In recent past, the emergence of superresolution fluorescence microscopy enabled the visualization of cellular structures with so far unmatched spatial resolution approaching virtually molecular dimensions. Despite these technological advances, currently super-resolution microscopy does not permit the same resolution level as provided by electron microscopy, impeding the attribution of a protein to an ultrastructural element. Here, we report a novel method of near-native expansion microscopy (UltraExM), enabling the visualization of preserved ultrastructures of macromolecular assemblies with subdiffraction-resolution by standard optical microscopy. UltraExM revealed for the first time the ultrastructural localization of tubulin glutamylation in centrioles. Combined with super-resolution microscopy, UltraExM unveiled the centriolar chirality, an ultrastructural signature, which was only visualizable by electron microscopy.

cell biology↗

bioRxiv2018

A cell separation checkpoint that enforces the proper order of late cytokinetic events

Eukaryotic cell division requires sequence dependency relationships in which late processes commence only after early ones are appropriately completed. We have discovered a system that blocks late events of cytokinesis until early ones are successfully accomplished. In budding yeast, cytokinetic actomyosin ring contraction and membrane ingression are coupled with deposition of an extracellular septum that is destroyed immediately after its completion by secreted enzymes. We find this secretion event is linked to septum completion and forestalled when the process is slowed. Delay of septum destruction requires Fir1, an intrinsically disordered protein localized to the cytokinesis site that is degraded upon septum completion but stabilized when septation is aberrant. Fir1 appears to protect cytokinesis in part by inhibiting a separation-specific exocytosis function of the NDR/LATS kinase Cbk1, a key component of a \"hippo\" signaling pathway that induces mother-daughter separation. We term this system \"enforcement of cytokinesis order\" (ECO), a checkpoint ensuring proper temporal sequence of mechanistically incompatible processes of cytokinesis.

cell biology↗

bioRxiv2018

Golgi stabilization, not its front-rear bias, is associated with EMT-enhanced fibrillar migration

Epithelial-to-mesenchymal transition (EMT) and maturation of collagen fibrils in the tumor microenvironment play a significant role in cancer cell invasion and metastasis. Confinement along fiber-like tracks enhances cell migration. To what extent and in what manner EMT further promotes migration in a microenvironment already conducive to migration is poorly understood. Here, we show that TGF{beta}-mediated EMT significantly enhances migration on fiber-like micropatterned tracks of collagen, doubling migration speed and quadrupling persistence relative to untreated mammary epithelial cells. Thus, cell-intrinsic EMT and extrinsic fibrillar tracks have non-redundant effects on motility. To better understand EMT-enhanced fibrillar migration, we investigated the regulation of Golgi positioning, which is involved in front-rear polarization and persistent cell migration. Confinement along fiber-like tracks has been reported to favor posterior Golgi positioning, whereas anterior positioning is observed during 2d wound healing. While EMT also regulates cell polarity, little is known about its effect on Golgi positioning. Here, we show that EMT induces a 2:1 rearward bias in Golgi positioning; however, positional bias explains less than 5% of single-cell variability in migration speed and persistence. Meanwhile, EMT significantly stabilizes Golgi positioning. Cells that enhance migration in response to TGF{beta} maintain Golgi position for 3-4 fold longer than untreated counterparts, irrespective of whether the Golgi is ahead or behind the nucleus. In fact, 35% of cells that respond to TGF{beta} exhibit a fully-committed Golgi phenotype with the organelle either in the anterior or posterior position for over 90% of the time. Furthermore, single-cell differences in Golgi stability capture up to 30% of variations in migration speed and persistence. These results lead us to propose that the Golgi is part of a core physical scaffold that distributes cell-generated forces necessary for migration. A stable scaffold more consistently, and therefore more productively, distributes forces over time, leading to efficient migration.

cell biology↗

bioRxiv2018

KCH kinesin drives nuclear transport and cytoskeletal coalescence for tip cell growth

Long-distance transport along microtubules (MTs) is critical for intracellular organisation. In animals, antagonistic motor proteins kinesin (plus end-directed) and dynein (minus end-directed) drive cargo transport. In land plants, however, the identity of motors responsible for transport is poorly understood, as genes encoding cytoplasmic dynein are missing. How other functions of dynein are brought about in plants also remains unknown. Here, we show that a subclass of the kinesin-14 family, KCH--which can also bind actin--drives MT minus end-directed nuclear transport in the moss Physcomitrella patens. When all four KCH genes were deleted, the nucleus was not maintained in the cell centre but was translocated to the apical end of protonemal cells. In the knockout (KO) line, apical cell tip growth was also severely suppressed. KCH was localised on MTs, including at the MT focal point near the tip where MT plus ends coalesced with actin filaments. MT focus was not persistent in KCH KO lines, whereas actin destabilisation also disrupted the focus despite KCH remaining on unfocused MTs. Functions of nuclear transport and tip growth were distinct, as a truncated KCH construct restored nuclear transport activity but not tip growth retardation of the KO line. Thus, our study identified KCH as a long-distance retrograde transporter as well as a cytoskeletal crosslinker, reminiscent of the versatile animal dynein.

cell biology↗

bioRxiv2018

The lipid droplet protein Pgc1 controls the subcellular distribution of phosphatidylglycerol

The biosynthesis of yeast phosphatidylglycerol (PG) takes place in the inner mitochondrial membrane. Outside mitochondria, the abundance of PG is low. Here we present evidence that the subcellular distribution of PG is maintained by locally controlled enzymatic activity of the PG-specific phospholipase, Pgc1. We document that the Pgc1 absence leads to spreading of PG over various cellular membranes. Fluorescently labeled Pgc1 protein strongly accumulates at the surface of lipid droplets (LD). We show, however, that LD are not only dispensable for Pgc1-mediated PG degradation, but even host no phospholipase activity of Pgc1. Our in vitro assays document the capability of LD-accumulated Pgc1 to degrade PG upon entry to membranes of the endoplasmic reticulum, mitochondria, and even of artificial phospholipid vesicles. FRAP analysis confirms continuous exchange of GFP-Pgc1 within individual LD in situ, suggesting that a steady-state equilibrium exists between LD and membranes to regulate immediate phospholipase activity of Pgc1. In this model, LD serve as storage place and shelter Pgc1 preventing untimely degradation, while both phospholipase activity and degradation of the enzyme occur in membranes.

cell biology↗

bioRxiv2018

Functional recruitment of dynamin requires multimeric interactions for efficient endocytosis

During clathrin mediated endocytosis (CME), membrane scission is achieved by the concerted action of dynamin and its interacting partners. Essential interactions occur between the proline/arginine-rich domain of dynamin (dynPRD) and the Src-homology domain 3 (SH3) of various proteins including amphiphysins. Here we show that multiple SH3 domains must bind simultaneously to dynPRD through three adjacent motifs for dynamins efficient recruitment and function. First, we show in dynamin triple knock-out cells that mutant dynamins modified in a single motif, including the central amphiphysin SH3 (amphSH3) binding motif, are partially capable of rescuing CME. However, mutating two motifs largely prevents that ability. To support this observation, we designed divalent dynPRD-derived peptides. These ligands bind multimers of amphSH3 with >100-fold higher affinity than monovalent ones in vitro. Accordingly, dialyzing living cells with these divalent peptides through a patch-clamp pipette blocks CME 2 to 3 times more effectively than with monovalent ones. Finally, the frequency of endocytic events decreases with competing peptides or hypomorphic rescue mutants but the kinetics of dynamin recruitment is unaffected. This suggests that PRD-SH3 interactions act upstream of dynamin accumulation at the neck of nascent vesicles. We conclude from these data that dynamin drives vesicle scission via multivalent interactions in vivo.

cell biology↗

bioRxiv2018

Ataxin-7 and non-stop coordinate SCAR protein levels, subcellular localization, and actin cytoskeleton organization

Ataxin-7 (Atxn7), a subunit of the SAGA chromatin remodeling complex, is subject to polyglutamine expansion at the amino terminus, causing spinocerebellar ataxia type 7 (SCA7), a progressive retinal and neurodegenerative disease. Within SAGA, the amino terminus of Atxn7 anchors the Non-stop deubiquitinase to the complex. To understand the consequences of Atxn7-dependent regulation of Non-stop, we sought substrates for Non-stop and discovered the deubiquitinase, dissociated from SAGA, interacts with Arp2/3 and WAVE regulatory complexes (WRC). Protein levels of WRC subunit suppressor of extracellular cAMP receptor (cAR) (SCAR) are regulated by a constant ubiquitination/proteasomal degradation mechanism. Loss of Atxn7 frees Non-stop from SAGA, leading to increased Non-stop interaction with SCAR and also increased SCAR protein levels. A Non-stop enzymatic pocket mutation that increases binding to ubiquitin increased interaction with SCAR, while an enzymatic pocket mutation reducing binding to ubiquitin also reduced binding to SCAR. Loss of Non-stop increased polyubiquitination of SCAR and reduced SCAR protein levels although SCAR protein levels were rescued by protease inhibition. Dependent on conserved WRC interacting receptor sequences (WIRS), Non-stop overexpression increased SCAR protein levels and directed subcellular localization of SCAR, leading to decreased cell area and decreased number of protrusions. In vivo, heterozygous mutation of Atxn7 rescued haploinsufficiency of SCAR to produce F actin, but heterozygous mutation of SCAR did not significantly rescue retinal axon mistargeting upon knockdown of Atxn7.\n\nSummarySAGA subunits Ataxin-7 and Non-stop regulate stability and subcellular localization of WRC subunit SCAR. Loss of Ataxin-7 increases, while loss of Non-stop decreases, SCAR protein levels and F-actin network assembly.

cell biology↗

bioRxiv2018

PP2C phosphatases promote autophagy by dephosphorylation of the Atg1 complex

Autophagy is orchestrated by the Atg1-Atg13 complex in budding yeast. Under nutrient-rich conditions, Atg13 is maintained in a hyperphosphorylated state by TORC1 kinase. After nutrient starvation, Atg13 is dephosphorylated, triggering Atg1 kinase activity and autophagy induction. The phosphatases that dephosphorylate Atg13 remain uncharacterized. We show that two redundant PP2C phosphatases, Ptc2 and Ptc3, regulate autophagy via dephosphorylating both Atg13 and Atg1. In the absence of these phosphatases, starvation-induced macroautophagy is inhibited, as is the cytoplasm-to-vacuole targeting (Cvt) pathway, and the recruitment of the essential autophagy machinery to phagophore assembly sites (PAS) is impaired. Despite prolongation of the DNA damage-induced checkpoint in ptc2{Delta} ptc3{Delta} cells, genotoxin-induced autophagy is also blocked. Creating a genomic atg13-8SA allele under its endogenous promoter, lacking key TORC1 phosphorylation sites, bypasses the autophagy defect in ptc2{Delta} ptc3{Delta} strains. Taken together, these results imply that PP2C type phosphatases promote autophagy by regulating Atg1 complex.

cell biology↗

bioRxiv2018

Claudin-4 Reconstituted in Unilamellar Vesicles is Sufficient to Form Tight Interfaces that Partition Membrane Proteins

Tight junctions have been hypothesized to act as molecular fences in the plasma membrane of epithelial cells, helping to form differentiated apical and basolateral domains. While this fence function is believed to arise from the interaction of four-pass transmembrane claudins, the complexity of tight junctions has made direct evidence of their role as a putative diffusion barrier difficult to obtain. Here we address this challenge by reconstituting claudin-4 into giant unilamellar vesicles using microfluidic jetting. We find that reconstituted claudin-4 is sufficient to form adhesive interfaces between unilamellar vesicles without accessory proteins present in vivo. By controlling the molecular composition of the inner and outer leaflets of jetted membranes, we show that claudin-4-mediated interfaces can drive partitioning of extracellular membrane proteins but not of inner or outer leaflet lipids. Our findings indicate that homotypic interactions of claudins and their small size can contribute to the polarization of epithelial cells.

cell biology↗

bioRxiv2018

An RNAi screen in human cell lines reveals conserved DNA damage repair pathways that mitigate formaldehyde sensitivity

Formaldehyde is a ubiquitous DNA damaging agent, with human exposures occuring from both exogenous and endogenous sources. Formaldehyde can also form DNA-protein crosslinks and is representative of other such DNA damaging agents including ionizing radiation, metals, aldehydes, chemotherapeutics, and cigarette smoke. In order to identify genetic determinants of cell proliferation in response to continuous formaldehyde exposure, we quantified cell proliferation after siRNA-depletion of a comprehensive array of over 300 genes representing all of the major DNA damage response pathways. Three unrelated human cell lines (SW480, U-2 OS and GM00639) were used to identify common or cell line-specific mechanisms. Four cellular pathways were determined to mitigate formaldehyde toxicity in all three cell lines: homologous recombination, double-strand break repair, ionizing radiation response, and DNA replication. Differences between cell lines were further investigated by using exome sequencing and Cancer Cell Line Encyclopedia genomic data. Our results reveal major genetic determinants of formaldehyde toxicity in human cells and provide evidence for the conservation of these formaldehyde responses between human and budding yeast.

cell biology↗

bioRxiv2018

Mechanistic Origins of Dynamic Instability in Filaments from the Phage Tubulin, PhuZ

A bacteriophage-encoded tubulin homologue, PhuZ, harnesses dynamic instability to position genomes of KZ-like bacteriophage at the midline of their Pseudomonas hosts, facilitating phage infectivity. While much has been learned about molecular origins of microtubule dynamics, how GTP binding and hydrolysis control dynamics in the divergent 3-stranded PhuZ filaments is not understood. Here we present cryo-EM reconstructions of the PhuZ filamentin a pre-hydrolysis (3.5[A]) and three post-hydrolysis states (4.2 [A], 7.3 [A] and 8.1 [A] resolutions), likely representing distinct depolymerization stages. Core polymerization-induced structural changes reveal similarities to {beta}-tubulin, suggesting broad conservation within the tubulin family. By contrast, GTP hydrolysis is sensed quite differently and is communicated by the divergent PhuZ C-terminus to the lateral interface, leading to PhuZ polymer destabilization. This provides a contrasting molecular description of how nucleotide state can be harnessed by the tubulin fold to regulate filament assembly, metastability and disassembly.

cell biology↗

bioRxiv2018

Spatiotemporally controlled Myosin relocalization and internal pressure cause biased cortical extension to generate sibling cell size asymmetry

Metazoan cells can generate unequal sized sibling cells during cell division. This form of asymmetric cell division depends on spindle geometry and Myosin distribution but the underlying mechanics are unclear. Here, we use atomic force microscopy and live cell imaging to elucidate the biophysical forces involved in the establishment of physical asymmetry in Drosophila neural stem cells. We show that the force driving initial apical membrane expansion is provided by hydrostatic pressure, peaking shortly after anaphase onset, and enabled by a relieve of actomyosin contractile tension on the apical cell cortex. The subsequent increase in contractile forces at the cleavage furrow, combined with the relocalization of basally located Myosin results in basal membrane extension and sustained apical expansion. We propose that spatiotemporally controlled actomyosin contractile tension and hydrostatic pressure enables stereotypic biased membrane expansion to generate sibling cell size asymmetry.

cell biology↗

bioRxiv2018

A non-canonical lysosome biogenesis pathway generates Golgi-associated lysosomes during epidermal differentiation

Keratinocytes maintain epidermis integrity and function including physical and antimicrobial barrier through cellular differentiation. This process is predicted to be controlled by calcium ion gradient and nutritional stress. Keratinocytes undergo proteome changes during differentiation, which enhances the intracellular organelle digestion to sustain the stress conditions. However, the molecular mechanism between epidermal differentiation and organelle homeostasis is poorly understood. Here, we used primary neonatal human epidermal keratinocytes to study the link between cellular differentiation, signaling pathways and organelle turnover. Upon addition of calcium chloride (2 mM) to the culture medium, keratinocytes increased their cell size and the expression of differentiation markers. Moreover, differentiated keratinocytes showed enhanced lysosome biogenesis that was dependent on ATF6-arm of UPR signaling but independent of mTOR-MiT/TFE transcription factors. Furthermore, chemical inhibition of mTOR has increased keratinocyte differentiation and relocalized the MiT/TFE TFs to the lysosome membranes, indicating that autophagy activation promotes the epidermal differentiation. Interestingly, differentiation of keratinocytes resulted in dispersal of fragmented Golgi and lysosomes, and the later organelles showed colocalization with Golgi-tethering proteins, suggesting that these lysosomes possibly originated from Golgi, hence named as Golgi-associated lysosomes (GALs). Consistent to this prediction, inhibition of Golgi function using brefeldin A completely abolished the formation of GALs and the keratinocyte differentiation. Thus, ER stress regulates the biogenesis of GALs, which maintains keratinocyte differentiation and epidermal homeostasis.

cell biology↗

bioRxiv2018

PLK4 is a microtubule-associated protein that self assembles promoting de novo MTOC formation

Summary statementPLK4 binds to microtubules and self assembles into supramolecular assemblies that recruit tubulin and trigger de novo MTOC formation in Xenopus laevis extracts.\n\nAbstractThe centrosome is an important microtubule-organizing center (MTOCs) in animal cells and it consists of two barrel-shaped structures (centrioles), surrounded by the pericentriolar material (PCM), which nucleates microtubules. PCM components form condensates, supramolecular assemblies that concentrate microtubule nucleators. Centrosomes can form close to an existing structure (canonical duplication) or de novo. How centrosomes form de novo is not known. PLK4 is a master driver of centrosome biogenesis, which is critical to recruit several centriole components. Here, we investigate the beginning of centrosome biogenesis, taking advantage of Xenopus egg extracts, where we and others have shown that PLK4 can induce de novo MTOC formation (Eckerdt et al., 2011; Zitouni et al., 2016). Surprisingly, we observe that in vitro, PLK4 can self-assemble into supramolecular assemblies that recruit /{beta}-tubulin. In Xenopus extracts, PLK4 supramolecular assemblies additionally recruit the PLK4 substrate STIL and the microtubule nucleator, {gamma}-tubulin, and form acentriolar MTOCs de novo. The assembly of these robust microtubule asters is independent of dynein, similarly to centrosomes. We suggest a new mechanism of action for PLK4, where it forms a self-organizing catalytic scaffold that recruits centriole components, PCM factors and /{beta}-tubulin, leading to MTOC formation.

cell biology↗

bioRxiv2018

Simu-dependent clearance of dying cells regulates macrophage function and inflammation resolution

Macrophages encounter and clear apoptotic cells during normal development and homeostasis, including at numerous sites of pathology. Clearance of apoptotic cells has been intensively studied, but the effects of macrophage-apoptotic cell interactions on macrophage behaviour are poorly understood. Using Drosophila embryos, we have exploited the ease of manipulating cell death and apoptotic cell clearance in this model to identify that the loss of the apoptotic cell clearance receptor Simu leads to perturbation of macrophage migration and inflammatory responses via pathological levels of apoptotic cells. Removal of apoptosis ameliorates these phenotypes, while acute induction of apoptosis phenocopies these defects and reveals that phagocytosis of apoptotic cells is not necessary for their anti-inflammatory action. Furthermore, Simu is necessary for clearance of necrotic debris and rentention of macrophages at wounds. Thus, Simu is a general detector of damaged self and represents a novel molecular player in controlling resolution of inflammation.

cell biology↗

bioRxiv2018

Amino acids stimulate the endosome-to-Golgi trafficking through Ragulator and small GTPase Arl5

The endosome-to-Golgi or endocytic retrograde trafficking pathway is an important post-Golgi recycling route. We made a novel discovery that the retrograde trafficking of cargos is inhibited and stimulated by the absence and presence, respectively, of amino acids (AAs), especially glutamine. By testing components of the AA-stimulated mTORC1 signaling pathway, we demonstrated that SLC38A9, v-ATPase and Ragulator, but not Rag GTPases and mTORC1, are essential for the AA-stimulated trafficking. Arl5, an ARF-like family small GTPase, interacts with Ragulator in an AA-regulated manner and both Arl5 and its effector, the Golgi-associated retrograde protein complex (GARP), are required for the AA-stimulated trafficking. We have therefore identified a mechanistic connection between the nutrient signaling and the retrograde trafficking pathway, whereby SLC38A9 and v-ATPase sense AA-sufficiency and Ragulator functions as a guanine nucleotide exchange factor to activate Arl5, which, together with GARP, a tethering factor, probably facilitates the endosome-to-Golgi trafficking.

cell biology↗

bioRxiv2018

Synthetic-evolution reveals that phosphoregulation of the mitotic kinesin-5 Cin8 is constrained

Cdk1 has been found to phosphorylate the majority of its substrates in disordered regions. These phosphorylation sites do not appear to require precise positioning for their function. The mitotic kinesin-5 Cin8 was shown to be phosphoregulated at three Cdk1 sites in disordered loops within its catalytic motor domain. Here, we examined the flexibility of Cin8 phosphoregulation by analyzing the phenotypes of synthetic Cdk1-sites that were systematically generated by single amino-acid substitutions, starting from a phosphodeficient variant of Cin8. Out of 29 synthetic Cdk1 sites that we created, eight were non-functional; 19 were neutral, similar to the phosphodeficient variant; and two gave rise to phosphorylation-dependent spindle phenotypes. Of these two, one site resulted in novel phosphoregulation, and only one site, immediately adjacent to a native Cdk1 site, produced phosphoregulation similar to wild-type. This study shows that, while the gain of a single phosphorylation site can confer regulation and modulate the dynamics of the spindle, to achieve optimal regulation of a mitotic kinesin-5 motor protein, phosphoregulation has to be site-specific and precise.

cell biology↗

bioRxiv2018

Misoprostol regulates Bnip3 repression and alternative splicing to control cellular calcium homeostasis during hypoxic stress.

The cellular response to hypoxia involves the activation of a conserved pathway for gene expression regulated by the transcription factor complex called hypoxia-inducible factor (HIF). This pathway has been implicated in both the adaptive response to hypoxia and in several hypoxic-ischemic related pathologies. Perinatal hypoxic injury, often associated with prematurity, leads to multi-organ dysfunction resulting in significant morbidity and mortality. Using a rodent model of neonatal hypoxia and several representative cell lines, we observed HIF1 activation and down-stream induction of the cell death gene Bnip3 in brain, large intestine, and heart which was mitigated by administration of the prostaglandin E1 analog misoprostol. Mechanistically, we determined that misoprostol inhibits full-length Bnip3 (Bnip3-FL) expression through PKA-mediated NF-{kappa}B (P65) nuclear retention, and the induction of pro-survival splice variants. We observed that the dominant small pro-survival variant of Bnip3 in mouse cells lacks the third exon (Bnip3{Delta}Exon3), whereas human cells produce a pro-survival BNIP3 variant lacking exon 2 (BNIP3{Delta}Exon2). In addition, these small Bnip3 splice variants prevent mitochondrial dysfunction, permeability transition, and necrosis triggered by Bnip3-FL by blocking calcium transfer from the sarco/endoplasmic reticulum to the mitochondria. Furthermore, misoprostol and Bnip3{Delta}Exon3 promote nuclear calcium accumulation, resulting in HDAC5 nuclear export, NFAT activation, and adaptive changes in cell morphology and gene expression. Collectively, our data suggests that misoprostol can mitigate the potential damaging effects of hypoxia on multiple cell types by activating adaptive cell survival pathways through Bnip3 repression and alternative splicing.

cell biology↗