Molecular Biology of the Cell

Contractile vacuole complexes (CVCs) are complex osmoregulatory organelles, with vesicular (bladder) and tubular (spongiome) subcompartments. The mechanisms that underlie their formation and maintenance within the eukaryotic endomembrane network are poorly understood. In the Ciliate Tetrahymena thermophila, six differentiated CORVETs (class C core vacuole/endosome tethering complexes), with Vps8 subunits designated A-F, are likely to direct endosomal trafficking. Vps8Dp localizes to both bladder and spongiome. We show by inducible knockdown that VPS8D is essential to CVC organization and function. VPS8D knockdown increased susceptibility to osmotic shock, tolerated in the wildtype but triggering irreversible lethal swelling in the mutant. The knockdown rapidly triggered contraction of the spongiome and lengthened the period of the bladder contractile cycle. More prolonged knockdown resulted in disassembly of both the spongiome and bladder, and dispersal of proteins associated with those compartments. In stressed cells where the normally singular bladder is replaced by numerous vesicles bearing bladder markers, Vps8Dp concentrated conspicuously at long-lived inter-vesicle contact sites, consistent with tethering activity. Similarly, Vps8Dp in cell-free preparations accumulated at junctions formed after vacuoles came into close contact. Also consistent with roles for Vps8Dp in tethering and/or fusion were the emergence in knockdown cells of multiple vacuole-related structures, replacing the single bladder. SynopsisIn the Ciliate Tetrahymena thermophila, VPS8D, which encodes a subunit of a non-conventional CORVET complex, is an essential determinant of the contractile vacuole complex (CVC). VPS8D knockdown results in retraction and dispersal of the spongiome, and disappearance of the bladder, reinforcing the view that CVCs arise from endosomal trafficking. Intermediate knockdown phenotypes and Vps8Dp localization support a role in homotypic tethering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/566071v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@b652c6org.highwire.dtl.DTLVardef@1f456f7org.highwire.dtl.DTLVardef@79ab8dorg.highwire.dtl.DTLVardef@1edbf74_HPS_FORMAT_FIGEXP M_FIG C_FIG

Spatial and temporal tracking of fluorescent proteins in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae. SIGNIFICANCE STATEMENT- Quantitative endocytic assays are required to characterize factors that regulate both ligand-dependent and constitutive endocytosis. - We optimize fluorogen-activating proteins (FAPs) technology for use as a live cell imaging probe in yeast that fluoresces in the far-red range for quantitative endocytosis assays. - The FAP tools and approaches generated will facilitate quantitative endocytic and protein recycling assays for yeast cell biologists.

Arf-like GTPases (Arls) regulate membrane trafficking and cytoskeletal organization. Genetic studies predicted a role for Arl15 in type-2 diabetes, insulin resistance, adiposity, and rheumatoid arthritis. Recent studies indicate a possible role for Arl15 in multiple physiological processes, including magnesium homeostasis. However, the molecular function of Arl15 is poorly defined. We evaluated the role of Arl15 in vesicular transport using techniques to quantify cargo trafficking, to mechanobiology. Fluorescence microscopy of stably expressing Arl15-GFP HeLa cells showed its localization to the Golgi and cell surface, including filopodia, and a cohort to recycling endosomes. The dissociation of Golgi, using small molecular inhibitors or the expression of Arf1 dominant-negative mutant, completely mislocalized Arl15 to the cytosol. Interestingly, site-directed mutagenesis analysis identified a novel V80A mutation in the GTP-binding domain that turns Arl15 into a dominant-negative form with reduced number of filopodia. Depletion of Arl15 in HeLa cells caused mislocalization of cargo, such as caveolin-2 and STX6, from the Golgi. Arl15 knockdown cells displayed reduced filopodial number, altered focal adhesion kinase organization, and enhanced soluble and receptor-mediated cargo uptake without affecting the TfR recycling. Arl15 knockdown decreased cell migration and enhanced cell spreading and adhesion strength. Traction force microscopy experiments revealed that Arl15 depleted cells exert higher tractions and generate multiple focal adhesion points during the initial phase of cell adhesion as compared to control cells. Collectively, these studies demonstrated a functional role for Arl15 in the Golgi, which includes regulating cargo transport to organize membrane domains at the cell surface. Key pointsO_LIArl15 primarily localizes to Golgi and plasma membrane, including filopodia C_LIO_LIMembrane localization of Arl15 is dependent on Golgi integrity or Arf1 activation C_LIO_LIArl15 knockdown mislocalizes STX6-dependent Golgi localized cargo required for cell surface organization and reduces the filopodial number C_LIO_LIArl15 is involved in cell spreading, adhesion, and migration C_LI

Activator of G-protein Signaling 3 (AGS3), a receptor independent activator of G-protein signaling, oscillates among different subcellular compartments in a regulated manner including punctate entities referred to as biomolecular condensates (BMCs). The dynamics of the AGS3 oscillation and the specific subcompartment within the cell is intimately related to the functional diversity of the protein. To further address the properties and regulation of AGS3 BMCs, we asked initial questions regarding a) the distribution of AGS3 across the broader BMC landscape with and without cellular stress, and b) the core material properties of these punctate structures. Cellular stress (oxidative, pHi, thermal) induced the formation of AGS3 BMCs in two cell lines (Hela, COS7) as determined by fluorescent microscopy. The AGS3-BMCs generated in response to oxidative stress were distinct from stress granules (SG) as defined by the SG marker protein G3BP1 and RNA processing BMCs defined by the P-body protein Dcp1a. Immunoblots of fractionated cell lysates indicated that cellular stress shifted AGS3 to the membrane pellet fraction, whereas the protein markers for stress granules (G3BP1) SG- BMCs remained in the supernatant. We next asked if the formation of the stress-induced AGS3 BMCs was regulated by protein binding partners involved with signal processing. The stress-induced generation of AGS3 BMCs was regulated by the signaling protein Gi3, but not by the AGS3 binding partner DVL2. Finally, we addressed the fluidity or rigidity of the stress-induced AGS3-BMCs using fluorescent recovery following photobleaching of individual AGS3-BMCs. The AGS3-BMCs indicated distinct diffusion kinetics that were consistent with restricted mobility of AGS3 within the stress-induced AGS3-BMCs. These data suggest that AGS3 BMCs represents a distinct class of stress granules that define a new type of BMC that may serve as previously unappreciated signal processing nodes. Summary statementAGS3 assembles into distinct biomolecular condensates in response to cell stress and this assembly is selectively regulated by AGS3 binding partners involved in signal transduction within the cell.

Tumor cell lines with elevated chromosome numbers frequently exhibit elevated expression of Mps1. These tumors are also dependent on high Mps1 activity for their survival. Mps1 is a conserved kinase involved in controlling aspects of chromosome segregation in mitosis and meiosis. The mechanistic explanation for the Mps1-addiction of aneuploid cells is unknown. To address this question, we explored Mps1-dependence in yeast cells with increased sets of chromosomes. These experiments revealed that in yeast, increasing ploidy leads to delays and failures in orienting chromosomes on the mitotic spindle. Yeast cells with elevated numbers of chromosomes proved vulnerable to reductions of Mps1 activity. Cells with reduced Mps1 activity exhibit an extended prometaphase with longer spindles and delays in orienting the chromosomes. One known role of Mps1 is in recruiting Bub1 to the kinetochore in meiosis. We found that the Mps1-addiction of polyploid yeast cells is due in part to its role in Bub1 recruitment. Together, the experiments presented here demonstrate that increased ploidy renders cells more dependent on Mps1 for orienting chromosomes on the spindle. The phenomenon described here may be relevant in understanding why high-ploidy cancer cells exhibit elevated reliance on Mps1 expression for successful chromosome segregation. AUTHOR SUMMARYLosing or gaining chromosomes during cell division leads to aneuploidy (an abnormal number of chromosomes) and can contribute to cancer and other diseases. Indeed, most cells in solid tumors carry abnormally elevated numbers of chromosomes. Mps1 is a regulator of the machinery that distributes chromosomes to daughter cells. In tumors with elevated chromosome numbers, the expression of Mps1 is often also elevated. In some aneuploid tumor cell lines these elevated Mps1 levels have been shown to be critical for tumor survival. To determine how cells with higher ploidy become dependent on Mps1, we explored Mps1-dependence in yeast cells with increased numbers of chromosomes. We report that yeast cells with elevated chromosome number are sensitive to reductions Mps1 expression. In cells with high ploidy and reduced levels of Mps1, the progression of the cell cycle is delayed and the ability of the cells to properly orient and segregate their chromosomes on the spindle is greatly reduced.

The Golgi apparatus comprises a connected ribbon of stacked cisternal membranes localized to the perinuclear region of most vertebrate cells. The position and morphology of this organelle depends upon interactions with microtubules and the actin cytoskeleton. In contrast, we know relatively little about the relationship of the Golgi apparatus with intermediate filaments. In this study we show that the Golgi is in close physical proximity to vimentin intermediate filaments (IFs) in cultured mouse and human cells. We also show that the trans-Golgi network coiled-coil protein GORAB can physically associate with IFs. Although loss of vimentin and/or GORAB does not have major effects upon Golgi morphology at steady-state, the Golgi undergoes more rapid disassembly upon chemical disruption with the drug brefeldin A, and slower reassembly upon drug washout, in vimentin knockout cells. Moreover, loss of vimentin causes reduced Golgi ribbon integrity when cells are cultured on high stiffness hydrogels, which is exacerbated by loss of GORAB. These results indicate that vimentin IFs contribute to the structural stability of the Golgi apparatus, and suggest a role for GORAB in this process.

Proper attachment of spindle microtubules to kinetochores is necessary to satisfy the spindle assembly checkpoint and ensure faithful chromosome segregation. Microtubules detach from kinetochores to correct improperly oriented attachments, and overall kinetochore-microtubule (k-MT) attachment stability is determined in response to regulatory enzymes and the activities of kinetochore-associated microtubule stabilizing and destabilizing proteins. However, it is unknown whether regulatory enzyme activity or kinetochore-associated protein localization respond to subtle changes in k-MT attachment stability. To test for this feedback response, we monitored Aurora B kinase activity and the localization of select kinetochore proteins in metaphase cells following treatments that subtly stabilize or destabilize k-MT attachments using low dose Taxol or UMK57 (an MCAK agonist), respectively. Increasing k-MT stability induced changes in the abundance of some kinetochore proteins. In contrast, reducing k-MT stability induced both increases in Aurora B kinase signaling and changes in the abundance of some kinetochore proteins. Thus, kinetochores dynamically respond to changes in the stability of their attached microtubules. This feedback control contributes to tuning k-MT attachment stability required for efficient error correction to facilitate faithful chromosome segregation. Summary StatementLive cell imaging demonstrates that kinetochore signaling responds to feedback from attached microtubules to tune their stability to ensure faithful chromosome segregation during cell division.

Glucose is the preferred carbon source for most eukaryotes, and the first step in its metabolism is phosphorylation to glucose-6-phosphate. This reaction is catalyzed by a family of enzymes called either hexokinases or glucokinases depending on their substrate specificity. The yeast Saccharomyces cerevisiae encodes three such enzymes, Hxk1, Hxk2 and Glk1. In yeast and mammals, some isoforms of this enzyme are found in the nucleus, suggesting a possible moonlighting function beyond glucose phosphorylation. In contrast to mammalian hexokinases, the yeast Hxk2 enzyme has been proposed to shuttle into the nucleus in glucose replete conditions where it reportedly moonlights as part of a glucose-repressive transcriptional complex. To achieve this role in glucose repression, Hxk2 reportedly binds the Mig1 transcriptional repressor, is dephosphorylated at serine 15 in its N-terminus, and requires an N-terminal nuclear localization sequence (NLS). In this study, we use high-resolution, quantitative, fluorescent microscopy of live cells to determine the conditions, residues, and regulatory proteins required for Hxk2 nuclear localization. In direct contradiction to previous yeast studies, our quantitative imaging demonstrates that Hxk2 is largely excluded from the nucleus under glucose replete conditions but is retained in the nucleus under glucose limiting conditions. Our data show that the Hxk2 N-terminus does not contain an NLS but instead comprises sequences necessary for nuclear exclusion and multimerization regulation. Amino acid substitutions of the phosphorylated residue, serine 15, disrupt Hxk2 dimerization but have no effect on its glucose-regulated nuclear localization. Substitution of alanine at the nearby residue, lysine 13, affects both dimerization and maintenance of nuclear exclusion under glucose replete conditions. Modeling and simulation provide insight into the molecular mechanisms of this regulation. In marked contrast to earlier studies, we find that the transcriptional repressor Mig1 and the protein kinase Snf1 have little effect on Hxk2 localization. Instead, the protein kinase Tda1 is a key regulator of Hxk2 localization. Finally, RNAseq analyses of the yeast transcriptome further dispel the idea that Hxk2 moonlights as a transcriptional repressor, demonstrating that Hxk2 has a negligible role in transcriptional regulation in both glucose replete and limiting conditions. Taken together, our studies provide a paradigm shift for the conditions, residues, and regulators controlling Hxk2 dimerization and nuclear localization. Based on our data, the nuclear translocation of Hxk2 in yeast occurs in glucose starvation conditions, a finding that aligns well with the nuclear regulation of mammalian orthologs of this enzyme. Our findings lay the foundation for future studies of Hxk2 nuclear activity.

Cell invasion through basement membrane (BM) extracellular matrix barriers is important during organ development, immune cell trafficking, and cancer metastasis. Here we study an invasion event, anchor cell (AC) invasion, which occurs during Caenorhabditis elegans development. The actin protrusion of the invading AC mechanically displaces the BM, but it is not known how forces are balanced to prevent the growing actin protrusion from pushing itself backward when confronted with a load. Here we observe that the distal end of the actin protrusion in the invading AC abuts the nucleus and deforms it. Further we show that there is a correlation between invasion efficiency and nuclear deformation: under mutant conditions where invasion is reduced, nuclear deformation is diminished. However, nuclear deformation and invasion are unaffected by interfering with the molecular connections between the actin and microtubule cytoskeletons and the nuclear envelope. Together these data suggest that the AC actin protrusion braces against the nucleus to apply forces during invasion, but that nucleus-cytoskeleton molecular connections are not necessary for this to occur. SUMMARY STATEMENTActin-based membrane protrusions in invading cells apply force to basement membrane (BM) barriers to help break through them. In cell motility in 2D, the actin protrusion uses cell-substrate adhesions for leverage to push forward against obstacles in what is known as the molecular clutch. The situation is different in 3D invasion, where the adhesive substrate is being effaced by the invading cell. It is not clear, in this case, why the growing actin protrusion doesnt push itself backwards instead of extending forwards through the BM. The data presented here provide evidence that the distal end of the invasive actin protrusion is braced against the stiff, immobile nucleus, allowing growth of the proximal end to apply force on the BM.

One emerging paradigm of cellular organization of RNA and RNA binding proteins is the formation of membraneless organelles (MLOs). Examples of MLOs include several types of ribonucleoprotein granules that form via phase separation. A variety of intracellular pH changes and post-translational modifications, as well as extracellular stresses can stimulate the condensation of proteins into granules. For example, the assembly of stress granules induced by oxidative stress, osmotic stress, and heat stress has been well-characterized in a variety of somatic cell types. In the germ line, similar stress-induced condensation of proteins occurs; however, less is known about the role of phase separation during gamete production. Researchers who study phase transitions often make use of fluorescent reporters to study the dynamics of RNA binding proteins during live-cell imaging. In this report, we demonstrate that certain conditions of live-imaging C. elegans can cause an inadvertent stress and trigger phase transitions of RNA binding proteins. We show that imaging stress stimulates decondensation of multiple germ granule proteins, and condensation of several P-body proteins. Proteins within larger RNP granules in meiotically-arrested oocytes do not appear to be as sensitive to imaging stress as proteins in diakinesis oocytes of young hermaphrodites, with the exception of the germ granule protein PGL-1. Our results have important methodological implications for all researchers using live-cell imaging techniques. The data also suggest that the RNA binding proteins within large RNP granules of arrested oocytes may have distinct phases which we characterize in our companion paper.

Human eosinophils activated in suspension with IL5 or IL33 undergo morphological change prior to adhesion. Refractive granules, which contain major basic protein-1 and other toxic proteins, move to one side of the cell, the granulomere, and the two nuclear lobes move to the other. How these features persist when eosinophils become adherent and migrate is not known. We now compare behavior of activated eosinophils on surfaces coated with ITGAM/ITGB2-integrin ligands fibrinogen or periostin using live cell imaging of reporters of tubulin/actin organization and cell viability. We find that unlike eosinophils activated with IL5, IL33-activated eosinophils undergo two stages of activation; a preliminary pear-like activation in which the cell develops polarity, followed by a flattening of the eosinophil into a thin pancake-like morphology with less discrete polarization. IL5-treated eosinophils migrated persistently for more than an hour with nucleopod in the back. In contrast, IL33-treated eosinophils moved more slowly and within 30 min transitioned to a flattened morphology with nuclear lobes in the center and dispersed motile granules. Loss of cell viability after an hour, although variable, in all comparisons was greater among IL33-treated eosinophils on periostin. We sought to understand how cytoskeletal elements may drive these differences in morphology. Cytoskeletal elements had similar responses when activated with IL5/IL33; vimentin collapsed from a web-like network at the periphery of the cell and condensed adjacent to the nucleopod/nuclear interface, f-actin was found in the granulomere as well as the tip of the nucleopod and forward periphery, and microtubules radiated from the microtubule organizing center (MTOC) spanning both the nucleopod and the granulomere. The dynamic formation of microtubules correlated with cellular locomotion, suggesting mesenchymal migration within these cells. These in vitro findings suggest that adhesion plays an important role in determining functional morphology and demonstrates new insights into IL33-activated eosinophils. This work suggests roles for activators and adhesive substrates in regulating the behavior of activated eosinophils in tissues.

The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprised of a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying the structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, while the lipid scramblase Scr7p co-localizes with Vps8Dp. The H+- ATPase subunit Vma4 is spongiome-specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools vs. lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. While the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome are isolated from bulk endocytic input. Summary statementIn the ciliate Tetrahymena thermophila, four proteins are shown to provide markers for different zones of the contractile vacuole complex. They shed light on its formation and maintenance by enabling in vivo analysis of its dynamics.

Cellular mechanoadaptation is a complex process involving multiple mechanotransduction pathways and mechanisms that operate in different cellular locations and organelles. Despite recent advances, the identity of all components and the molecular mechanisms of these pathways remain poorly understood. Here, we describe a strategy to identify previously unrecognized mechanotransduction components throughout the cell. Using this approach, we identify several candidate proteins involved in mechanotransduction in cellular organelles. A screen of selected candidates identified DANGER as a nuclear envelope component required for nuclear mechanoadaptation and stability. DANGER is distributed in discrete regions of the nuclear envelope. Notably, DANGER is highly enriched in bent and stretched regions of the nuclear envelope, a feature not observed for other nuclear envelope proteins associated with mechanotransduction pathways. Upon increased nuclear tension, either induced by osmotic swelling or integrin-mediated nuclear deformation, DANGER responds by forming larger clusters, suggesting that DANGER can sense changes in the nuclear envelope induced by mechanical cues. Furthermore, DANGER-depleted nuclei have a larger area, are more elongated, and are more prone to forming blebs, consistent with DANGER localizing to regions under higher tension. Together, these findings identify DANGER as a key nuclear envelope component regulating nuclear shape and nuclear envelope stability, and provide proof of concept that our gene expression correlation-based strategy can identify previously unrecognized mechanotransduction components.

Myosins are required for clathrin-mediated endocytosis, but their precise molecular roles in this process are not known. This is, in part, because the biophysical properties of the relevant motors have not been investigated. Myosins have diverse mechanochemical activities, ranging from powerful contractility against mechanical loads to force-sensitive anchoring. To better understand the essential molecular contribution of myosin to endocytosis, we studied the in vitro force-dependent kinetics of the Saccharomyces cerevisiae endocytic type I myosin called Myo5, a motor whose role in clathrin-mediated endocytosis has been meticulously studied in vivo. We report that Myo5 is a low-duty-ratio motor that is activated [~]10-fold by phosphorylation, and that its working stroke and actin-detachment kinetics are relatively force-insensitive. Strikingly, the in vitro mechanochemistry of Myo5 is more like that of cardiac myosin than like that of slow anchoring myosin-1s found on endosomal membranes. We therefore propose that Myo5 generates power to augment actin assembly-based forces during endocytosis in cells. SummaryPedersen, Snoberger et al. measure the force-sensitivity of the yeast endocytic the myosin-1 called Myo5 and find that it is more likely to generate power than to serve as a force-sensitive anchor in cells. Implications for Myo5s role in clathrin-mediated endocytosis are discussed.

Yeast use a G-protein coupled receptor (GPCR) signaling pathway to detect mating pheromone, arrest in G1, and direct polarized growth towards the potential mating partner. The primary negative regulator of this pathway is the regulator of G-protein signaling (RGS), Sst2, which induces G GTPase activity and subsequent inactivation of all downstream signaling including a MAPK cascade. The MAPK Fus3 phosphorylates the RGS in response to pheromone, but the role of this modification is unknown. We set out to examine the role of RGS phosphorylation during the pheromone response. We found that RGS phosphorylation peaks early in the pheromone response and diminishes RGS localization to the polarity site and focuses MAPK complexes there. At later time points, RGS is predominantly unphosphorylated, which promotes RGS localization to the polar cap and broadens the distribution of MAPK complexes relative to the Cdc42 polarity machinery. Surprisingly, we found that phosphorylation of the RGS is required for the completion of cytokinesis prior to pheromone induced growth. The completion of cytokinesis in the presence of pheromone is promoted by the formin Bnr1 and the kelch-repeat protein, Kel1, both proteins previously found to interact with the RGS.

The nucleus vacuole junction NVJ in yeast is a multifunctional contact site between the nuclear ER membrane and the vacuole with diverse roles in lipid metabolism, transfer and storage. Adaptation of NVJ functions to metabolic cues is mediated by a striking remodeling of the size and the proteome of the contact site, but the extent and the molecular determinants of this plasticity are not fully understood. Using microscopy-based screens, we monitored NVJ remodeling in response to glucose availability. We identified Pex31, Nsg1, Nsg2, Shr5, and Tcb1 as NVJ residents. Glucose starvation typically results in an expansion of the NVJ size and proteome. Pex31 shows an atypical behavior, being specifically enriched at the NVJ at high glucose conditions. Loss of Pex31 uncouples NVJ remodeling from glucose availability, resulting in recruitment of glucose starvation-specific residents and NVJ expansion at glucose replete conditions. Moreover, PEX31 deletion results in alterations of sterol ester storage and a remodeling of vacuolar membranes that phenocopy glucose starvation responses. We conclude that Pex31 has a role in metabolic adaptation of the NVJ. SUMMARY STATEMENTUsing microscopy-based screens in yeast, we identified Pex31, Nsg1, Nsg2, Shr5 and Tcb1 as residents of the nucleus vacuole junction NVJ. Pex31 has a role in NVJ adaptation to glucose availability.

Apical constriction is a cell shape change that drives key morphogenetic events during development, including gastrulation and neural tube formation. The forces driving apical constriction are primarily generated through the contraction of apicolateral and/or medioapical actomyosin networks. In the Drosophila ventral furrow, the medioapical actomyosin network has a sarcomere-like architecture, with radially polarized actin filaments and centrally enriched non-muscle myosin II and myosin activating kinase. To determine if this is a broadly conserved actin architecture driving apical constriction, we examined actomyosin architecture during C. elegans gastrulation, in which two endodermal precursor cells internalize from the surface of the embryo. Quantification of protein localization showed that neither the non-muscle myosin II NMY-2 nor the myosin-activating kinase MRCK-1 is enriched at the center of the apex. Further, visualization of barbed- and pointed-end capping proteins revealed that actin filaments do not exhibit radial polarization at the apex. Taken together with observations made in other organisms, our results demonstrate that diverse actomyosin architectures are used in animal cells to accomplish apical constriction. SummaryThrough live-cell imaging of endogenously-tagged proteins, Zhang, Medwig-Kinney, and Goldstein show that the medioapical actomyosin network driving apical constriction during C. elegans gastrulation is organized diffusely, in contrast to the sarcomere-like architecture previously observed in the Drosophila ventral furrow.

Primary cilia are sensory cellular organelles crucial for organ development and homeostasis. Ciliogenesis in polarized epithelial cells requires Rab19-mediated clearing of apical cortical actin to allow the cilium to grow from the apically-docked basal body into the extracellular space. Loss of the lysosomal membrane-tethering HOPS complex disrupts this actin-clearing and ciliogenesis, but it remains unclear how ciliary function of HOPS relates to its canonical function in regulating late endosome-lysosome fusion. Here, we show that disruption of HOPS-dependent lysosomal fusion indirectly impairs actin-clearing and ciliogenesis by disrupting the targeting of Rab19 to the basal body. We also find that Rab19 functions in endolysosomal cargo trafficking apart from its previously-identified role in ciliogenesis. In summary, we show that inhibition of lysosomal fusion abnormally accumulates Rab19 on late endosomes, thus depleting Rab19 from the basal body and thereby disrupting Rab19-mediated actin-clearing and ciliogenesis. Summary statementLoss of HOPS-mediated lysosomal fusion indirectly blocks apical actin clearing and ciliogenesis in polarized epithelia by trapping Rab19 on late endosomes and depleting Rab19 from the basal body.

Tubulin post-translational modifications regulate microtubule dynamics; among these, -tubulin acetylation has been linked to microtubule stability. We generated a Chlamydomonas mutant lacking the acetyltransferase TAT1, which completely abolished -tubulin K40 acetylation. Surprisingly, the lengths of normally acetylated structures, axonemes and rootlets, were largely unaffected. TAT1 localized to the flagellar tip, suggesting that it is the primary site of acetylation. Loss of acetylation caused an increase in axonemal tubulin turnover, as revealed by dikaryon-fusion assays. Unexpectedly, the tat1 mutant displayed an increased number of dynamic cytoplasmic microtubules and could regenerate long flagella after amputation, even when protein synthesis was inhibited. Despite these cytoskeletal changes, steady-state flagellar length, cell growth, and cell division remained essentially normal. These findings suggest that acetylation modulates microtubule behavior by regulating axonemal tubulin turnover and cytoplasmic microtubule dynamics, while cellular morphology is buffered against variations in microtubule content.

In the cytoplasm, filamentous actin (F-actin) plays a critical role in cell regulation, including cell migration, stress fiber formation, and cytokinesis. Recent studies have shown that actin filaments that form in the nucleus are associated with diverse functions. Here, using live imaging of an F-actin-specific probe, superfolder GFP-tagged utrophin (UtrCH-sfGFP), we demonstrated the dynamics of nuclear actin in zebrafish (Danio rerio) embryos. In early zebrafish embryos up to around the high stage, UtrCH-sfGFP increasingly accumulated in nuclei during the interphase and reached a peak during the prophase. After nuclear envelope breakdown (NEBD), patches of UtrCH-sfGFP remained in the vicinity of condensing chromosomes during the prometaphase to metaphase. When zygotic transcription was inhibited by injecting -amanitin, the nuclear accumulation of UtrCH-sfGFP was still observed at the sphere and dome stages, suggesting that zygotic transcription may induce a decrease in nuclear F-actin. The accumulation of F-actin in nuclei may contribute to proper mitotic progression of large cells with rapid cell cycles in zebrafish early embryos, by assisting in NEBD, chromosome congression, and/or spindle assembly. Summary statementFilamentous actin accumulates in the nucleus of zebrafish early embryos and forms patches associating with condensing chromosomes during prophase.