Molecular Biology of the Cell

The spindle midzone, an array of overlapping, antiparallel microtubules, contributes to chromosome segregation and cytokinesis. As cells exit mitosis, midzone microtubules reorganize to form the midbody, the location of cell abscission. The mechanisms governing microtubule dynamics during this transition remain incompletely understood. The microtubule depolymerase, Kif2a, has been shown to contribute to midzone microtubule length control (Uehara et al., 2013), but how the depolymerase is regulated is not understood. Since CAMSAPs govern minus-end microtubule dynamics, we examined their role in midzone microtubule behavior. CAMSAP2, the major CAMSAP in HeLa cells, localized to the minus-ends of midzone microtubules and cells depleted of CAMSAP2, showed similar phenotypes as cells depleted of Kif2a, including elongated and bent midzones and enlarged asters. Next, we localized Kif2a in CAMSAP2-depleted cells and vice versa. CAMSAP2 remained present and extended along elongated midzone microtubules in Kif2a-depleted cells. In contrast Kif2a localization was no longer present at microtubule minus-ends but retained at plus-ends in CAMSAP2-depleted cells. In long-term live-cell movies of CAMSAP2-depleted cells abscission at the midbody was not detected, although two daughter cells formed. Markers for abscission including ESCRT-III component CHMP2A and Spastin were mislocalized, and midzone overlap zones, marked by PRC1, were extended. Together, our results demonstrate that CAMSAP2 is essential for midzone microtubule organization and dynamics, ultimately impacting cell abscission.

Cell migration depends on coordinating cell shape changes with force generation, yet how these processes are integrated remains unclear. Here, we combine live-cell imaging with traction force microscopy and computational analysis to quantify cell morphology, motility and force generation in migrating fibroblasts. We find that traction force magnitudes display a multimodal distribution, suggesting discrete migratory regimes. Using a Hidden Markov Model, we identify distinct force states that exhibit differences in shape and motion metrics, and show that individual cells transition between force states over time. To test the role of cytoskeletal organization in establishing the identified states, we analyzed cells lacking Arpc2, which disrupts branched actin assembly. Despite reduced forces and altered morphology, these cells also exhibit three migratory states. State transitions occur more frequently in cells lacking Arpc2 and unlike normal cells their protrusion geometry is force dependent. Together, our findings show that cell migration is organized into discrete mechanical states that couple morphology, motility and force generation. SUMMARY STATEMENTFibroblast motility involves distinct migratory states. These states exist independent of branched actin. However, state transition frequencies, traction force magnitudes and protrusion geometry are branched actin dependent.

Signaling pathways often share components yet produce highly specialized biological responses. How signaling specificity is achieved between pathways utilizing common components is a fundamental question. In budding yeast, the same transmembrane mucin, Msb2, regulates two Mitogen-Activated Protein Kinase (MAPK) pathways controlling filamentous growth (fMAPK) and the response to osmotic stress (HOG). How this shared sensor distinguishes between stimuli and regulates different pathways is not clear. Using structure-guided analysis, we identified a conserved SEA (Sea urchin sperm protein, Enterokinase, Agrin) domain in fungal mucins and found that mutations disrupting protein folding selectively impair one pathway (fMAPK) but were tolerated by another (HOG). Mechanistically, these differences revealed distinct modes of signal transmission. The fMAPK pathway required an intact SEA domain and the cytosolic tail, consistent with a cis signaling mechanism that required structural coupling across the membrane. In contrast, the HOG pathway functioned independently of the cytosolic tail and tolerated misfolded SEA domain variants, consistent with trans signaling mediated by extracellular domains of interacting partners. The HOG pathway may detect misfolding as part of its sensing mechanism, as stressors that induce protein misfolding required Msb2 for survival. This work reveals how differential tolerance to protein deformation confers signaling specificity and identifies sensor deformation as a general feature of mechanosensory pathways that respond to environmental stress. HIGHLIGHTSO_LISignaling pathways differ in tolerance to misfolding of a sensory domain C_LIO_LIMisfolded SEA domains retain function in a stress pathway (HOG) pathway but not a cell differentiation pathway (fMAPK) O_LIMisfolded SEA domain variants showed altered protein levels, mis-localization in the secretory pathway, and turnover by ERAD C_LIO_LINon-functional variants lacked residues that stabilize the structure through intramolecular bonds C_LI C_LIO_LIDifferential tolerance for misfolding revealed distinct modes of signaling O_LITrans signaling predominated in the HOG pathway and did not require proper SEA domain folding or the mucin cytosolic tail O_LIA dominant hyperactive variant next to the SEA domain revealed basal interactions with the CR domain of tetraspanin C_LIO_LIAlphaFold modeling showed distinct interactions occur between the SEA domain and tetraspanin in the basal and activated states C_LI C_LIO_LICis signaling predominated in the fMAPK pathway O_LIRequired a properly folded SEA domain and conformational coupling to the cytosolic tail C_LIO_LIYapsin processing was required for SEA domain activation and turnover of the mucin cytosolic tail C_LI C_LI C_LIO_LIHOG pathway may sense protein misfolding as part of its activation mechanism. C_LIO_LISEA domains are conserved throughout fungal mucins and mammalian glycoprotein sensors suggesting a generalizable mechanism C_LIO_LIProtein deformation may provide information to survival pathways about environmental stress. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/723240v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1cd30f3org.highwire.dtl.DTLVardef@48c96corg.highwire.dtl.DTLVardef@9fffc2org.highwire.dtl.DTLVardef@504b1d_HPS_FORMAT_FIGEXP M_FIG C_FIG Signaling pathways often share components yet activate different effector processes through mechanisms that remain unclear. The same mucin regulates two MAPK pathways (red and green), and the discovery of a conserved SEA domain provided insights into specificity mechanisms. In the fMAPK pathway that regulates filamentous growth, the mucin works in a classical manner, where an external signal (in this case underglycosylation by glucose limitation) transduces a signal to the cytosolic domain in cis. By comparison, the HOG pathway that responds to osmotic stress displayed a remarkable tolerance for mucin and SEA domain deformation. Protein variants that caused SEA domain misfolding, mislocalization, and degradation by ERAD retained function in the HOG pathway. Truncations that removed the cytosolic tail and transmembrane anchor were also functional. These phenotypes support a trans activation mechanism with external partners that was preferential for activation of the HOG pathway. SEA domain deformation may be induced by environmental stress as a trigger for the HOG pathway. Cells may detect misfolding of protein domains to gain information about environmental stress.

Physical interactions among cells and their processes are critical for intercellular communication and the generation of ordered tissue patterns. Primary cilia projecting from the cell surface have recently been shown to form contacts with the processes of diverse cell types, as well as with other cilia, in the brain and other organs. Whether these ciliary contacts are established in an instructive manner or are formed passively due to physical proximity is unclear. Ultrastructural analyses previously showed that the cilia of a subset of sensory neurons in the head amphid organs of C. elegans exhibit interciliary contacts within a glia-defined channel. Here we show that these ciliary contact patterns are stereotyped and can be re-established in the adult in the absence of neighboring cilia, indicating that these associations may not simply reflect relative positioning within the amphid channel. We show that mutations in a subset of genes implicated in ciliary protein trafficking, ciliary membrane phospholipid composition, and cilia-cell interactions disrupt both cilia structure and interciliary contacts. However, in a subset of mutants, cilia with altered morphologies can nevertheless establish correct contacts, implying that these contacts may be established via a regulated process. Together, our findings suggest that cilia-cilia interactions within a sense organ are established via defined mechanisms and raise the possibility that cilia-mediated intercellular communication may modulate cellular functions.

Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells. Here, we utilize mouse embryonic fibroblast CRISPR knock-in lines with labeled ARP2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that ARP2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of ARP2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility through cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity.

Lipophagy is an important microautophagic process that degrades lipid droplets (LDs) to mobilize stored lipids as an energy source during nutrient starvation. However, the molecular mechanisms regulating lipophagy in response to nutrient starvation remain poorly understood. We found that budding yeast mutants defective in glycosylphosphatidylinositol (GPI) lipid remodeling exhibited aberrant accumulation of lipid droplets (LDs) and neutral lipids under glucose starvation. Our data suggest that the accumulation results from a failure of vacuolar liquid-ordered (Lo) domain-mediated lipophagy. Furthermore, we demonstrated that glycosylphosphatidylinositol-anchored proteins (GPI-APs) localize to vacuoles in response to glucose depletion and that a mutant defective in endocytosis has defects in both vacuolar Lo domain formation and lipophagy. These results imply that GPI lipid remodeling is required for Lo domain-mediated lipophagy upon glucose starvation. We propose that endocytosis functions to supply the lipid portion of GPI-APs, remodeled to C26 diacylglycerol, to the vacuolar membrane for Lo domain formation. Summary StatementOur data suggest that the endocytic transport of GPI-APs remodeled with C26 diacylglycerol to the vacuole is required for vacuolar Lo domain formation and subsequent lipophagy in response to glucose deprivation. This reveals the essential role of GPI lipid remodeling in ensuring lipophagy to adapt to changes in nutrient availability.

The microtubule and actin cytoskeletons form dynamic, interconnected networks that are critical for eukaryotic cell function. These networks govern intracellular organization, cargo transport, cell migration, and tissue morphogenesis. Microtubules and actin filaments are regulated by diverse binding proteins that control many aspects of their function. However, identifying cytoskeletal-interacting proteins has been challenging due to the transient and weak nature of many interactions and the disruption of native architecture by conventional biochemical approaches. These limitations suggest that numerous physiologically relevant cytoskeletal regulators remain undiscovered. Identifying these factors requires novel and sensitive methodologies that can capture cytoskeletal interactions under native cellular conditions. Here, we present MT-ID and Act-ID, powerful proximity-labeling tools for identifying microtubule and actin-interacting proteins, respectively. MT-ID employs the microtubule-binding domain of MAP7 (EMTB) fused to TurboID, a highly active promiscuous biotin ligase. Act-ID utilizes the actin-binding domain of ITPKA (F-tractin) similarly fused to TurboID. We validate both approaches by successfully identifying numerous known cytoskeletal regulators and discovering potentially novel interacting proteins. Functional characterization reveals that LIMCH1 is a previously unrecognized microtubule-associated protein whose depletion increases microtubule density. Additionally, we identify FBXO30 as a novel actin-interacting protein, with its loss promoting increased focal adhesion formation. MT-ID and Act-ID will be useful not only to identify cytoskeletal interacting proteins but also to define changes to the cytoskeletal interactome when cells are exposed to changing physiological conditions.

Eukaryotic cells control their size by coordinating growth and division. Fission yeast divide at a reproducible cell size due to regulated activation of the cyclin-dependent kinase Cdk1. The nuclear concentration of mitotic cyclin Cdc13 increases in a time-dependent manner to promote Cdk1 activation as cells grow. Here, we show that interphase Cdc13 is stable against degradation and nuclear export, but is diluted by cell growth. Low glucose reduced cell growth rate but not time-dependent accumulation of Cdc13. Uncoupling the rates of cell growth and Cdc13 accumulation resulted in higher concentrations of nuclear Cdc13 despite reduced cell size. This change coincided with reduced activating phosphorylation of Cdk1-T167 and occurred dynamically during abrupt changes in glucose concentration. Mathematical modeling and experiments showed that cells maintain size homeostasis under these conditions. In contrast to low glucose, poor nitrogen reduced both cell growth rate and Cdc13 accumulation rate. Therefore, Cdc13 accumulation is independent of cell growth rate but can be altered by nutrient-specific mechanisms.

LIN-12/Notch signaling regulates C. elegans vulval development via cell fate specifications in the gonad and epidermis. In the somatic gonad LIN-12/Notch activity specifies the anchor cell (AC) versus ventral uterine cell (VU) fates, with VU receiving more signal. The AC secretes epidermal growth factor (EGF) which induces the underlying vulval precursor cells (VPCs) to adopt vulval fates. In the VPCs the secondary vulval fates are specified by LIN-12/Notch activity. We previously reported that the AGEF-1, an Arf GEF homologous to ArfGEF1 and ArfGEF2, the ARF-1 GTPase, and the adaptor protein complex 1 (AP-1) inhibit LET-23/EGF receptor (EGFR) signaling in the VPCs by antagonizing LET-23/EGFR basolateral localization. Here we report that AGEF-1, ARF-1 and AP-1 regulate LIN-12/Notch signaling during somatic gonad and vulval development. The lin-12(n302) partial gain-of-function causes a potent Vulvaless phenotype due to a lack of AC specification. We demonstrate that loss of AGEF-1, ARF-1 or AP-1 restored the AC fate in lin-12(n302) animals, indicating that AGEF-1/ARF-1/AP-1 promotes LIN-12/Notch signaling in the somatic gonad. Interestingly, loss of AGEF-1, ARF-1 or AP-1 also induced ectopic vulval secondary fates in lin-12(n302) animals, indicating that AGEF-1/ARF-1/AP-1 inhibits LIN-12/Notch in the VPCs. Using a LIN-12/Notch biosensor we demonstrate that loss of UNC-101/AP-1 results in decreased signaling in the VU cell and increased signaling in the VPCs that correspond with decreased expression levels of LIN-12/Notch and LAG-1/DSL ligand in the presumptive AC and VU while also causing increased apical localization of LIN-12/Notch in the VPCs. We hypothesize that the differential regulation of LIN-12/Notch signaling could reflect different trafficking pathways in epithelial cells (VPCs) versus non-epithelial cells (AC and VU). Our results indicate that the AGEF-1/ARF-1/AP-1 trafficking pathway maintains the VPC cell fate patterning by limiting both LET-23/EGFR and LIN-12/Notch signaling. Author summaryCell signaling and membrane trafficking are highly interconnected processes whereby membrane trafficking can regulate signal transduction pathways and vice versa. We previously demonstrated that the ARF-1 GTPase, the downstream AP-1 clathrin adaptor and upstream activator AGEF-1 antagonize the membrane trafficking of the Epidermal Growth Factor Receptor (EGFR) and hence signaling during C. elegans vulva induction. Strong loss of the ARF-1 GTPase pathway resulted in ectopic vulval induction. Here we demonstrate that the ARF-1 GTPase pathway differentially regulates Notch signaling to regulate vulva induction. In the somatic gonad it promotes Notch signaling to regulate the specification of the anchor cell which secretes the inductive signal. In the vulva precursor cells, the ARF-1 GTPase pathway antagonizes Notch signaling which cooperates with EGFR signaling to induce the vulval cell fates. We hypothesize that the differential regulation of Notch signaling by the ARF-1 GTPase pathway could be a result of more complex membrane trafficking pathways in polarized epithelial cells (vulva precursors) versus non-epithelial cells in the developing somatic gonad. Thus, the AGEF-1/ARF-1/AP-1 antagonizes both EGFR and Notch signaling in ensuring that only three of the six vulval precursor cells adopt are induced.

The endoplasmic reticulum (ER)-Golgi interface is a dynamic trafficking hub maintained in part by TANGO1, a scaffolding protein that coordinates proteins and membranes at ER exit sites (ERES). TANGO1 has two isoforms: TANGO1L, which has a lumenal SH3 domain, and TANGO1S, which lacks this domain but retains the transmembrane and cytoplasmic coiled-coil (CC), TEER, and PRD domains common to both forms. We showed previously that loss of both isoforms disrupts ER-Golgi organization more severely than TANGO1L loss alone, indicating TANGO1S is functional and can compensate. Here we dissect the role of each TANGO1 cytoplasmic domain in maintaining secretory pathway organisation by expressing TANGO1S domain-deletion mutants in TANGO1L-/S-knockout cells. We show that TANGO1 loss causes cis-Golgi vesiculation that cannot be rescued by TANGO1S, suggesting the lumenal domain of TANGO1L is essential in supporting Golgi architecture. Meanwhile, the TEER domain is essential for the organisation of the ER, whilst the TEER, CC2 and PRD domain are required for a defined ERGIC. All constructs partially rescue COPII recruitment. This study represents an advance towards a domain-level resolution of TANGO1S function. Summary statementIn this study we perform rescue experiments in TANGO1 knockout cells to dissect the role of the TANGO1 cytoplasmic domains in maintaining the ER-ERGIC-Golgi continuum.

Cell size in a proliferating cell population generally varies over a limited range ([~]2-4-fold). Within such populations, organelle content increases with cell size maintaining a relatively constant organelle density (amount per cell volume). However, cells of different types can differ greatly in cell size as well as in organelle composition. In such cases, it is often unclear to what degree, if any, the differences in organelle composition are due to the difference in cell size. In principle, this issue could be resolved by examining situations where a proliferating population of cells of the same cell type exhibit much greater size variation. Here we characterize how organelle content scales with cell volume in the polymorphic fungus, A. pullulans, whose proliferating cells span a [~]100-fold size range. We find that mitochondria and ER content increases in proportion to cell volume, while this is not the case for vacuoles and peroxisomes. Thus, organelle composition is affected by cell size in this system.

Phosphatidylinositol (4,5)-bisphosphate (PIP2), the most abundant cellular poly-phosphoinositide (PPI) class of phospholipid, is a central plasma membrane (PM)-associated signaling hub that controls many cellular processes. In this study, we demonstrate that either deletion of the gene encoding actin-binding protein profilin1 (Pfn1) or disruption of Pfn1-actin interaction leads to downregulation of PM PIP2 content in cells. This is also phenocopied when F-actin is depolymerized implying that Pfn1-dependent PIP2 alteration is related to its actin-regulatory function. Phospholipase C (PLC) activity is critical for Pfn1-deficient cells to exhibit the PIP2-related phenotype. These findings, taken together with biochemical signatures of elevated PIP2 hydrolysis (higher baseline PM diacylglycerol-to PIP2 ratio and protein kinase C activity) exhibited by Pfn1-deficient cells, imply that PLC-mediated PIP2 hydrolysis plays a role in Pfn1-dependent regulation of PM PIP2. Furthermore, we unexpectedly found that Pfn1 loss leads to dramatic alterations in several other important forms of lipids, revealing a previously unrecognized role of Pfn1 as a broad regulator of cellular lipid environment that extends beyond PPI control. In conclusion, our study establishes Pfn1 as an important regulator of cellular lipid homeostasis. SUMMARY STATEMENTThis study uncovers a mechanism of how functional loss of Profilin1, a key regulator of actin cytoskeleton, can trigger downregulation of plasma membrane content of PIP2, an important class of phospholipid, in cells.

Subcellular organelles must undergo periodic fission to be evenly distributed during cell division. These division events are mediated by protein members of the dynamin family, including dynamin-related proteins. Protozoan parasites, including trypanosomatids such as Trypanosoma brucei, have several single-copy organelles, suggesting tightly regulated systems for organelle fission and segregation. However, trypanosomatid genomes typically encode only one dynamin-like protein (DLP), which in T. brucei has multiple roles including endocytosis and mitochondrial fission. How DLPs are recruited to different membranes, and how their fission activity is regulated, are unknown. We used tandem-affinity purification in the related trypanosomatid Crithidia fasciculata to identify interacting partners of DLP. Surprisingly, we found that CfDLP co-purified with multiple proteins predicted to localize to glycosomes, peroxisome-related glycolytic organelles. Using expansion microscopy, we confirmed the localization of CfDLP to glycosomes, specifically those that appear to be undergoing division. To see if changes in the levels of DLP could alter glycosome morphology, we conducted RNAi-mediated knockdown and inducible overexpression experiments in T. brucei. TbDLP knockdown causes subtle changes in glycosome size, while overexpression of TbDLP1 causes an increase cytoplasmic vesicles and altered permeability of glycosomal membranes. These results suggest that the multifunctional DLP of trypanosomatids plays a role in glycosome maintenance. Author SummaryTrypanosomatids are eukaryotic parasites that cause devastating diseases in humans and animals. Like all eukaryotic cells, they must maintain their subcellular compartments through organelle division and other membrane remodeling events. Dynamin-like proteins are enzymes that work with other proteins to apply mechanical force to membranes. The dynamin-like proteins of Trypanosoma brucei, the causative agent of human African trypanosomiasis, have been implicated in endocytosis and mitochondrial division, although how these activities are regulated is not known. We have used a model trypanosomatid, the mosquito parasite Crithidia fasciculata, to look for dynamin-interacting proteins. In addition to proteins of unknown function, we show that dynamin-like protein associates with proteins found on glycosomes, trypanosomatid-specific organelles that contain enzymes required for breakdown of sugars. Knockdown and overexpression of dynamin-like proteins in T. brucei causes changes in glycosomes, supporting a role in organelle maintenance. Dynamin-like proteins likely regulate organelle structure and function, allowing parasites to adapt to different energetic requirements during their life cycle.

Nucleus shape is a sensitive indicator of cell state, influenced by numerous bio-chemical and physiological factors. While prior work has cataloged how perturbations alter nucleus morphology, we address the inverse: inferring underlying molecular changes from nucleus shape alone. We previously developed a mechanical model yielding two nondimensional parameters: flatness index and scale factor, which are surrogate measures for cortical actin tension and nuclear envelope compliance respectively. In this study, we apply these parameters to investigate the dynamics in cellular mechanics during confined migration. We fabricated polydimethylsiloxane (PDMS) microchannels with widths of 3 {micro}m (high confinement) and 10 {micro}m (low confinement) and tracked cells migrating through them. We captured high-frequency 3D nucleus shapes via double fluorescence exclusion microscopy and custom image analysis. Fitting the model and estimating flatness index and scale factor to time-resolved shapes revealed dynamic regulation in 3 {micro}m channels: actin tension decreased and nucleus compliance increased immediately before nucleus entry into the constriction, with rapid restoration to baseline upon exit. No such changes occurred in 10 {micro}m channels, indicating active, confinement-dependent cytoskeletal adaptation. Immunostaining for YAP and lamin-A,C confirmed these model inferences. Our results uncover mechanostasis, active mechanical homeostasis, during confined migration and establish the combination of double fluorescence exclusion microscopy and nondimensional nucleus shape parameters as a powerful, non-invasive tool for single-cell mechanobiology studies.

Intracellular calcium signaling plays a vital role in regulating various cellular processes including gene regulation, motility, metabolism and cell death. Inositol 1,4,5-trisphosphate receptors (IP3R) on the Endoplasmic Reticulum (ER) are a major cation channel that regulates stimulus-induced calcium release from the ER. While several molecular players regulate activity of IP3R, its regulation by actin filaments were uncharacterized. Here we show that actin filaments polymerized by a specific actin nucleator INF2 facilitates agonist-induced IP3R activity. Our results demonstrate that INF2-mediated actin filaments regulate formation and/or stability of IP3R clusters on the ER that have been previously shown to be hotspots of ER calcium release. Using cell-biological and biochemical techniques we further show that INF2 physically interacts with IP3R isoforms, often at IP3R clusters. While INF2-IP3R interaction is independent of INF2-activity, the ability of INF2 to mediate IP3R clusters is dependent on its actin polymerization activity. Finally, we demonstrate that in addition to its calcium mobilization activity, INF2 on ER specifically regulates IP3R cluster positioning to mediate ER-mitochondrial contacts and facilitate ER to mitochondrial calcium transfer. Overall, these results reveal an actin-dependent step in regulation of IP3R activity both in terms of ER calcium release and modulation of ER-mitochondrial contacts. HighlightsO_LIINF2-mediated actin filaments potentiate agonist-induced IP3R-mediated ER calcium release without affecting the ER calcium stores per se. C_LIO_LIER-localization of INF2 is dispensable for its role on IP3R activity. Moreover INF2-mediated actin filaments affect the activity of all IP3R isoforms. C_LIO_LIINF2 interacts with IP3R in an activity and actin filament independent manner through its C-terminal region. C_LIO_LIINF2 regulates IP3R cluster formation in actin-filament dependent manner and thereby regulates IP3R activity. C_LIO_LIFurther we show that ER-localized INF2 specifically regulate IP3R cluster positioning thereby promoting ER to mitochondrial contact and calcium transfer. C_LI

The tight junction (TJ) protein cingulin binds directly to nonmuscle myosin 2B (NM2B) through sequences in its C-terminal rod-tail region and recruits it to tight junctions (TJ) to control membrane cortex mechanics, epithelial morphogenesis and cingulin conformation. However, the minimal sequence required for cingulin-NM2B interaction and how this interaction is regulated is not known. Here we identify a 19-aminoacid sequence at the hinge between the cingulin rod and tail that is required for cingulin-NM2B interaction, and we investigate the role of phosphorylation of Ser residues within this region in regulating this interaction. Immunofluorescence microscopy localization of NM2B in cingulin-KO cells rescued with mutant cingulin constructs shows that phospho-mimetic but not dephospho-mimetic cingulin mutants inhibit NM2B recruitment to junctions and downstream regulation of cingulin conformation and TJ tortuosity, correlating with cingulin-NM2B interaction, as determined by GST pulldown analysis. In contrast, either phospo-or dephospho-mimetic mutants of Ser residues within the cingulin head domain do not affect either NM2B recruitment to TJ, or cingulin conformation and localization in cells, or TJ membrane tortuosity. Finally, Ser residues within the hinge display the consensus sequence for protein kinases CK1 and CK2, and, through in vitro phosphorylation, site mutation analysis and use of inhibitors, we identify a complex interplay between CGN phospho-sites, with a prominent negative role of Ser1162 phosphorylation in the regulation of cingulin-NM2B interaction. In summary, we show that cingulin-NM2B interaction is regulated by cingulin phosphorylation within the hinge and identify a potential role for CK1 and CK2 kinases in cingulin phosphorylation.

Cells experience mechanical loading across a broad range of loading rates, from low strain rates that are generated during morphogenesis and tissue remodelling, to high and injurious strain rates that are sustained during ventilation-induced lung injury, blast-induced injury, and impact-induced traumatic brain injury. Cell survival under high strain rate loading conditions depends on the ability of the cytoskeleton and plasma membrane to sustain mechanical load without permanent damage. The activity of different cytoskeletal and membrane regulatory proteins could therefore modulate cell susceptibility to injury, but the underlying mechanisms of injury at high strain rate are poorly understood. Tau is a microtubule-associated protein best known for its role in stabilizing microtubules in neurons and as a marker of neurodegenerative disease. Here, we investigated how Tau expression, phosphorylation, and microtubule binding modulates cell viscoelastic behaviour and membrane integrity during high strain-rate uniaxial stretch. We show that Tau expression and de-phosphorylation stabilize microtubules and causes increases in cell stiffness, suppresses cytoskeletal fluidity, and heightens susceptibility to stretch-induced membrane poration. Interestingly, we also find that these effects cannot be explained by microtubule stabilization by Tau alone. Actin architecture acts as a key determinant of injury vulnerability at high strain rate, highlighting the importance of cytoskeletal fluidity and microtubule-actin crosstalk for rapid force dissipation. Significance StatementCells must rapidly adapt to mechanical stress during high strain rate deformation. This study shows that Tau, a microtubule-associated protein, modulates cellular mechanics by increasing stiffness, decreasing viscoelastic fluidity, and enhancing susceptibility to membrane poration under rapid stretch. While Tau-mediated microtubule stabilization contributes to these effects, actin architecture and microtubule-actin crosstalk are also critical determinants of injury vulnerability in Tau-expressing cells.

Myosin 3A (MYO3A) is an unconventional myosin involved in the formation and maintenance of hair-cell stereocilia of the sensory epithelia in the inner ear. The kinase domain has been implicated in phosphoregulation of MYO3A activity through intermolecular autophosphorylation. Previous studies using mass spectrometry identified two potential phosphorylation sites in the motor domain. To investigate the regulatory roles of these sites, we generated glutamic acid point mutations in our mchr-MYO3A{Delta}K construct to mimic phosphorylation and assayed the constructs for their ability to tip-localize and influence filopodial density via transfection into COS7 cells. The phosphomimic constructs were less able to generate filopodia when compared to wildtype constructs. To gain a better understanding of the phosphoregulation of MYO3A, we transfected COS7 cells with mchr-MYO3A{Delta}K in combination with GFP-tagged full-length MYO3A (GFP-MYO3AFL), or GFP attached to just the kinase domain of MYO3A (GFP-MYO3AKIN). Coexpression of mchr-MYO3A{Delta}K with either construct resulted in decreased mchr-MYO3A levels at the tips of filopodia and fewer filopodia at the edge of the cell, compared to cells expressing mchr-MYO3A{Delta}K alone. This implies that the kinase domain does not require motor activity to contribute to phosphoregulation of MYO3A, and that MYO3A phosphoregulation may be influencing filopodia initiation. Informatic analyses and structural predictions suggest that the two phosphorylation sites in the motor domain inhibit actin/MYO3A interactions. Taken together, these analyses link MYO3A phosphorylation with the regulation of its ability to create actin protrusions such as filopodia and stereocilia.

The Ezrin, Radixin, and Moesin (ERM) family of proteins anchors the actin cytoskeleton to the plasma membrane for the purpose of either stabilizing or altering cell shape. In Caenorhabditis elegans, ERM-1, is essential for cell polarity, signaling, intestine development, and larval viability. Interestingly, ERM-1 proteins are produced by erm-1 mRNA transcripts that concentrate at the plasma membrane in embryos. The localization of erm-1 mRNA to the plasma membrane occurs in a 3UTR-independent, translation-dependent manner, directed by the PH-subdomain within ERM-1s N-terminal FERM domain. This has led to the model that erm-1 mRNA, its associated ribosome, and its emerging nascent peptide are all transported together to the plasma membrane as a complex. Here, we characterize the transport mechanism. Using a microscopy approach, we observed that the localizations of erm-1 mRNA and ERM-1 protein to the plasma membrane were disrupted by nocodazole treatment, illustrating a microtubule role. Furthermore, erm-1 mRNA and ERM-1 protein localized to the plasma membrane independently of myosin and dynein motors, but dependent on the kinesin bmk-1 (bmk-1), a plus-end-directed, Kinesin-5 family motor protein. Loss of bmk-1 did not reduce the total number of erm-1 mRNA molecules in the cell, arguing against a diffusion- and protection-based mechanism of mRNA localization. Together, these findings suggest that erm-1 mRNA is localized via an active transport pathway mediated by a plus-end-directed kinesin adapter. Interestingly, loss of bmk-1 led to diffuse localization of ERM-1 protein along the plasma membrane and reduced ERM-1 protein levels at the site of abscission, the midbody, and the midbody remnant. This suggests that ERM-1 local translation at the plasma membrane is critical for its proteins ultimate spatial patterning in the cell.

UBQLN2 is a ubiquitin-binding shuttle protein that undergoes phase separation in vitro and localizes to stress-induced cellular condensates including stress granules. The central region of UBQLN2 contains two chaperone- and substrate-binding STI1 domains (STI1-I, STI1-II) and disordered linkers; the individual contributions of these domains and linkers to cellular condensate partitioning remain poorly characterized. Here we use live-cell imaging and immunofluorescence experiments to systematically examine domain requirements for UBQLN2 puncta formation in cultured human cells. We show that in vitro phase separation propensity largely correlates with puncta formation in transfected cells. Importantly, STI1-II and UBA domains are each required for baseline puncta formation in cells, but not STI1-I. In contrast, both STI1 domains are required for heat stress-induced puncta formation. Removal of STI1-II abrogates this stress response, and STI1-I deletion substantially attenuates it. Using N-terminal truncation constructs, we demonstrate that STI1-I strongly promotes both phase separation and puncta formation in the absence of the N-terminal region containing the UBL domain. Together, our findings demonstrate that the two STI1 domains of UBQLN2 have distinct roles in puncta formation and condensate partitioning, with STI1-II essential under all conditions.