Journal of Cell Science

Gap junction communication is reduced during mitosis as the junction protein connexin-43 (Cx43) is redistributed from gap junction plaques on the plasma membrane to cytoplasmic annular vesicles and actin-based mitotic nanotubes that transiently connect mitotic cells to neighboring cells. However, the dynamic details of Cx43 redistribution during cell entry into and exit from mitosis, and the roles of mitotic nanotubes and associated Cx43 in intercellular communication, remain poorly understood. Here, using confocal live-cell imaging, we show that as cells enter mitosis, plaque-derived Cx43 structures are transferred to mitotic nanotubes. Over time, these structures fragment and migrate along the length of the nanotubes, either being transferred to the cytoplasm of adjacent cells or being positioned at the nanotube ends where they could potentially enable communication. Functionally, mitotic nanotubes indeed facilitate gap junction-dependent intercellular communication, though at reduced rates compared interphase cells. Interestingly, knockdown of Cx43 resulted in impaired nanotube formation and intercellular communication while inhibition of Rho kinase (ROCK) with Y-27632 prevented mitotic cell rounding and nanotube elongation, and increased cell-cell communication during mitosis, suggesting that nanotube function is influenced by Cx43 expression and trafficking as well as actin remodeling via ROCK. Overall, these findings provide valuable insights into the mechanisms that regulate Cx43 and mitotic nanotube dynamics and reveal a novel role for mitotic nanotubes in facilitating cell-cell communication during cell division.

Centrosomal and microtubule-associated proteins such as CEP170 and WDR62 are essential in regulating mitotic spindle formation and pole orientation during cell division. MAPKBP1, a paralog of WDR62, is also a centrosomal protein, but its function is currently unclear. We have shown here that MAPKBP1 is localised to the subdistal appendages of the mother centriole, the pericentriolar material (PCM) of the centrosomes and the mitotic spindles during metaphase. Furthermore, MAPKBP1, WDR62 and CEP170 exists as a complex, where MAPKBP1 is recruited to the centrosomes by WDR62 and CEP170, and CEP170-MAPKBP1 interaction is mediated by WDR62. In addition, MAPKBP1 depletion leads to mitotic spindle defects and delayed mitosis that were further exacerbated with WDR62 knockout, indicating a possible redundancy between MAPKBP1 and WDR62. MAPKBP1 loss also leads to PCM fragmentation, which supports its role as a subdistal appendages protein vital in maintaining centrosome structure and PCM cohesion for proper anchoring of mitotic spindles. This study provides insight into how subdistal appendages and centrosome and microtubule associated proteins co-operate to tightly regulate mitotic spindle formation and stability.

Lamin B receptor (LBR) is an inner nuclear membrane (INM) protein that plays crucial roles in maintaining nuclear architecture and organization of peripheral heterochromatin. Lamins and LBR both contribute to chromatin tethering at the nuclear periphery, and the expression of LBR and A-type lamins is tightly regulated during development to ensure a faithful transition between different chromatin tethering modalities. Despite its well-established association with B-type lamins, the contributions of individual lamin isoforms to LBR localization and anchorage have not been systematically examined. Here, we used mouse embryonic fibroblasts (MEFs) lacking all endogenous lamins (triple lamin knockout: TKO) to assess how specific lamin isoforms and domains regulate LBR subcellular localization and mobility. Whereas ectopic expression of either lamin B1 or lamin B2 was sufficient to tether LBR to the nuclear envelope in TKO cells, expression of lamin A increased the lateral mobility of LBR at the nuclear membrane, resulting in its displacement from the nuclear envelope to the ER. The lamin A-induced displacement of LBR was mediated by phosphorylation of LBR. Overexpression of lamin A in wild-type MEFs similarly increased LBR phosphorylation and promoted its displacement from the nuclear envelope. Collectively, these findings define isoform-specific and antagonistic roles for A-type and B-type lamins in regulating LBR anchorage at the nuclear envelope. In addition, they indicate a lamin A-dependent mechanism that may reflect a broader developmental process, since LBR and lamin A sequentially tether peripheral heterochromatin during development.

Cell morphology, dictated by the filamentous actin (F-actin) cytoskeleton, is fundamental to cell migration during wound healing and cancer metastasis. Cell morphology is shaped by the extracellular matrix (ECM), which provides mechanical cues in the form of ECM stiffness. These mechanical cues regulate the assembly of the F-actin cytoskeleton which in turn controls cell morphology and cell migration. Formins are key regulators of linear F-actin, assembling it into stress fibers, yet the specific roles of individual formins in controlling distinct stress fiber subpopulations to control cell morphology and migration remain poorly defined. Here, we characterize formin expression across different cell types and leverage the inherent expression and cell morphology differences to identify FHOD3 and DIAPH3 as strongly correlated with cell elongation. We demonstrate that these formins regulate complementary but distinct stress fiber networks. In contractile, but less motile cells, FHOD3 knock-down shifts the balance towards stress fibers oriented perpendicular to the long axis of the cell. In contrast, DIAPH3 knock-down shifts the balance towards stress fibers oriented parallel to the long axis of the cell. However, in less contractile and highly motile cells, knockdown of either formin significantly impairs cell migration speed, suggesting both F-actin fiber networks are necessary for cell migration. Our work establishes a model where FHOD3 and DIAPH3 function through non-overlapping mechanisms to control the F-actin architecture that governs cell shape and motility.

Macrophages are innate immune cells contributing to tissue homeostasis and various pathologies. Signals from their environment can lead macrophages to adapt distinct functional phenotypes, a process called polarization. Because macrophages have been previously shown to degrade the nuclear envelope proteins lamin A/C upon pro-inflammatory polarization, and lamins are considered key determinants of nuclear deformability, we aimed to address the effect of pro-inflammatory stimulation on nuclear mechanics. We present the surprising finding that polarized bone marrow-derived macrophages have less deformable nuclei than unpolarized macrophages, despite their reduced lamin A/C levels. Furthermore, pro-inflammatory macrophages exhibited altered chromatin dynamics relative to unpolarized macrophages, including redistribution of trimethylated histone H3K9 (H3K9me3) from the nuclear periphery to the interior and increased chromatin compaction. Our findings suggest a model in which pro-inflammatory stimulation of macrophages induces chromatin changes that drive nuclear stiffening, and that in these cells, chromatin, rather than the nuclear lamina, is the major driver for resisting nuclear deformation. These findings may have functional relevance for the physiological function of polarized macrophages, as the mechanical properties of the nucleus can influence how these cells adapt and respond to their environments in the context of cell migration or inflammatory disease pathologies.

Centriolar satellites are dynamic pericentrosomal structures implicated in centrosomal protein homeostasis and ciliogenesis. Centriolar satellites have been identified in vertebrates and were only recently described in flies. In C. elegans similar pericentriolar structures were reported for the Sjogrens Syndrome Nuclear Antigen 1 (SSNA-1). However, whether these foci have characteristics resembling centriolar satellites of vertebrates, has not been explored. We show that Spindle Assembly-1 (SAS-1), the interaction partner of SSNA-1 forms similar satellite-like structures that localize to a pericentrosomal space in a cell cycle-dependent manner. SAS-1 satellite-like structures associate with and are dependent on the microtubule cytoskeleton. Furthermore, we demonstrate that they form in a dose dependent manner, are dynamic and sensitive to agents disrupting weak hydrophobic interactions, characteristics of biomolecular condensates. We conclude that C. elegans has bona fide centriolar satellites highlighting their evolutionary conservation and importance across species, and at the same time opening new avenues for future mechanistic studies.

Primary cilia exhibit conserved organization and contain structural and functional domains of unique molecular composition. The inversin compartment (InvC), which is found in the proximal ciliary segment of a subset of vertebrate and invertebrate cell types, concentrates different classes of signaling molecules. Mutations in genes encoding resident proteins of the InvC manifest in ciliopathies, highlighting the importance of the InvC in cilia biology. We previously showed that a chaperone of G proteins RIC-8 localizes to the InvC of C. elegans channel cilia; however, the mechanisms that regulate RIC-8 targeting to this ciliary sub-domain or RIC-8 function in the InvC remain unknown. Here, we build on our prior work to demonstrate that RIC-8 becomes restricted to the InvC during larval development and show that, while the RVxP motif and intact transition zone are required for its proper intraciliary distribution, RIC-8 localization to the cilium depends on intraflagellar transport. Using the ASH neuron as a model, we establish that RIC-8 functions in channel cilia to modulate chemosensory responses. Finally, we demonstrate that human RIC8A and RIC8B proteins are required for ciliogenesis in RPE-1 cells. Collectively, our results define ciliary trafficking mechanisms and novel cell-specific functions for a highly conserved signaling protein. AbbreviationsInvC, WT, TZ, IFT, PCMC, KD, RT, s

Kinesin-3 motor proteins are increasingly recognized for their important roles in cilia. The mammalian kinesin-3 motor KIF13B moves bidirectionally in primary cilia and regulates ciliary content, but its relationship to the intraflagellar transport (IFT) machinery is unclear. Here, we combine quantitative live-cell imaging with a new kymograph analysis based on dynamic mode decomposition (DMD) to separate mobile from immobile protein populations in primary cilia. This approach simplifies extraction of molecular velocities from kymographs and reveals that a KIF13B deletion mutant retaining only the motor domain and part of the forkhead-associated domain does not alter steady-state IFT velocity or frequency. However, when retrograde dynein-2 function is inhibited by Ciliobrevin D, both anterograde and retrograde IFT velocities decrease in parental cells, as expected, but remain unchanged in KIF13B mutant cells. Structured illumination, confocal, and STED microscopy further show that KIF13B localizes to the ciliary membrane and concentrates at the periciliary membrane region and the centriolar subdistal appendages, below the distal appendage marker FBF1. Our improved kymograph approach provides new insight into KIF13B ciliary function and simplifies the quantitative analysis of ciliary protein transport.

Cell division is a crucial process that ensures proper development of multicellular organisms. Cell division ends in abscission, a process in which the intercellular bridge between two sister cells is cut. Although abscission usually happens shortly after chromosome segregation, abscission is severely delayed in mouse embryos and embryonic stem cells (mESC). The regulation of the duration of abscission influences cell fate transitions but how cell state and abscission dynamics crosstalk remains unknown. Here, we show that a key pluripotency pathway, the Wnt signalling pathway, controls abscission dynamics. Upon deactivation of Wnt signalling in naive mESCs, abscission becomes faster. Wnt signalling regulates abscission dynamics in mESCs through two mechanisms. First, Wnt signalling keeps the amount of Aurora B high at the intercellular bridge, probably by preventing Aurora B degradation. In turn, high Aurora B activity at the bridge delays bridge abscission. Second, a key component of Wnt signalling, the kinase GSK-3{beta} localizes at the intercellular bridge with microtubules and their associated proteins (MAPs). In pluripotent cells, inactivation of GSK-3{beta} leads to an increase of stable microtubules at the bridge stable which causes delayed abscission. Crucially, inhibition of GSK-3{beta} after cells have exited naive pluripotency accelerates abscission, demonstrating that cell state influences the output of the abscission signalling pathway. The permissive function of canonical Wnt on cell state is thought to be mediated by reinforcement of existing pluripotency network; altogether, our work shows that non-canonical Wnt is also context dependent.

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.

Automated transmission electron microscopy (TEM) generates large datasets that challenge traditional qualitative analysis of cellular ultrastructure. Quantitative assessment of structural differences between different samples remains difficult due to structural variability in thin sections of organelles. Here we applied supervised machine learning (sML) to segment, quantify and compare cellular structures -including nuclei, chromatin, mitochondria, rough endoplasmic reticulum, and endocytic vesicles- in large TEM images of wild-type macrophages versus those with altered cellular physiology due to deficiency in O-GlcNAc Transferase (OGT). sML revealed that OGT knockout macrophages are larger and more oval, with increased euchromatin, nucleoli size, and relative mitochondrial and rER surface areas. Comparison with six TEM experts showed sML provides more objective and sensitive quantification of subtle differences, while expert consensus is only achieved for larger structural variations. These findings demonstrate that sML enhances quantitative TEM analysis and complements human expertise in ultrastructural studies.

Intraflagellar transport (IFT) is a conserved trafficking system in eukaryotes that moves proteins along microtubules. It is best known for its essential role in building and maintaining cilia and flagella. Intriguingly, several IFT components are still found in organisms that no longer possess flagella, raising important questions about their original functions and how they may have been repurposed during evolution. The filamentous alga Klebsormidium nitens, positioned at the base of the streptophyte lineage, offers a valuable model for exploring this transition. Here, we investigate the IFT machinery in K. nitens and its relationship with the blue-light photoreceptor phototropin. Comparative genomic analyses show that key IFT-A and IFT-B components are retained, despite the complete loss of flagella in vegetative state. Cellular detection and immunofluorescence studies revealed the presence and localisation of IFT components, interestingly, their co-localization with phototropin. Notably, IFT-139 and IFT-20 strongly co-localize with phototropin at plasma membrane-associated regions. Phototropin overlapping localization (plasma membrane associated) with conserved phospho-adaptor protein 14-3-3, pointing to a phosphorylation-dependent signaling network. Unlike in Chlamydomonas reinhardtii, where these proteins localize to flagella, their interaction in K. nitens occurs independently of cilia presence. Together, these results evidenced that IFT components were retained and repurposed early in streptophyte evolution and might support phototropin localization and signalling, revealing an ancestral, non-ciliary role for the IFT system.

The nuclear pore complex (NPC) is the only gateway that connects the nucleus with the cytoplasm in eukaryotic cells. Its nucleoplasmic face is decorated by the nuclear basket, a filamentous structure with important roles in mRNA export and chromatin organization. In contrast to major parts of the nuclear pore scaffold, the architecture and organization of the nuclear basket remain poorly defined. In this study, we investigate the interaction network required for formation of the nuclear basket in vivo using budding yeast. We demonstrate that the filamentous Mlp1 protein relies on coiled-coil segments outside its previously characterized NPC-binding region to stably interact with the NPC. Furthermore, our results reveal that Mlp1s paralogue, Mlp2, plays a central role in nuclear basket architecture. Specifically, Mlp2 associates with the NPC independently of Mlp1 and together with Mlp1 is essential for the efficient recruitment of Pml39 and additional Mlp1 subunits. Our findings allow us to propose a refined model of nuclear pore basket architecture and organization.

The nucleus-vacuole junction (NVJ) is a membrane contact site between the nuclear envelope and the vacuole in yeast that undergoes dynamic remodeling in response to nutrient starvation. Here, we report that Msc1 is a glucose starvation (GS)-responsive NVJ factor. GS strongly induced Msc1 expression and promoted its accumulation at the NVJ. Although Msc1 is not essential for NVJ formation itself, loss of Msc1 impaired GS-dependent functional maturation of the NVJ, including stabilization and recruitment of multiple NVJ-associated proteins. Notably, GS-induced transcriptional activation of NVJ1 was markedly attenuated in msc1{Delta} cells, suggesting that proper NVJ remodeling contributes to the execution of stress-responsive transcriptional programs. Together, these findings establish Msc1 as an upstream regulator linking GS to functional remodeling of the NVJ and associated transcriptional responses.

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.

SUN5 is a testis-specific SUN domain protein essential for connecting the sperm tail to the nucleus. However, until now, its precise localization, intracellular dynamics, and membrane topology during spermiogenesis have remained controversial. To address these discrepancies, we applied ultrastructure expansion microscopy (U-ExM) to systematically track SUN5 redistribution throughout spermiogenesis. This approach enabled a detailed reconstruction of SUN5 localization across developmental stages and revealed previously undescribed enrichment at the perinuclear ring (PNR) and the microtubule manchette, suggesting secondary functions at the PNR or a potential role in intra-manchette transport (IMT). Complementary immunogold labelling using the Tokuyasu method, together with biochemical assays, demonstrated that SUN5 adopts a membrane localization and topology consistent with that of classical SUN domain proteins. Quantitative measurements of the nuclear envelope architecture at the head-to-tail coupling apparatus (HTCA) further enabled us to present a refined structural model of SUN5 positioning at the head-tail junction. Overall, our findings resolve previous discrepancies in the field and provide a coherent framework for understanding SUN5 organization and its role in mammalian spermiogenesis. Summary StatementIn the presented study, we analyzed the dynamic redistribution of SUN5 during mammalian spermiogenesis and resolved its topology in developing spermatids to gain insights concerning the proteins molecular function in head-tail coupling.

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.

Membrane trafficking is essential to maintain cellular homeostasis, enabling cells and organelles to exchange molecular components via vesicle transport. Therefore, it is tightly regulated, including by RAB GTPases. Among these, RAB21, which is primarily associated with early endosomes, plays a central role in coordinating endocytosis, sorting, and degradation. Like other RABs, it cycles between GTP- and GDP-bound forms. Although three specific guanine exchange factors (GEFs) for RAB21 have been identified, surprisingly, no GTPase-activating proteins (GAPs) have been found to directly modulate RAB21. Here, we describe a genetic modifier screen in Drosophila that identified Tre/Bub2/Cdc16 (TBC) domain family member 25 (TBC1D25) as a potential negative regulator of RAB21. We confirmed the RAB21-TBC1D25 interaction using co-immunoprecipitation and proximity ligation assays and further demonstrated that their association depends on the catalytic activity of TBC1D25. Genetic interaction studies revealed a functional link between TBC1D25 and RAB21 in autophagy and cargo sorting. Collectively, our results indicate that TBC1D25 negatively regulates RAB21, potentially by serving as a RAB21-specific GAP.

The biogenesis of integral membrane proteins is complex, as revealed by an ever-growing number of cellular components shown to be dedicated to the insertion, folding, surveillance, rectification, or quality control of specific client membrane proteins. The zinc metalloprotease ZMPSTE24 and its yeast homolog Ste24 have well-established roles in the proteolytic maturation of the nuclear scaffold protein lamin A and yeast a-factor, respectively. Additionally, Ste24 has been implicated through yeast genetic screens in a variety of membrane processes, including ER- associated degradation (ERAD), Sec61 translocon "unclogging," the unfolded protein response (UPR), and potentially as a membrane protein topology determinant. Recently, an interaction was demonstrated between ZMPSTE24 and the antiviral interferon induced transmembrane protein IFITM3, although the functional significance of this interaction is not well-understood. IFITM3 is a tail-anchored protein with a cytoplasmic N-terminus, a single transmembrane span, and a lumenal/exocellular C-terminus. Here, we show that a catalytic-dead version of ZMPSTE24, ZMPSTE24E336A, exhibits enhanced binding to IFITM3, and this bound species of IFITM3 is hypo-palmitoylated. Using a split fluorescence topology reporter, we demonstrate that ZMPSTE24E336A "traps" and stabilizes a subpopulation of IFITM3 molecules with an atypical membrane topology, whose C-terminus is cytosolic instead of lumenal. Such inverted forms of IFITM3 are also detected in the presence of ERAD inhibitors when ZMPSTE24E336A is absent. We hypothesize the ZMPSTE24E336A trap mutant reveals a normally transient isoform of IFITM3 whose transmembrane span is inverted and that ZMPSTE24 is involved in the quality control of IFITM3 topology, either inverting, correcting or assisting in removal of aberrant IFITM3 molecules.

Cardiac function is dependent upon the ability of cardiomyocytes to adapt their contractions to meet the demands of the body. Increased preload lengthens the sarcomere, altering the efficiency of contraction. The surface topography of cardiomyocytes is distinct from other cell types. T-tubules are key membrane structures which protrude into the cell body, aligned with the edges of the sarcomere. These are key signalling domains which ensure efficient and adaptable excitation-contraction coupling. It has been shown that T-tubules are dynamic structures which deform during the contraction cycle however, how the T-tubule structure adapts to increased preload has not been realised. Here we demonstrate a methodology for the measurement of the surface topography and sub-cellular signalling of isolated adult cardiomyocytes under diastolic stretch. We track individual T-tubule openings, showing that increased load causes them to shift, increase diameter and become stiffer. Future applications of this system include experimental modelling of preload-reducing therapies, for the treatment of acute and chronic heart failure.