Journal of Cell Science

Cilia are assembled by intraflagellar transport (IFT), which relies on two protein complexes: IFT-A and IFT-B. It is generally assumed that IFT-B and IFT-A are critical for anterograde and retrograde transport, respectively. However, full deletion of IFT-A genes in several organisms suggests a possible contribution to anterograde transport. In many species, cilia collapse when IFT is altered, hindering functional studies. Here, we investigated the role of IFT-A in the protist Trypanosoma brucei, where IFT is not required for cilium maintenance. Following the inducible knockdown of IFT88 (an IFT-B member) or IFT140 (an IFT-A member), we monitored the fate of several IFT proteins in preassembled cilia using live imaging and evaluated the consequences on train formation by volumetric electron microscopy. Surprisingly, both IFT88 and IFT140 turned out to be essential for anterograde train assembly. Their depletion initially led to the formation of shorter trains and subsequently to an inhibition of train injection. We propose a model to reconcile the diverging phenotypes reported in the literature.

Kinesins are molecular motor proteins essential for organizing and remodeling the cytoskeleton during neuronal development and maintenance. One key regulator is kinesin family binding protein (KIFBP), which inhibits a subset of kinesins by blocking motor-microtubule interactions. Homozygous mutations in KIFBP cause Goldberg-Shprintzen Syndrome (GOSHS), a neurodevelopmental disorder characterized by intellectual disability, microcephaly, and axonal neuropathy. Although loss of KIFBP has been linked to reduced neurite length and microtubule disorganization, the specific kinesins underlying these phenotypes remain unclear. Here we use a CRISPR-Cas9 generated KIFBP knockout Neuro-2a cell line to demonstrate that KIFBP is required for neurite extension and use inducible GFP-KIFBP to define the KIFBP interactome during neuronal differentiation. Immunoprecipitation coupled with mass spectrometry identified both known and novel KIFBP-associated kinesins. Single molecule TIRF microscopy confirmed direct inhibition of a subset of kinesins that co-immunoprecipitated with KIFBP. Notably, we identified KIF5A and KIF18B as previously unrecognized regulatory targets with potential roles in neuronal development. Together, these findings establish Neuro-2a cells as a model for studying KIFBP function and provide new insight into the regulation of kinesin activity and cytoskeletal dynamics in neurons.

Centrioles are highly organised microtubular scaffolds which grow and mature progressively during successive cell cycles. Their molecular organisation is extensively characterized, yet the contribution of several components to centriole assembly, maturation or stability is incompletely understood. Here, using ultrastructure expansion microscopy and transmission electron microscopy, we show that ALMS1, the protein mutated in Alstrom syndrome, is required for proper centriole architecture. In absence of ALMS1, RPE1 cells exhibit shorter centrioles with defects in the microtubular wall, including broken or missing triplets or open B/C tubules. These structural defects arise after procentriole assembly. We show that ALMS1 loss selectively reduces the proximal region proteins CCDC77 and CEP44, leaving intact central and distal ones. ALMS1 is further required for the recruitment of the proximal CEP135 cap and the clearance of the {gamma}-tubulin/GCP2 pool present at the procentriole base. Our findings thus identify ALMS1 as a key organiser of the centriole proximal domain and required for remodelling and stabilising the proximal end of centrioles during cell cycle progression.

Clathrin mediated endocytosis (CME) is a vital cellular process that mediates cell signaling by controlling the internalization of extracellular cargo and activated receptors. Arp2/3-branched actin provides force to assist in membrane invagination and scission in CME. Loss of Arp2/3-branched actin in conditional Arpc2 KO fibroblasts results in an increase of large arrested clathrin lattices (LACLs) visualized by live-cell TIRF imaging. Additional structural details of LACLs were revealed using electron microscopy and include an increase in arrested clathrin lattices of various curvatures. Numerous CME proteins have heightened levels of tyrosine phosphorylation at these LACLs in Arpc2 KO cells. We identified the non-receptor tyrosine kinase ACK (Activated Cdc42-Associated Kinase) as a key upstream regulator of LACL turnover. CRISPR KO of ACK abrogates LACL tyrosine phosphorylation and impairs cells capacity to resolve LACLs. Our results support a model where ACK recruitment and activation at LACLs drives branched actin formation to help resolve large accumulations of arrested CME structures.

Adherens junctions physically connect neighboring cells and are built around classical cadherins, homophilic transmembrane proteins that link to the actin cytoskeleton. Classical cadherins can organize into ordered arrays in vitro, but whether they do so in cells remains to be established. Here, we use fluorescence polarization microscopy to show that the classical cadherin N-cadherin is orientationally ordered at cardiomyocyte cell-cell junctions. Whereas the desmosomal cadherin desmoglein 2 was similarly ordered across junction types, N-cadherin order was spatially heterogeneous. Order was lowest where organized myofibrils terminate at high-load, vinculin-enriched axial junctions and highest at low-load, vinculin-poor lateral junctions. This inverse relationship between order and mechanical load suggests that robust cadherin-mediated adhesion does not require ectodomain order. Our findings provide evidence that a classical cadherin is orientationally ordered in cells and show that mechanically active adhesions adopt distinct organizational strategies according to local mechanical demands. Summary StatementAt cardiomyocyte junctions, N-cadherin is ordered where mechanical load is low but disordered where load is high, suggesting that cadherin organization adapts to local force conditions.

Classical immunolabeling approaches can achieve homogeneous and continuous labeling of cellular membranes and organelles at wide-field and confocal resolution. In super-resolution and expansion microscopy, however, the lack of high-density labels hampers the localization of membrane proteins and protein complexes within their membrane context. Here we show that secondary antibodies coupled to the lipophilic dyes ATTO 647N or ATTO 550 brightly label the nuclear envelope, mitochondria, and endoplasmic reticulum of fixed, permeabilized cells, and that graded labelling intensities allow selective visualization of organelles and precise segmentation of mitochondria. Using state-of-the-art super-resolution and expansion microscopy, we achieve high-density labelling of nuclear and mitochondrial membranes, with targeting and density comparable to existing membrane-labelling approaches and a signal that can be further amplified with additional secondary antibodies. Finally, we show that these dye-conjugated IgG allow to resolve mitochondria-ER contacts and mitochondrial ultrastructure as well as precise visualization of the nuclear envelope and its invaginations. This study demonstrates that secondary antibodies conjugated to lipophilic fluorophores represent stable, convenient and affordable tools for organelle visualization in conventional microscopy and for high-density labeling of membranes in super-resolution and expansion microscopy.

One of the first genes identified to regulate the spacing of nuclei in the multinucleated myofiber was Kinesin-1. However, the mechanism by which Kinesin-1 or other kinesins regulate myonuclear spacing is not known. Critically, the myofiber lacks centrosomes, and the many myonuclei act as the primary microtubule organizing centers of the cell. Because of this unique re-structuring, we hypothesized that the kinesins that drive centrosomes apart during mitotic spindle elongation may play a similar role in spacing myonuclei. We found that the bipolar Kinesin-5 (Klp61f) and the (-)-end directed Kinesin-14 (ncd) were both necessary for myonuclear spacing at different times, with both being necessary during embryogenesis, but only ncd being necessary in the fully differentiated myofiber. To investigate the shared mechanisms during embryogenesis, we used live-imaging and found that, similar to the mitotic spindle, Klp61f acts as an accelerator for myonuclear movement, whereas ncd acts as a brake contrary to this movement. To investigate these mechanisms and test the hypothesis that this is dependent on microtubule-sliding, we used super-resolution microscopy to visualize and quantify the microtubule network in animals with disrupted Klp61f or ncd. We found that in both cases, there was a decrease in the amount of microtubule overlap between neighboring myonuclei. Furthermore, we found that disrupting ncd led to severe changes in microtubule network organization, supporting our hypotheses that microtubule-sliding is necessary to space myonuclei, and that ncd likely functions through a unique mechanism in the differentiated myofiber to maintain myonuclear spacing. Together, our data supports a model where myonuclear spacing is regulated by a counteracting force generated by different kinesins during embryonic development. Furthermore, one kinesin, ncd, is repurposed in the differentiated myofiber to dynamically crosslink microtubules, a function necessary to anchor nuclei in place. Thus, kinesin motors regulate myonuclear spacing across developmental time by leveraging opposing forces through diverse mechanisms.

Msc1 is a yeast nuclear envelope (NE) protein that facilitates DNA double-strand break repair. In its absence, cells exhibit abnormal nuclear morphologies and maldistribution of nuclear pore complexes (NPCs). Msc1 is not uniformly distributed across the NE but concentrates onto dynamic patches that often coincide with blebs or herniations. Here, we report that Msc1 abundance and the number of patches dramatically increase after the diauxic shift. Msc1 patches are devoid of NPCs and fully colocalize with nucleus-vacuole junctions (NVJs), which are involved in piecemeal micronucleophagy. In the absence of Msc1, abnormal NPC aggregates accumulate adjacent to vacuoles, both at and outside the NE. We conclude that Msc1 is the key factor that maintains NPC homeostasis as cells prepare to enter quiescence.

Bipolar spindle assembly is essential for accurate chromosome segregation. KIFC1, a conserved Ran- regulated minus-end-directed kinesin-14 motor, accumulates in the nucleus during interphase and promotes chromatin-mediated spindle assembly during mitosis and meiosis. In human oocytes, reduced KIFC1 levels destabilize meiotic spindles, a defect that can be rescued by increasing KIFC1 expression. However, how KIFC1 expression levels affect mitotic spindle stability during cleavage divisions in vertebrates remains unclear. Here, we show that whereas an approximately 50% reduction in KIFC1 causes no detectable defects in spindle assembly, approximately 10-fold overexpression of KIFC1 induces monopolar spindle formation, leading to chromosome mis-segregation and embryonic lethality in medaka early embryos. KIFC1 overexpression results in ectopic centrosomal localization during interphase, impairing the separation of duplicated centrosomes before mitotic entry. Analyses of KIFC1 mutants demonstrated that these centrosome separation defects require KIFC1s microtubule-binding and motor activities and are further enhanced by deletion of KIFC1s nuclear localization sequences. Together, our findings demonstrate that tight regulation of KIFC1 expression and its nuclear sequestration is essential for the proper separation and positioning of duplicated centrosomes before mitotic entry, thereby ensuring efficient bipolar spindle assembly during the rapid cleavage divisions of vertebrate embryos. HighlightsO_LIKIFC1 accumulates in the nucleus and at the embryonic spindle midplane via the Ran pathway. C_LIO_LIPartial KIFC1 depletion does not impair spindle assembly in medaka early embryos. C_LIO_LIKIFC1 overexpression induces monopolar spindles by preventing centrosome separation. C_LIO_LICentrosome separation defects require KIFC1 microtubule-binding and motor activity. C_LI

Cell migration through confined tissue environments requires precise coordination between the nucleus and the cytoskeleton. Translocation of the nucleus through small pores is a limiting step in confined cell migration. Work in immune and cancer cells has illuminated a key role for actin polymerization and Myosin-II contractility in facilitating nucleus translocation through confinement. However, the microtubule cytoskeleton can also adaptively stabilise under confinement. Microtubule-dependent nucleokinesis constitutes an evolutionarily conserved cellular program ensuring directional nucleus translocation by deployment of cytoplasmic dynein motors. However, whether microtubule-driven nucleokinesis can operate during cell migration through confinement remained so far unexplored. The zebrafish neural crest (NC) provides a unique in vivo system to address this question. NC cells are a highly migratory, multipotent precursors of the vertebrate peripheral nervous system, which traverse diverse environments along the anterior-posterior axis of the embryo. In the head, cranial neural crest (cNC) cells migrate through loosely organised tissues, whereas trunk neural crest (tNC) cells navigate narrowly confined tissue spaces. Here, we show that tNC cells engage a microtubule motor-driven program of nuclear deformation and translocation during confined migration in vivo. Under confinement, tNC cells reorganize their microtubules from a perinuclear meshwork into a polarized, centrosome-associated bundle positioned ahead of the nucleus. Laser ablation reveals that this microtubule array actively pulls and deforms the nucleus. Using pharmacological and genetic approaches, we discover that dynein-dependent pulling forces enable nucleus translocation through confinement. Strikingly, this process occurs independently of Rho/ROCK/myosin II-mediated contractility, revealing a novel neuronal-like mode of nucleokinesis operating in confined tissue environments. Together, our findings uncover a conserved nuclear translocation program deployed during neural crest migration and suggest that microtubule-based nucleokinesis represents a fundamental strategy for navigating tissue confinement in vivo during both central and peripheral nervous system development.

Actin is critical for cell integrity and nuclear processes, but methods to visualise how environmental and intracellular signals affect cellular monomeric (G-) actin levels are lacking. We present the GASPs (G-actin sensing proteins), genetically encoded G-actin FRET sensors. Using cytoplasmic- and nuclear-localised GASPs, we show that in fibroblasts integrity of focal adhesions plays a critical role in maintaining low G-actin levels, and that cytoplasmic and nuclear G-actin levels increase at high cell densities and when spreading is restricted. Upon serum stimulation, it is depletion of the cytoplasmic G-actin pool that correlates with rapid nuclear accumulation of the MRTF transcription factors, whose subcellular localisation is controlled by G-actin. Stimuli that induce rapid increases or decreases in cytoplasmic G-actin level induce similar but slower changes in nuclear G-actin level, indicating that fluctuations in nuclear G-actin levels are largely subservient to cytoplasmic actin dynamics.

Organelle membranes employ diverse lipids to relay key signals for efficient coordination of cellular processes. Diacylglycerol (DAG) is a simple yet critical lipid secondary messenger, but the regulatory mechanisms and functional implications for its distribution remain poorly understood. We have recently shown that Protein Kinase C (Pkc1) activation is driven by selective DAGs (C36:0, C36:1), whose levels are governed by Disco-interacting protein 2 (Dip2) (Shambhavi et al., 2025). Here, through genetic, chemical, and lipidomic screens, we show that the absence of Dip2 leads to specific DAG accumulation on the mitochondrial membrane and impacts its morphology, function, and quality control in yeast. Remarkably, the elevated DAGs in{Delta} dip2 promote translocation of Pkc1 to mitochondria via its DAG-binding C1 domain, but not the HR1 domain, suggesting functional partitioning between the regulatory domains. We also show that only specific DAGs, not the bulk DAGs, are required for Pkc1s targeting and inactivating Phospholipase C (Plc1) restores Pkc1 localisation and the associated mitochondrial defects. In addition, we identify that respiratory growth triggers specific DAG (C36:1) accumulation in the mitochondria, thereby promoting Pkc1 recruitment. Furthermore, we establish that the Psi1-Plc1-Dip2 axis is required for survival under respiratory growth conditions. Taken together, our study uncovers a novel, Dip2-mediated, unconventional Pkc1 signalling axis for maintaining mitochondrial homeostasis under nutrient transition and highlights how distinct lipid fingerprints enable precise control over organellar homeostasis.

Polarized epithelia are essential for organ function, and disruption of epithelial polarity is a hallmark of many diseases, including cancer. We previously showed that the RhoG-specific guanine nucleotide exchange factor SGEF interacts with the Scribble polarity complex to regulate epithelial junction assembly in 2D monolayers. However, its role in epithelial morphogenesis and lumen formation in 3D remains unknown. Here, we combined quantitative morphometric analysis with long-term live-cell imaging to investigate the role of SGEF during MDCK cyst development. SGEF KD disrupted normal lumenogenesis, producing enlarged cysts with multiple collapsed lumens accompanied by reduced E-cadherin, {beta}-catenin, and ZO-1 expression. Loss of SGEF also altered the distribution of the actomyosin network. Re-expression of WT SGEF restored the normal phenotype, whereas restoration of E-cadherin and ZO-1 partially rescued lumen architecture, identifying the loss of junction integrity as a key driver of the morphogenetic defects. Unexpectedly, live-cell imaging revealed increased motility and frequent cyst fusion in SGEF-KD cysts. Restoring E-cadherin levels abolished cyst migration, while inhibition of matrix metalloproteinases markedly restored normal cyst volume and lumen architecture, identifying extracellular matrix remodeling as an additional contributor to the SGEF-deficient phenotype. Together, these findings identify SGEF as a key regulator of epithelial morphogenesis, coordinating junction integrity, actomyosin organization, lumen formation, and collective migration.

Microtubule dynamic instability, driven by GTP turnover, allows microtubules in cells to re-organise themselves adaptively. In some models of dynamic instability, GTP-tubulin is selectively captured at the tips of microtubules. In others, GTP- and GDP-tubulin are both captured, but GTP-tubulin is selectively retained. To investigate, we mutated the interprotofilament interface in human 1b{beta}3 and 1b{beta}4b tubulins, whose sequences diverge markedly in this region. We find that transplanting the 1b{beta}3 M-loop or its binding pocket into 1b{beta}4b tubulin creates tubulins that assemble in 1 mM GDP. In co-assembly experiments in GTP, such hyper-assembler mutants can recruit hypo-assembler mutants into a mosaic lattice, under conditions in which the hypo-assembler alone does not polymerise. We propose that GTP- and GDP-tubulins are captured equivalently at the tips of microtubules, but then differentially retained, based on their differing abilities to form stable interprotofilament bonds. This biased retention mechanism allows mosaic lattices to be built and dynamic instability to be tuned.

Programmed cell death is essential for organismal development. When dysregulated, it leads to neurodegenerative diseases and cancer. Although apoptotic pathways are well studied, the role of mitochondrial import translocases in regulating cell death remains unclear. Our study reveals a unique apoptotic pathway controlled by the TIM22 complex, an inner mitochondrial membrane translocase. This pathway involves a multiprotein complex formed by Tim22 and Nde1, a part of the respiratory electron transport chain. Under stress, the cytosol-exposed Nde1 isoform, a pro-apoptotic factor, is stabilised by the TIM22 complex, which includes the Tim18 subunit and Tim22s transmembrane segments. Notably, impairing the TIM22 complex and deleting Nde1 suppresses apoptosis and restores mitochondrial health. Beyond its role in import, our study uncovers a moonlighting function of the TIM22 complex in regulating mitochondria-dependent apoptotic cell death.

RASAL3 acts as a negative regulator of small cellular GTPases in hematopoietic cells. In immune cells, it primarily modulates the RAS/MAPK signaling pathway and affects cellular events including proliferation, differentiation, survival, and migration. Due to its inhibitory role in T cells, RASAL3 may represent a potential modulatory target for improving therapeutic strategies such as cell-based immunotherapy. However, most existing knowledge about RASAL3 function is derived from murine models, and its role in human T-cell signaling remains insufficiently characterized. To address this gap, we systematically investigated the function of RASAL3 in human primary T cells and T-cell line. For this purpose, we employed RASAL3 overexpression, CRISPR/Cas9-mediated deletion, and siRNA-mediated knockdown to thoroughly analyze the effects of RASAL3 on T-cell signaling, proliferation, and migration. Our data demonstrate that RASAL3 modulates primarily CDC42 and RAC1/RAC2 GTPases activity, SAPK/JNK phosphorylation, c-Fos and c-Jun expression, and IL-2 gene promoter activation. In addition, RASAL3 regulates actin polymerization and T-cell migration. Notably, loss of RASAL3 increases Jurkat T cells motility in vivo and potentiates their homing to the spleen. Collectively, these findings identify RASAL3 as an important regulator of human T-cell activation and motility and highlight its application potential for improving CAR-T cell therapy.

Transfection of cells with DNA plasmids typically involves the uptake of lipoparticles by endocytosis, followed by the inefficient escape of these particles from endosomes into the cytoplasm. We found that the expression of transfected plasmids was reduced in cells depleted of either SEPTIN 9 or proteins in the endosomal sorting complexes required for transport (ESCRT) pathway. The reduction in plasmid expression could not be fully explained by effects on endocytosis. SEPTIN 9 depletion appeared to reduce the acidification of plasmid-containing compartments, suggesting that it primarily affects the pH-sensitive escape of plasmids from endosomes. Depletion of the ESCRT proteins VPS36 or ALIX resulted in especially dramatic reductions in transfected plasmid expression, which were accompanied by reduced colocalization between the transfected DNA and CHMP4, an ESCRT protein important for endosomal membrane remodeling during intraluminal vesicle formation. Finally, transfected plasmid DNA was strongly colocalized with LC3B, suggesting that the default pathway for transfected material is autophagy.

Gasdermins (GSDMs) are a family of pore forming protein that trigger pyroptosis by permeabilizing cell membranes. Pyroptotic cells often release the proinflammatory cytokines interleukin-1{beta} (IL-1{beta}), and IL-18, thereby promoting an inflammatory response. GSDMs are typically cleaved by caspases or granzymes, which enable their translocation to the membrane. Here, we showed GSDMA and GSMDB localize to the cytoskeletal fraction of keratinocytes. Disruption of cell junctions causes gasdermin A and B (GSDMA and GSDMB) to translocate to the membrane fraction in the absence of cleavage. Cell junction disrupted keratinocytes release post-translationally modified keratins, but not IL-1{beta} or IL-18. These events depend on endocytic mechanisms associated with recycling of cell junctional proteins. Our study suggests that cell junction disruption can drive translocation of GSDMA and GSDMB from cytoskeleton to plasma membrane in keratinocytes, however there may be a subsequent trigger that causes the confirmational change allowing these gasdermins to form open pores.

Nuclear pore complexes (NPCs) control molecular exchange across the nuclear envelope, but how they tailor their selective permeability to meet the needs of specific cell types and/or environments remains poorly understood. We demonstrate that the strength of the NPC diffusion barrier differs across cell types, is particularly stringent in cultured neurons, and correlates with the O-linked N-acetylglucosamine (GlcNAc) modification of nucleoporins. Using conditional tools that specifically control nucleoporin GlcNAcylation, we show that GlcNAc modulates NPC permeability. Interestingly, nucleoporin GlcNAcylation is mechanosensitive, increasing in cells plated on stiff substrates, a condition where nuclear pores dilate. Indeed, we demonstrate that increasing or decreasing GlcNAcylation dilates and constricts NPCs, respectively. Further, O-linked N-acetylglucosamine transferase is recruited to modify NPCs upon their acute constriction during osmotic shock. Thus, cells employ GlcNAc to modulate steady-state NPC permeability in response to mechanical inputs and to counteract critical changes to their osmotic environment.

The polarization of motile cilia requires that the centrioles, from which cilia are formed, display rotational asymmetry. This property is manifested in the presence of asymmetrically distributed appendages and relies on evolutionary conserved mechanisms. These mechanisms are also at play in cells that form primary cilia despite the lack of ciliary motility and asymmetric centriole appendages in this context. Here, we find that a complex consisting of CCDC61, KIAA1328 (K1328), and Centlein (CNTLN) contributes to the establishment of centriole rotational asymmetry. In cells with a primary cilium, this complex is required for assembling a linker that repositions the daughter centriole close to and orthogonal to the proximal end of the mother centriole/basal body. The CCDC61/K1328/CNTLN complex also triggers the asymmetric recruitment of pericentriolar matrix components around newly assembled centrioles, which likely facilitates the later attachment of the basal body-daughter centriole linker. Overall, our results establish that rotational asymmetry relies on the coordinated recruitment of asymmetric landmarks along centrioles and is necessary for positioning the centrioles in a configuration that is widely conserved in ciliated cells.