Journal of Cell Science

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.

The axonemes of eukaryotic cilia and flagella display high tubulin glutamylation heterogeneity, yet the functional significance of this variation remains elusive. We previously showed that long-chain polyglutamylation is crucial for ciliary motility in Chlamydomonas. However, the respective contributions of long-chain polyglutamylation versus short-chain species to motility remain unclear, as existing mutants did not allow for a clear functional dissection of these two modification states. Here, we generated mutants deficient in deglutamylases, cytosolic carboxypeptidases (CCPs) 1, 2, and 5. Importantly, CCP5 is known to remove the branch-point glutamate residue, the final step in deglutamylation. While axonemal polyglutamylation levels remained largely unaffected in these mutants, abundance of short-chain glutamylation was significantly increased in both the axonemal and cytoplasmic microtubules of ccp5-1, consistent with CCP5s role as a branch-point deglutamylase. Although each single mutant exhibited slightly reduced swimming velocity, the loss of CCP5 in the tpg1 background lacking long polyglutamate side chains resulted in a significant restoration of motility. These findings indicate that the abundance of short-chain species, regulated by CCP5, plays a distinct role in modulating ciliary motility, particularly in the absence of long polyglutamate side chains. This suggests that even minimal glutamylation can functionally support dynein-driven microtubule sliding.

Subcellular architecture is tightly controlled and contributes to the maintenance of cells homeostasis. Organelles are regulated in size, shape, number and position which respond to changes in extracellular environment. Lysosomes are of particular interest as they integrate various functions in the cells (nutrient sensing, metabolism, cell migration and adhesion), serving as signaling hubs. Their function is tightly linked to their subcellular position and deregulation of lysosome homeostasis leads to several diseases including cancer. Therefore, methods allowing precise analysis of organelle subcellular distribution can aid in fundamental, diagnostic and therapeutic approaches. Here, we provide a versatile image analysis pipeline using ImageJ and CellProfiler. This workflow allows to quantify subcellular lysosome distribution in living and fixed melanoma cells, and is applicable to other subcellular compartments and to various cell types.

The axon initial segment (AIS), situated within the first 20-60 {micro}m of the axon, is essential for action potential generation and maintenance of axonal identity. Its structure relies on the beta ({beta})-IV-spectrin/AnkyrinG (AnkG) scaffold arranged periodically underneath the plasma membrane, harbouring diverse membrane proteins. Although a [~]190-nm cytoskeletal periodic organization is well established, the precise stoichiometry and spatial arrangement of AIS proteins within the [~]190-nm spatial period remain rudimentary, mostly for lack of sufficient spatial resolution and labelling efficiency. Here, using expansion microscopy and cryo-electron tomography, which overcome these technical limitations, we present data on the organization of the AnkG-associated complex within the [~]190-nm spatial period. We demonstrate that exactly two AnkG molecules with their C-termini separated by [~]80 nm are situated within each period. By contrast, the AnkG-associated cell-adhesion protein neurofascin-186 appears in clusters of varying sizes that are consistent with the periodic organisation of AnkG pairs, yet suggest a more complex molecular arrangement between the two molecules. Altogether, our novel approach provides new insights into AIS molecular organisation and protein stoichiometry.

Heterotrimeric kinesin 2 is the canonical motor protein for anterograde intraflagellar transport (IFT), driving movement of protein complexes towards the tip of cilia and flagella. Here, we show that all members of the Euglenozoa group lack genes for heterotrimeric kinesins and instead possess a variable number of genes for two homodimeric kinesins termed KIN2A and KIN2B. When expressed in vitro, both Trypanosoma brucei kinesins form homodimers and move processively along brain microtubules, KIN2A being faster than KIN2B. Studies in T. brucei and Leishmania mexicana show anterograde and retrograde IFT of both kinesins, with KIN2A travelling throughout the whole length of the flagellum, while KIN2B is concentrated at its base. In the proximal portion of the flagellum, most KIN2B molecules travel without IFT proteins, except for a few particles that are associated with IFT proteins and reach the tip. Surprisingly, the absence of KIN2A has mild effects on IFT and flagellum assembly, whereas KIN2B is essential for both. Investigation of trypanosome flagella deprived of KIN2B revealed that IFT proteins do not access these flagella but that KIN2A can still circulate. These results support a division-of-labour model where KIN2B is responsible for the import of IFT proteins while KIN2A is responsible for most of the anterograde transport.

Nucleoli and centromeres play essential roles in cellular proliferation and homeostasis, and are structurally and functionally interconnected. Centromeres frequently cluster around nucleoli, and some centromere assembly factors are known to reside in the nucleoli. To investigate the spatial and temporal relationships between these nuclear domains, we examined their dynamics in living cells. We imaged HeLa cells stably expressing mCherry-NPM1 and GFP-CENP-A using time-lapse microscopy. The results show that a subset of centromeres exhibits dynamic behavior during interphase, migrating over micrometer-scale distances within two hours. On average, 40-50% of centromeres maintain an association with nucleoli throughout interphase, with some cells displaying nucleolar-centromere association and dissociation within hours. Upon entry into mitosis, nucleoli are disassembled, and NPM1 localizes to the periphery of mitotic chromosomes. Nucleolar-centromere interactions are re-established in early G1, coinciding with the assembly of new centromeres. Treatment with actinomycin D, an inhibitor of RNA polymerase I, significantly reduces nucleolar size, nucleolar-centromere interactions, and centromere dynamics. Furthermore, post-mitotic nucleolar reformation is impaired. These findings highlight the dynamic nature of centromeres in interphase nuclei and their interactions with nucleoli. This behavior is partially dependent on rDNA transcription and nucleolar integrity, underscoring the critical roles of nucleoli, centromeres, and their interaction in 4D genome organization.

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.

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.

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

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.

Accurate chromosome segregation relies on proper centromere and kinetochore formation and phospho-regulation. We previously demonstrated that a pluripotent state confers a low fidelity of chromosome segregation, however it is unknown how a pluripotent state impacts centromere and kinetochore function. Here, we demonstrate that both centromere and kinetochore structural organization and phosphorylation in mitosis are developmentally regulated. CENP-A, CENP-C, and HEC1 protein abundance is reduced at mitotic centromeres and kinetochores of human pluripotent stem cells (hPSCs) compared to isogenic somatic cells; however, elevating their levels does not improve chromosome segregation fidelity. Rather, we find that reduced phosphorylation of kinetochores is responsible for their low fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs compared to isogenic somatic cells at Cyclin B/Cdk1 and Aurora kinase phospho-sites. Inhibiting PP2A phosphatase activity or differentiation increases HEC1 phosphorylation at hPSC kinetochores decreasing chromosome segregation errors. Thus, mitotic fidelity in non-transformed human cells depends on the developmental regulation of the kinase and phosphatase networks controlling kinetochore phosphorylation. SummaryGalaviz Sarmiento et al show that the developmental regulation of kinetochore phosphorylation governs mitotic fidelity. HEC1 is hypophosphorylated at kinetochores of hPSCs during mitosis contributing to their high rate of chromosome segregation errors. While differentiation increases HEC1 phosphorylation improving chromosome segregation fidelity.

Cadmium, being a highly toxic metal, perturbs cellular homeostasis by forming stable complexes with numerous thiol-active proteins, ultimately leading to severe liver and lung damage. Despite its well-documented toxicity, the molecular mechanisms governing cadmium export remain poorly understood. Given the chemical similarity between cadmium and copper, we investigated whether the canonical copper-exporting ATPases, ATP7A and ATP7B participate in cadmium handling. Upon Cd treatment in hepatocytes, ATP7B undergoes trafficking to lysosomes via the retromer complex, as also observed in the case of elevated copper, accompanied by the upregulation of acidic lysosomal populations. In contrast, ATP7A expressed in lung adenocarcinoma cells, though exhibit vesicular redistribution upon Cd exposure, does not mediate lysosomal sequestration, suggesting distinct deployment of late secretory pathways by the two copper ATPases in response to cadmium. We have also observed that ATP7B-/- hepatocytes exhibit increased sensitivity to Cd exposure compared to wild-type cells. Whereas, overexpressing the ATP7B amino-terminal copper-binding domain in bacteria alleviates cadmium-induced stress, indicating its capacity to sequester Cd. Caenorhabditis elegans lacking copper-ATPase cua-1, displayed increased Cd sensitivity, while mutants (glo-1-/-), deficient in lysosome-related organelles (LRO), and (lmp-1-/-), deficient in lysosomal membrane glycoprotein, showed reduced resistance to cadmium toxicity. Treatment of the worm with cadmium increases the abundance of lysosomes marked by elevation in lysosomal biogenesis and functional genes, reinforcing the importance of lysosomal pathways in cadmium detoxification. To summarise, we delineated the non-canonical role of copper ATPases and lysosomes in cadmium-induced cellular toxicity.

The integrity of the axoneme - the microtubule (MT)-based core of the cilium - and intraflagellar transport (IFT) are interdependent. The mechanisms determining axoneme structure and dynamics have remained largely unknown, especially in primary cilia with a more variable architecture and longer MT singlet parts. Using fluorescence imaging in the phasmid neurons of C. elegans, we here demonstrate that {beta}-tubulin isotype TBB-4 diffuses through the dendrite and employs a combination of anterograde IFT and diffusion to reach the sites of incorporation in the steady-state axoneme. Disrupting tubulins ability to bind to the IFT significantly reduces its share in the axoneme. We suggest that, in phasmid cilia, a constant supply of tubulin by IFT is required for steady-state length maintenance, in order to elevate soluble tubulin concentration near the axonemal tips and to promote MT stability.

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.

Proper connection between the sperm head and tail is critical for fertility and is mediated by the head-tail coupling apparatus (HTCA). Recent evidence suggests that the nuclear pore complex (NPC) may be important in male fertility, though a specific role at the HTCA has not been described. To investigate this, we performed a testis-specific RNAi screen targeting nucleoporins of the NPC. We identified Nup133 and Nup107 as regulators of HTCA development. We found that Nup133 and Nup107 were required to form the initial connection between the nucleus and centriole during HTCA establishment. We determined that failure to build the HTCA following Nup133 and Nup107 depletion was due to loss of nuclear envelope dynein/dynactin. Finally, we showed that loss of the NPC cytoplasmic filament component Nup358 results in the most severe centriole detachment phenotype, thus potentially functioning as the dynein anchor. Together, our data indicate that NPCs are critical regulators of early HTCA establishment and are required to recruit dynein to the nuclear envelope to bring the nucleus and centriole together during spermiogenesis.

Copines are a family of calcium-dependent phospholipid-binding proteins found in most eukaryotes. The expression of multiple copine genes is dysregulated in various types of human cancers. Despite this, a common mechanistic function for copines remains unknown. We are studying copines in Dictyostelium discoideum, which has six copine genes (cpnA-cpnF). Cells lacking cpnA or cpnC (cpnA- and cpnC-, respectively) exhibit many phenotypes, including defects in development, chemotaxis, adhesion, and contractile vacuole (CV) function. To further characterize the function of copines, this study tested the hypothesis that CpnD is responsible for cellular functions distinct from CpnA and CpnC. In this study, we obtained two cpnD mutants that were generated via restriction enzyme-mediated integration (REMI) mutagenesis; one in the first exon (cpnD(i291)), and one in the second exon (cpnD(i459)) of the endogenous cpnD gene. Throughout our experiments, we found that cpnD mutants had increased cellular proliferation in both axenic and bacterial cultures. Additionally, we found that cpnD mutants exhibited precocious development and had significantly larger fruiting bodies than the parental cell line. We further investigated the morphology of cpnD mutants and found that they were significantly larger than parental cells and exhibited decreased cell-substrate adhesion. cpnD mutants also had increased activated Ras compared to the parental cell line, along with significantly smaller CVs, a phenotype that was rescued after PI3K inhibition. Finally, we found that GFP-tagged CpnD localizes to the leading edge of both randomly migrating cells and in cells responding to folate. This study is the first to describe copine proteins as having a regulatory function in Ras activation and downstream signaling effects. Additionally, this study further supports our hypothesis that copines act as nonredundant cellular proteins in Dictyostelium to regulate numerous processes.

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.

T cell activation is associated with, and dependent upon, the upregulation of amino acid uptake from the extracellular environment. Uptake of the non-essential amino acid asparagine (Asn) is mediated via amino transporters such as Slc1a5 whilst Asn can be synthesized within cells that express asparagine synthetase (ASNS). Previous work demonstrated that initial activation of CD8+ T cells is perturbed in the absence of Asn, whereas effector cytotoxic T cells cells upregulate ASNS and lose their dependence on Asn uptake. By contrast, less is known of the role of Asn uptake and ASNS in CD4+ T cell responses. Here we demonstrate that CD4+ T cells are more reliant than CD8+ T cells on Asn uptake for initial activation, differentiation, metabolic reprogramming and regulation of autophagy. These phenotypes are associated with enhanced expression of ASNS in CD8+ as compared to CD4+ effector T cells.