Journal of Cell Science

Fibroblastic migration is of key importance in wound healing. While the intermediate filament (IF) protein vimentin is required for normal wound healing, we examined whether vimentin-mediated regulation of fibroblast migration could be involved. In wound healing assays triggering cell polarization and directed migration, we observed that vimentin-deficient mouse and rat embryonic fibroblasts lost their directional persistence. We show that vimentin maintains directionality by guiding focal adhesions (FAs) in fibroblasts. Detailed analysis showed that vimentin stabilizes FAs and regulates their disassembly rate. The destabilization of Vim-/- FAs was reflected by smaller FAs. Live cell and super-resolution imaging demonstrate that vimentin interacts dynamically with the key molecules of FAs and, importantly, with FAK, which is crucial for the maturation of FAs. These results demonstrate that vimentin IFs control the maturation, stability, dynamics, arrangement, and overall orientation of FAs, with a net effect on FA coordination during migration.

The small G-protein CDC42 is an evolutionary conserved polarity protein and a key regulator of numerous polarized cell functions, including directed cell migration. In vertebrates, alternative splicing gives rise to two CDC42 proteins: the ubiquitously expressed isoform (CDC42u) and the brain isoform (CDC42b), whose specific roles are not fully elucidated. The two isoforms only differ in their carboxy-terminal sequence, which includes the CAAX motif essential for CDC42 interaction with membrane. Here we show that these divergent sequences do not directly affect the range of CDC42s potential binding partners, but indirectly influence CDC42-driven signaling by controlling the specific subcellular localization of the two isoforms. In astrocytes and neural precursors, which naturally express both variants, CDC42u is mainly cytosolic and associates with the leading-edge plasma membrane of migrating cells where it recruits the Par6-PKC{zeta} complex to fulfill its polarity function. In contrast, CDC42b mainly localizes to intracellular membrane compartments, where it interacts with N-WASP. CDC42b does not participate in cell polarization but embodies the major isoform regulating endocytosis. Both CDC42 isoforms act in concert by contributing their specific functions to promote chemotaxis of neural precursors, demonstrating that the expression pattern of the two isoforms is decisive for the tissue-specific behavior of cells.

Laminin N-terminus 31 (LaNt 31), a member of the laminin superfamily, expressed at low levels in intact epithelium but upregulated during wound repair. Increased expression of LaNt 31 reduced migration rate of corneal keratinocytes through an unknown mechanism. Here, we investigated whether LaNt 31 influences cell behaviour through modulating laminin-mediated processes. Adenoviral delivery of LaNt 31 into corneal epithelial cells led to reduced migration speed and increased cell spreading and changed laminin 332 organisation from diffuse arcs to tight clusters. Enhanced recruitment of collagen XVII and bullous pemphigoid antigen 1e to {beta}4 integrin, indicating early maturation of hemidesmosomes, and changed focal adhesion distribution were also identified. LaNt 31 and laminin {beta}3 co-immunoprecipitated from doubly transduced cells and were deposited together in live imaging experiment. Moreover, LaNt 31 expression led to increased matrix metalloproteinase (MMP) activity and proteolytic processing of laminin 3, and the inhibition of MMP activity rescued the laminin and hemidesmosome phenotypes. Provision of cell-derived extracellular matrix rescued the cell spreading and motility effects. These findings reveal LaNt 31 as a new player in regulating cell-to-matrix adhesion through its ability to influence laminin organisation and proteolytic processing.

Cell invasion and metastasis is a multi-step process, initialised through the acquisition of a migratory phenotype and the ability to move through differing and complex 3D extracellular environments. In this study we set out to identify the parameters required for invasive cell migration in 3D environments. Cells interact with the extracellular matrix via transmembrane-spanning integrin adhesion complexes, which are well characterised in cells plated on 2D surfaces, yet much less is known about them in cells embedded in 3D matrices. We establish a technique to determine the composition of cell matrix adhesion complexes of invasive breast cancer cells in 3D matrices and on 2D surfaces and we identify an interaction complex enriched in 3D adhesive sites required for 3D invasive migration. Depletion of {beta}-PIX-Myosin18A (Myo18A) abolishes cancer cell invasion, without negatively affecting matrix degradation, Rho GTPase signalling, or protrusion formation in collagen matrices. Instead, in a mechanism only seen in cells moving through 3D matrix, {beta}-PIX and Myo18A drive the polarised recruitment of non-muscle Myosin 2A (NM2A) to the tips of protrusions. This recruitment of NM2A is required for the creation of an NM2A-NM2B isoform gradient, which ranges from the protrusion to the nucleus. We observe a requirement for active force transmission to the nucleus during invasive migration that is needed to pull the nucleus forward. We postulate that the establishment of the NM2A-NM2B actomyosin gradient facilitates the coupling of cell-matrix interactions at the protrusive cell front with nuclear movement, enabling effective invasive migration and front-rear cell polarity.

MT1-MMP plays a crucial role in promoting the cellular invasion of cancer cells by degrading the extracellular matrix to create a path for migration. During this process, its localization at the leading edge of migrating cells is critical, and it is achieved by targeted transport of MT1-MMP-containing vesicles along microtubules by kinesin superfamily proteins (KIFs). Here we identified three KIFs involved in MT1-MMP vesicle transport: KIF3A, KIF13A, and KIF9. Knockdown of KIF3A and KIF13A effectively inhibited MT1-MMP-dependent collagen degradation and invasion, while knockdown of KIF9 increased collagen degradation and invasion. Our data suggest that KIF9 competes with KIF3A/KIF13A to bring MT1-MMP vesicles to different locations in the plasma membrane. Live-cell imaging analyses have indicated that KIF3A and KIF13A coordinate to transport the same MT1-MMP-containing vesicles. Taken together, we have identified a unique interplay between three KIFs to regulate leading edge localization of MT1-MMP and MT1-MMP-dependent cancer cell invasion.

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

Cell adhesion to the substrate influences a variety of cell behaviors and its proper regulation is essential for migration, although details of the molecular pathways regulating cell adhesion during migration are lacking. Rap1 is a small GTPase that regulates adhesion in mammalian cells, as well as in Dictyostelium discoideum social amoeba, which is an established model for studying directed cell migration. In Dictyostelium, Rap1 controls adhesion via its effects on adhesion mediator talin and Ser/Thr kinase Phg2, which inhibits myosin II function. Kinase responsive to stress B (KrsB), a homolog of mammalian tumor suppressor MST1/2 and Drosophila Hippo, also regulates cell adhesion and migration, although the molecular mechanism of KrsB action is not understood. Since KrsB has been shown to interact with active Rap1 by mass spectroscopy, we investigated the genetic interaction between Rap1 and KrsB. Cells lacking KrsB have increased adhesion to the substrate, which leads to reduced movement. Expression of constitutively active Rap1 G12V increased cell spreading and adhesion even in the absence of KrsB, suggesting that Rap1 does not require KrsB to mediate cell adhesion. In contrast, dominant negative Rap1 S17N completely reversed the over-adhesive phenotype of KrsB-null cells and impaired KrsB phosphorylation, suggesting that KrsB activation and function in adhesion requires Rap1. Even though Rap1 did not require KrsB for its function in adhesion, KrsB negatively regulates Rap1 function as seen by increased cortical localization of Rap1 in KrsB-null cells. Consistent with this, chemoattractant-induced activation of downstream effectors of Rap1, TalB and Phg2, was increased in the absence of KrsB. Taken together, these findings suggest that Rap1 leads to activation of KrsB, which inhibits Rap1 and its downstream targets, shutting off adhesion. The existence of a negative feedback loop between Rap1 and KrsB may contribute to the dynamic regulation of cell adhesion that is necessary for rapid amoeboid-type migration.

The small GTPase Rho5 acts as a central hub to mediate the yeasts response to adverse environmental conditions, including oxidative stress, with the concomitant induction of mitophagy and apoptosis. A proper cellular stress response has been correlated with the rapid translocation of the GTPase to the mitochondria, which depends on its activating dimeric GDP/GTP exchange factor (GEF). Here, the small ALFA tag was attached to Rho5 or the GEF subunits Dck1 and Lmo1 to efficiently trap the functional fusion proteins to specific cellular membranes, i.e. the plasma membrane, the mitochon-drial outer membrane, or the nuclear membrane, via fusions of integral membrane proteins residing in these compartments with an ALFA nanobody. The trapped components were subjected to life-cell fluorescence microscopy in combination with GFP fusions of the GTPase or its GEF subunits to investigate their interaction in vivo. We found that the dimeric GEF tends to auto-assemble and form stable dimers independent of its intracellular localization. On the other hand, GFP-Rho5 does not stably colocalize with the trapped GEF, attributed to its transient interaction. Phenotypic analyses of strains with the misslocalized proteins indicate that for a proper oxidative stress response Lmo1 needs to associate with the plasma membrane. In contrast, Rho5 only exerts its role at the mitochondrial surface when it is there in its active conformation. These data underline the importance of the proper spatio-temporal distribution of Rho5-GTP during oxidative stress response.

Unc-51-like kinase (ULK) family serine-threonine protein kinase homologs have been linked to the function of motile cilia in diverse species. Mutations in Fused/STK36 and ULK4 in mice resulted in hydrocephalus and other phenotypes consistent with ciliary defects. How either protein contributes to the assembly and function of motile cilia is not well understood. Here we studied the phenotypes of ULK4 and Fused gene knockout (KO) mutants in the flagellated protist Leishmania mexicana. Both KO mutants exhibited a variety of structural defects of the flagellum cytoskeleton. Biochemical approaches indicate spatial proximity of these proteins and indicates a direct interaction between the N-terminus of LmxULK4 and LmxFused. Both proteins display a dispersed localisation throughout the cell body and flagellum, with enrichment near the flagellar base and tip. Fused/STK36 was previously shown to localise to mammalian motile cilia and we demonstrate here that ULK4 also localises to the motile cilia in mouse ependymal cells. Taken together these data suggest a model where the pseudokinase ULK4 is a positive regulator of the kinase Fused/STK36 in a pathway required for stable assembly of motile cilia. Summary StatementKnockout phenotypes in Leishmania, and confirmation of ULK4 ciliary localisation in mouse, show ULK4 and Fused/STK36 interact in a conserved pathway for stable assembly of motile cilia.

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.

Outer dynein arms (ODAs) are responsible for ciliary beating in eukaryotes. They are assembled in the cytoplasm and shipped by intraflagellar transport (IFT) before attachment to microtubule doublets via the docking complex. The LRRC56 protein has been proposed to contribute to ODAs maturation. Mutations or deletion of the LRRC56 gene lead to reduced ciliary motility in all species investigated so far, but with variable impact on dynein arm presence. Here, we investigated the role of LRRC56 in the protist Trypanosoma brucei, where its absence results in distal loss of ODAs, mostly in growing flagella. We show that LRRC56 is a transient cargo of IFT trains during flagellum construction and surprisingly, is required for efficient attachment of a subset of docking complex proteins present in the distal portion of the organelle. This relation is interdependent since the knockdown of the distal docking complex prevents LRRC56s association with the flagellum. Intriguingly, lrrc56-/- cells display shorter flagella whose maturation is delayed. Inhibition of cell division compensates for the distal ODAs absence thanks to the redistribution of the proximal docking complex, restoring ODAs attachment but not the flagellum length phenotype. This work reveals an unexpected connection between LRRC56 and the docking complex.

Controlling cell polarity and the directionality of cell motility is critical for effective cell migration during wound healing. NDR (nuclear dbf2-related) kinase pathways have roles in cell morphogenesis that are conserved from yeast to humans. Here, we reveal that knockdown of NDR1/2 kinases significantly alters cell size, shape, and the actin cytoskeleton, while reducing migration persistence and impairing cell polarization in wound healing assays. Mechanistically, we find that NDR1/2 kinases regulate the spatial and temporal dynamics of Cdc42 GTPase. Reduced NDR kinase levels increase Cdc42 GTPase activity and disrupt Pard3 subcellular location. NDR kinases phosphorylate Pard3 at Serine144, and overexpressing Pard3 can partially restore wound healing in NDR-depleted cells, an effect lost when Serine144 is mutated. Finally, we determine that NDR1 knockdown significantly impairs wound closure in human skin ex vivo wound healing assays, highlighting NDR kinase physiological importance. Collectively, this study demonstrates that NDR kinases modulate cell motility and polarization through the control of Pard3 and Cdc42 signaling in human fibroblasts.

Contractile actomyosin bundles play crucial roles in various physiological processes, including cell migration, morphogenesis, and muscle contraction. The intricate assembly of actomyosin bundles involves the precise alignment and fusion of myosin II filaments, yet the underlying mechanisms and factors involved in these processes remain elusive. Our study reveals that LUZP1, a leucine zipper protein, plays a central role in orchestrating the formation of thick actomyosin bundles. Loss of LUZP1 caused abnormal cell morphogenesis, migration, and the ability to exert forces on the environment. Importantly, knockout of LUZP1 results in significant defects in the concatenation and persistent association of myosin II filaments, severely impairing the assembly of myosin II stacks. The disruption of these processes in LUZP1 knockout cells provides mechanistic insights into the defective assembly of thick ventral stress fibers and the associated cellular contractility abnormalities. Overall, these results significantly contribute to our understanding of the molecular mechanism involved in actomyosin bundle formation and highlight the essential role of LUZP1 in this process.

Vinculin is a cytoskeletal linker strengthening cell adhesion. The Shigella IpaA invasion effector binds to vinculin to promote vinculin supra-activation associated with head-domain mediated oligomerization. Our study investigates the impact of mutations of vinculin D1D2 subdomains residues predicted to interact with IpaA VBS3. These mutations affected the rate of D1D2 trimer formation with distinct effects on monomer disappearance, consistent with structural modeling of a "closed" and "open" D1D2 conformer induced by IpaA. Notably, mutations targeting the closed D1D2 conformer significantly reduced Shigella invasion of host cells as opposed to mutations targeting the open D1D2 conformer and later stages of vinculin head-domain oligomerization. In contrast, all mutations affected the formation of focal adhesions (FAs), supporting the involvement of vinculin supra-activation in this process. Our findings suggest that IpaA-induced vinculin supra-activation primarily reinforces matrix adhesion in infected cells, rather than promoting bacterial invasion. Consistently, shear stress studies pointed to a key role for IpaA-induced vinculin supra-activation in accelerating and strengthening cell matrix adhesion.

Cell migration requires spatiotemporally coordinated activities of multicomponent structures including the actomyosin cortex, plasma membrane, adhesion complexes and the polarity proteins. How they function together to drive this complex dynamic process remains an outstanding question. Here, we show that a member of the protocadherin family, PCDH7 displays a polarized localization in migratory cells with a dynamic enrichment at the leading and rear edges. Perturbation of PCDH7 interferes with the migration of nontransformed retinal pigment epithelial cells and the invasion of cancer cells. The overexpression of PCDH7 enhances the migration capability of cortical neurons in vivo. PCDH7 interacts with the myosin phosphatase subunits MYPT1 and PP1c{beta}. Ectopic expression of PCDH7 enhances the MYPT1 inhibitory phosphorylation levels and the phosphorylation of the myosin regulatory light chain and ERM at the polarized cortex. The chemical inhibition of phosphatase activity recovers migration phenotypes of PCDH7 knockout cells. We propose that PCDH7 regulates phosphorylation thus the activity of myosin and ERM at the polarized cortex through its interaction with myosin phosphatase. Collectively, our study suggests a new component for the spatial coordination of the plasma membrane and the cortex during cell migration.

Construction of cilia and flagella relies on Intraflagellar Transport (IFT). Although IFT proteins can be found in multiple locations in the cell, transport has only been reported along the axoneme. Here, we reveal that IFT concentration at the base of the flagellum of Trypanosoma brucei is required for proper assembly of IFT trains. Using live cell imaging at high resolution and direct optical nanoscopy with axially localized detection (DONALD) of fixed trypanosomes, we demonstrate that IFT proteins are localised around the 9 doublet microtubules of the proximal portion of the transition zone, just on top of the transition fibres. Super-resolution microscopy and photobleaching studies reveal that knockdown of the RP2 transition fibre protein results in reduced IFT protein concentration and turnover at the base of the flagellum. This in turn is accompanied by a 4- to 8-fold drop in the frequency of IFT train injection. We propose that the flagellum base provides a unique environment to assemble IFT trains.

Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear if these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the Dictyostelium discoideum lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of Dictyostelium and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages.

Cell-matrix adhesion regulates Golgi organization through Arf1-mediated dynein recruitment, maintaining its juxtanuclear localization. On loss of adhesion, Arf1 activation drops, causing loss of dynein, promoting differential disorganization of cis- vs trans-Golgi along microtubules. Golgi regulates microtubule nucleation and stability. In fibroblasts, acetylated tubulin levels drop on loss of adhesion, recovering on re-adhesion with time. Active Arf1 overexpression in preventing Golgi disorganization sustains microtubule acetylation, also seen in T24 bladder cancer cells. Active Arf1 binds KIF5B, recruiting it to the Golgi. KIF5B and dynein knockdown disorganize the Golgi as ministacks, with cis- and trans-Golgi. Dynein knockdown disrupts MTOC positioning, causing ministacks to disperse, preventing Golgi reorganization upon re-adhesion. Dispersed ministacks interestingly maintain microtubule acetylation in adherent and non-adherent cells. The joint KIF5B-dynein knockdown causes the Golgi to lose its ribbon morphology, becoming compact while keeping cis- and trans-Golgi together. This also causes a change in spreading, aspect ratio and migration of knockdown cells, which could be regulated by their Golgi phenotype. In evaluating adhesion-dependent Golgi organization, we reveal the Arf1-KIF5B-dynein crosstalk to regulate Golgi-dependent tubulin acetylation and cell function. SummaryKIF5B and dynein are vital microtubule-associated motors that drive organelle positioning and organization. Adhesion-dependent Arf1 activation mediates KIF5B and dyneins recruitment to the Golgi, regulating its organization and position. This, in turn, regulates microtubule acetylation levels, localization, and cellular functions.

In order to invade 3D tissues, cancer cells dynamically change cell morphology in response to geometric and mechanical cues in the environment. But how cells determine their shape in 3D versus 2D environments is poorly understood. Studying 2D versus 3D single cell shape determination has historically been technically difficult due to the lack of methodologies to directly compare the two environments. We developed an approach to study cell shape in 2D versus 3D by measuring cell shape at different depths in collagen using stage-scanning oblique plane microscopy (ssOPM). We find characteristic shape changes occur in melanoma cells depending on whether a cell is attached to a 2D surface or 3D environment, and that these changes can be modulated by Rho GTPase regulatory proteins. Our data suggest that regulation of cell protrusivity undergoes a switch of control between different Rho GTPase regulators depending on the physical microenvironment.

Minus-end directed transport along microtubules in eukaryotes is primarily mediated by cytoplasmic dynein and its cofactor dynactin. Significant advances have been made in recent years characterizing human dynein-dynactin structure and function using in vitro assays, however, there is limited knowledge about the motile properties and functional organization of dynein-dynactin in living human cells. Total internal reflection fluorescence microscopy (TIRFM) of CRISPR-engineered human cells is employed here to visualize fluorescently tagged dynein heavy chain (DHC) and p50 with high spatio-temporal resolution. We find that p50 and DHC exhibit indistinguishable motility properties in their velocities, run lengths, and run times. The dynein-dynactin complexes are fast ([~]1.2 m/s) and run for several microns ([~]2.7 m). Quantification of the fluorescence intensities of motile puncta reveals that dynein-dynactin runs are mediated by at least one DHC dimer while the velocity is consistent with that measured for double dynein (two DHC dimers) complexes in vitro.