European Journal of Cell Biology

The polyadenylation binding protein nucleus 1 (PABPN1), a multifactorial regulator of mRNA processing, regulates muscle wasting and atrophy. Previously, we elucidated the PABPN1-dependent proteome and found that levels of structural proteins, sarcomeric and cytoskeletal, were highly altered. We identified MURC, a plasma membrane-associated protein, to be affected by the cytoskeletal stability and suggest that MURC is a novel marker for impaired regeneration in muscles. We also studied the spatial organization of muscle structural proteins in 2D and 3D cell models with reduced PABPN1 levels (named here as shPAB). We show that dysregulation of cytoskeletal proteins in the shPab proteome is associated with a cytoskeleton lacking a polarized organization in muscle cells. We show that consequently, the cell mechanical features as well as myogenic differentiation are significantly reduced. We then show that restoring cytoskeletal stability, by actin overexpression in shPAB was beneficial for cell fusion and for the expression of sarcomeric proteins in shPAB models. We suggest that poor cytoskeleton mechanical features are caused by altered expression levels and contribute to aging-associated muscle wasting and atrophy.

Various heterozygous cytoskeletal {gamma}-actin mutations have been shown to cause Baraitser-Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal {gamma}-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin {beta}4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q-filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q-filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin-affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover contribute to the observed disease phenotype.

Living cells require a dynamic and precisely regulated actin cytoskeleton to carry out normal cellular functions. In addition to well-established actin cytoskeleton regulators, such as nucleators, capping proteins, and bundlers, cells likely have uncharacterized modulators that regulate cytoskeleton dynamics, the detailed functions of which are not yet fully understood. In this study, we conducted biochemical exploration to identify the actin-regulatory activity of Kaptin (KPTN), a protein known to co-localize with actin-rich structures at the cells periphery. Using single-molecule assays, we demonstrated that KPTN inhibits actin nucleation. Our results revealed that KPTN possesses a novel barbed-end capping activity, which stabilizes and bundles actin filaments. Structural modeling, based on AlphaFold, suggests that KPTN is a member of the WD-repeat-containing protein family. Furthermore, we identified a crucial cationic residue in the putative N-terminal beta-propeller region of KPTN that plays a critical role in modulating actin dynamics. In summary, our data unveil the mechanistic underpinning functions of KPTN and establish its novel role as a regulator of the actin cytoskeleton.

Muscle stem cells (satellite cells) retain their identity and function through expression of the paired homeobox transcription factor PAX7. PAX7 is able to both stimulate satellite cell proliferation and activate target genes involved in establishing myogenic identity, including myogenic factor 5 (MYF5) and the other myogenic regulatory factors (MRFs). Upregulation of the MRFs promotes commitment to the muscle lineage by initiating withdrawal from the cell cycle, upregulating expression of muscle-specific transcripts, and directing myoblast fusion. We have previously shown that knockout of the N-terminal methyltransferase NRMT1 in C2C12 mouse myoblasts results in significantly decreased Pax7 expression, an inability of the cells to differentiate into myotubes, and abnormal upregulation of osteogenic markers. Here, we use RNA-sequencing to more comprehensively determine how loss of NRMT1 affects the transcriptional profile of proliferating and differentiating C2C12 myoblasts. We see that upon inducing differentiation, NRMT1 knockout cells can downregulate cell cycle, DNA replication, and histone gene expression. Though they also have significantly downregulated Pax7 and Myf5 expression, other muscle-specific transcripts are significantly increased over wild type, indicating the muscle transcriptional program is not completely inhibited. However, signaling pathways involved in the differentiation of other types of mesenchymal and hematopoietic lineages are also increased with NRMT1 loss and expression of chemotactic genes is downregulated. Together, these data indicate that NRMT1 knockout cells can upregulate genes needed for cell cycle withdrawal and muscle specification but fail to suppress markers of other lineages and activate normal chemotactic signaling, which may lead to the observed differentiation phenotypes.

The actin cytoskeleton is a potent regulator of tenocyte homeostasis. However, the mechanisms by which actin regulates tendon homeostasis are not entirely known. This study examined the regulation of tenocyte molecule expression by actin polymerization via the globular (G-) actin-binding transcription factor, myocardin-related transcription factor-a (MRTF). We determined that decreasing the proportion of G-actin in tenocytes by treatment with TGF{beta}1 increases nuclear MRTF. These alterations in actin polymerization and MRTF localization coincided with favorable alterations to tenocyte gene expression. In contrast, latrunculin A increases the proportion of G-actin in tenocytes and reduces nuclear MRTF, causing cells to acquire a tendinosis-like phenotype. To parse out the effects of F-actin depolymerization from regulation by MRTF, we treated tenocytes with cytochalasin D. Similar to latrunculin A treatment, exposure of cells to cytochalasin D increases the proportion of G-actin in tenocytes. However, unlike latrunculin A treatment, cytochalasin D increases nuclear MRTF. Compared to latrunculin A treatment, cytochalasin D led to opposing effects on the expression of a subset of genes. The differential regulation of genes by latrunculin A and cytochalasin D suggests that actin signals through MRTF to regulate a specific subset of genes. By targeting the deactivation of MRTF through the inhibitor CCG1423, we verify that MRTF regulates Type I Collagen, Tenascin C, Scleraxis, and -smooth muscle actin in tenocytes. Actin polymerization status is a potent regulator of tenocyte homeostasis through the modulation of several downstream pathways, including MRTF. Understanding the regulation of tenocyte homeostasis by actin may lead to new therapeutic interventions against tendinopathies, such as tendinosis.

Extracellular matrix (ECM) is the main component of cartilage, making it an ideal environment to study cell-matrix interactions. Among ECM constituents, heparan sulfate (HS)-carrying proteoglycans (PGs) are of particular interest since they are not only structural components but are also involved in cell matrix adhesion and signalling processes. We previously demonstrated that transgenic mice with a clonal loss of HS synthesis in chondrocytes (Col2-rtTA-Cre;Ext1e2fl/e2fl) develop clusters of enlarged cells in the articular cartilage (AC), which are surrounded by a glycosaminoglycan (GAG)-rich ECM. This led to the questions how HS regulate the molecular composition and mechanical properties of the ECM, how they sense alterations in the HS structure and how they respond to it. We stained tissue sections of Col2-rtTA-Cre;Ext1e2fl/e2f animals and detected increased levels of chondroitin sulfate (CS), Aggrecan (Acan), Perlecan (Pcan), Matrilin (Matn)-3 and-4, Collagen type II (Col2) and Col9, while Col12 was abolished in the HS-deficient clusters. We assessed the stiffness of the mutant matrix by Atomic Force Microscopy (AFM) and found that it was markedly softer than the surrounding, HS-containing tissue. Likely in response to this altered texture, HS-deficient clones showed increased protein levels of Integrin pathway components. To model a loss of HS-function in vitro, we treated murine embryonic fibroblasts (MEFs) with the HS-antagonist Surfen. Treatment during cell adhesion resulted in impaired cell-substrate adhesion, increased formation of filopodia-like membrane protrusions, decreased cell polarisation and migration, reduced formation of FA and SF, and a translocation of YAP into the cytoplasm. Similarly, we observed reduced cell polarisation in HS-deficient CHO pgsD-667 cells, which could not be rescued by external presentation of HS. When MEFs were treated with Surfen after the completion of the initial cell adhesion process, inhibition of HS-function led to an increased formation of FA and SF, in line with the increased levels of Integrin pathway components observed in HS-deficient chondrocytes in vivo. We detected high levels of Yes1-associated protein (YAP) in the HS-deficient clusters, and we investigated the effect of YAP modulation on high density micromass cultures from primary murine chondroprogenitors. YAP activation induced an increased GAG synthesis similar to Surfen, while YAP inactivation partially abolished the effect of Surfen, showing that YAP acts downstream of HS function and controls GAG synthesis. Taken together, we demonstrated that HS-function is essential for Integrin-dependent cell-matrix interactions. Information on the impaired cell matrix adhesion upon loss of HS is conveyed into the nucleus via YAP, which at least partially controls the synthesis of GAGs in chondrocytes.

Tunneling nanotubes (TNTs) are thin cytoplasmic protrusions involved in long-distance cellular communication. The presence of TNTs has been found in vivo and in vitro studies in non-small cell lung cancer (NSCLC). Cancer cells transport a range of organelles and signalling molecules along TNTs, to confer a survival phenotype for the recipient cell, contributing toward chemoresistance and malignancy. Despite its important role in cancer progression, the molecular mechanisms underlying TNT formation is not well defined. Within the tumour microenvironment (TME) of NSCLC, hepatocyte growth factor (HGF) and its receptor, c-Met, are mutationally upregulated causing growth, and invasion. In this study, we report a novel crosstalk between HGF/c-Met and {beta}1-integrin involved in the formation of functional TNTs in A549 cells. Through pharmacological inhibitor studies, we discovered Arp2/3 complex, MAPK and PI3K pathways were activated downstream of this crosstalk signalling axis. Furthermore, paxillin was recruited during this key process, localising at the protrusion site of HGF-induced TNTs, and therefore serving as the central link between the upstream and downstream regulators involved. Overall, these results demonstrate a novel strategy to inhibit TNT formation in NSCLC through targeting the HGF/c-Met and {beta}1-integrin signalling axis, thus highlighting the importance of personalised multi-drug targeting in NSCLC.

The cytoplasm exhibits viscoelastic properties, displaying both solid and liquid-like behavior, and can actively regulate its mechanical attributes. The cytoskeleton is a major regulator among the numerous factors influencing cytoplasmic mechanics. We explore the interdependence of various cytoskeletal filaments and the impact of their density on cytoplasmic viscoelasticity. The heterogeneous distribution of these filaments give rise to polarised mechanical properties of the cytoplasm along the apicobasal axis. Actin filament (F-actin) disassembly softens the basal cytoplasm while stiffening the mid-cytoplasm due to increased vimentin filament assembly. Disruption of microtubules (MT) or depletion of vimentin softens both the basal and mid-cytoplasm. Cyto D treatment results in localised increase of vimentin assembly in the mid cytoplasm which is dependent on the cytolinker plectin. Nocodazole treatment has a negligible effect on F-actin distribution but significantly alters vimentins spatial arrangement. We demonstrate that Cyto D treatment upregulates vimentin expression via ROS-mediated activation of NF-{kappa}B. This manuscript investigates how different cytoskeletal filaments influence the rheological characteristics of various cytoplasmic regions.

Cell migration is a fundamental process pertaining to many critical physiological events. The ability to form and release adhesion structures is necessary for cell migration. The Calpain family of cysteine proteases are known to target adhesion proteins as their substrates and modulate adhesion dynamics. The two best studied Calpains, Calpain 1 and Calpain 2 form catalytically active holoenzymes through heterodimerization with a common non-catalytic regulatory small subunit known as Calpain 4. In previous studies, we determined that calpains are important in the production of traction forces and in the sensing of localized mechanical stimulation from the external environment. We found that perturbation of either Calpain 1 or 2 had no effect on the generation of traction forces. However, traction forces were weak when Calpain 4 was silenced. On the other hand, silencing of Calpain 1, 2, or 4 resulted in deficient sensing of external mechanical stimuli. These results together suggest that Calpain 4 functions independent of the catalytic large subunits in the generation of traction forces but functions together with either catalytic subunit in sensing external mechanical stimuli. The small subunit Calpain 4 contains 268 a.a. and is composed of 2 domains, the N-terminal domain V and C-terminal domain VI. Domain VI is a calmodulinlike domain containing five consecutive EF-hand motifs, of which the fifth one heterodimerizes with a large subunit. Moreover, domain V contains the common sequence GTAMRILGGVI that suggests cell membrane interactions. Given these attributes of domain V and VI of Calpain 4, we speculated that an individual domain might provide the functional properties for either traction or sensing. Therefore, each domain was cloned and expressed individually in Capn4-/- cells and assayed for traction and sensing. Results revealed that over-expression of domain V was sufficient to rescue the traction forces defect in Capn4-/- cells while overexpression of domain VI did not rescue the traction force. Consistent with our hypothesis, overexpression of domain VI rescued the sensing defect in Capn4-/- cells while overexpression of domain V had no effect. These results suggest that individual domains of Calpain 4 do indeed function independently to regulate either traction force or the sensing of external stimuli. We speculate that membrane association of Calpain 4 is required for the regulation of traction force and its association with a catalytic subunit is necessary for mechanosensing.

Vinculin is a protein associated to linking adhesion receptors facing the outside of cells and reinforcing them by linking its intracellular domain of those receptors or, in the case of Cell-Matrix adhesions, to bind to a first level adaptor protein such as talin. The structural organization of vinculin allows it to bind on one part to specific amphipathic motifs collectively designated as vinculin binding sites (VBS), to a set of different vinculin coactivators or actin regulators, and finally a domain responsible to constantly bind to F-actin in a catch bond manner. However, the ability of vinculin to effectively bind all of those intracellular partners, is highly dependent on its structural organization. Which is critically dependent on its ability to respond to mechanical tension on the molecule itself and not necessarily to its binding capacity to VBSs and complementary activators. This is recognized as the combinatorial model of activation. Nonetheless, Shigellas IpaA effector protein is able to mimic the conformational changes associated with the ones associated with the mechanical deformation of the molecule. This model of vinculin activation is designated as the non-combinatorial model, as the presence of a single activation-partner is enough to get the same effect. This work is devoted to dig in further to develop the previous work from this lab, as we have been able to characterize the in vitro and in vivo effects of Shigellas IpaA-Cterm region as the one responsible for both inducing conformational changes in solution, as well as the formation of super-stable adhesion, associated to maturity markers as VASP and alpha actinin. Additionally the IpaA-Cterm transfection renders those cells with the ability to maintain the adhesion structures stable and even resist the action of actomyosin relaxing molecules. Which renders them as mechanically-independent adhesions. We found that residue substitution at the surface of D1 and D2 interphase, (as well as residues maintaining the D2 domain helical bundles folded), might participate in the maintaining the structural integrity and interdomain interaction during force dependent as revealed by its ability to form protein complexes in vitro and under force-independent settings, as the morphology of cellular adhesions is altered in a way different from the previously reported targeting only the D1-D5 interaction.

Traction force and mechanosensing (the ability to sense the mechanical attributes of the environment) are two key factors that enable a cell to modify its behavior during migration. Previously, it was determined that the calpain small subunit, calpain 4 (CapnS1), regulates the production of traction force independent of its proteolytic holoenzyme. A proteolytic enzyme is formed by calpain 4 binding to either of its catalytic partners, calpain 1 and 2. To further understand how calpain 4 regulates traction force, we used two-hybrid analysis to identify more components of the traction pathway. We discovered that basigin, an integral membrane protein and a documented inducer of matrix-metalloprotease (MMP), binds to calpain 4 in two-hybrid and pull-down assays. Traction force was deficient when basigin was silenced in MEF cells, and this deficiency was also reflected in the defect in substrate adhesion strength. Unlike Capn4-/- MEF cells, the cells deficient in basigin had normal mechanosensing abilities. Together, these results implicate basigin in the pathway in which calpain 4 regulates traction force independent of the catalytic large subunits.

Amyotrophic lateral sclerosis (ALS) is an irreversible neurodegenerative disease caused by the degeneration of motor neurons, and cytoskeletal instability is considered to be involved in neurodegeneration. A hexanucleotide repeat expansion of the C9orf72, one of the most common causes of familial ALS, produces toxic proline:arginine (PR) poly-dipeptides. PR poly-dipeptides binds polymeric forms of low complexity sequences and intracellular puncta, thereby altering intermediate filaments (IFs). However, how PR poly-dipeptides affect the cytoskeleton, including IFs, microtubules and actin filaments, remains unknown. Here we performed a synthetic PR poly-dipeptide treatment on mammalian cells and investigated how it affects morphology of cytoskeleton and cell behaviors. We observed that PR poly-dipeptide treatment induce the degradation of vimentin bundles at perinucleus and dissociation of {beta}-tubulin network. PR poly-dipeptides also lead to alteration of actin filaments toward to cell contours and strength cortical actin filaments via activation of ERM (ezrin/radixin/moesin) proteins. In addition, we found that PR poly-dipeptides promote phosphorylation of paxillin and recruitment of vinculin on focal adhesions, which lead to maturation of focal adhesions. Finally, we evaluated the effects of PR poly-dipeptides on mechanical property and stress response. Interestingly, treatment of PR poly-dipeptides increased the elasticity of the cell surface, leading to maladaptive response to cyclic stretch. These results suggest that PR poly-dipeptides cause mechanically sensitive structural reorganization and disrupt the cytoskeleton architecture.

Integrins bind to extracellular matrix proteins and, upon clustering, form multimolecular integrin adhesion complexes (IACs) that connect to and regulate the cell cytoskeleton, influencing various aspects of normal and tumour cell behaviour. Alongside well-characterized nascent adhesions, focal adhesions (FAs), fibrillar adhesions (FBs) and hemidesmosomes, a new class of IACs, reticular adhesions (RAs), have been identified. RAs, initially described as flat clathrin lattices formed by integrin V{beta}5, lack association with actin and are devoid of FAs markers. The physiological role of RAs in normal and tumor cells is still incompletely understood and requires further investigation. Previously, we analysed IACs of two melanoma cell lines, MDA-MB-435S and RPMI-7951, grown under long term culture conditions, and demonstrated that both cell lines preferentially use integrin V{beta}5 for adhesion. Here we present a comprehensive analysis of RAs in these two melanoma cell lines that differ in their ability to form FBs. To determine RAs composition, we treated cells with actin polymerisation inhibitor cytochalasin D (CytoD) which disrupts FAs, allowing isolation of RAs, which were analysed by MS-based proteomics, Western blotting and immunofluorescence. Known RA-associated proteins, including the AP-2 adaptor complex, disabled homolog 2 (DAB2) and Numb were identified in both lines, along with talin2. Notably, we also detected the presence of KN motif and ankyrin repeat domains protein (KANK2) in RA isolates. Proximity ligation analysis following CytoD-induced actin disruption confirmed the proximity of KANK2 and talin2 in RAs. We then investigated the effect of talin2 or KANK2 knockdown on RAs composition. While both talin2 and KANK2 are located in RAs, neither is essential for RA formation. Talin2 knockdown led to a reduction in RA components abundance in both cell lines. In MDA-MB-435S cell line, KANK2 produced a similar effect, mirroring the functional interaction of talin2 and KANK2 in FAs. However, in RPMI-7951 cells, KANK2 knockdown had no significant effect on RA components abundance. This discrepancy likely reflects the preferential localization of KANK2 in FBs and underscores the differing roles of talin2 and KANK2 in V{beta}5-mediated FAs across the two cell lines. These findings underscore the complexity of adhesion signalling and highlight the importance of adhesion crosstalk in regulating cellular function.

Formin1 (FMN1), a member of the non-diaphanous formin family, is essential for development and neuronal function. Unlike diaphanous-related formins, FMN1 is not subject to canonical autoinhibition through the DID and DAD domains, nor is it activated by Rho GTPase binding. Recent studies suggest that formins also play roles in the nucleus, influencing DNA damage response and transcriptional regulation. However, the mechanisms regulating nuclear formins particularly non-diaphanous ones like FMN1 remain poorly understood. Our previous research identified the interaction between FMN1 and FNBP4, prompting further investigation into its functional role in regulating actin dynamics. Results reveal that FNBP4 inhibits FMN1-mediated actin assembly in vitro. It is shown that FNBP4 prevents FMN1 from displacing the capping protein CapZ at the growing barbed end of actin filaments. Additionally, FNBP4 inhibits FMN1s bundling activity in a concentration-dependent manner. Further analysis indicates that FNBP4 interacts with the FH1 domain and the interdomain connector between the FH1 and FH2 domains, creating spatial constraints on the FH2 domain. We propose that FNBP4 acts as a stationary inhibitor of FMN1. In addition, we identify a monopartite nuclear localization signal (NLS) in FNBP4, and subcellular localization studies show that FNBP4 colocalizes with FMN1. This study provides new insights into the regulatory role of FNBP4 in modulating FMN1-mediated actin dynamics, suggesting that FNBP4 may function as a nuclear inhibitor of actin polymerization and shedding light on regulatory mechanisms specific to non-diaphanous formins.

The inhibitors, CK-666 and CK-869, are widely used to probe the function of actin nucleation by the Arp2/3 complex in vitro and in cells. However, in mammals, the Arp2/3 complex consists of 8 iso-complexes, as three of its subunits (Arp3, ArpC1, ArpC5) are encoded by two different genes. Here, we used recombinant Arp2/3 with defined composition to assess the activity of CK-666 and CK-869 against iso-complexes. We demonstrate that both inhibitors prevent linear actin filament formation when ArpC1A- or ArpC1B-containing complexes are activated by SPIN90. In contrast, inhibition of actin branching depends on iso-complex composition. Both drugs prevent actin branch formation by complexes containing ArpC1A, but only CK-869 can inhibit ArpC1B-containing complexes. Consistent with this, in bone marrow-derived macrophages which express low levels of ArpC1A, CK-869 but not CK-666, impacted phagocytosis and cell migration. CK-869 is also only able to inhibit Arp3-but not Arp3B-containing iso-complexes. Our findings have important implications for the interpretation of results using CK-666 and CK-869, given that the relative expression levels of ArpC1 and Arp3 isoforms in cells and tissues remains largely unknown.

In response to external mechanical stimuli, cells remodel their actin cytoskeleton. Solo, a Rho guanine nucleotide exchange factor (RhoGEF), is involved in mechanical stress responses. Using BioID, we identified PDZ-RhoGEF (PRG), a member of the RGS-RhoGEF family (regulator of G protein signaling domain-containing RhoGEFs, as a Solo-interacting protein. Moreover, we found that Solo regulates PRG during the mechanical stress response. Furthermore, we identified leukemia-associated RhoGEF (LARG), another RGS-RhoGEF member, as a Solo-interacting protein; however, the functional role of this interaction remains unknown. Therefore, in this study, we investigated the interaction between Solo and LARG and found that LARG localizes to Solo accumulation sites at the basal plane and that LARG is required for Solo-induced actin polymerization. Additionally, Solo is required to maintain LARG activity in cells, and this interaction is related to actin regulation in response to substrate stiffness. We further investigated the relationship between LARG and PRG as a function of Solo. We noted that although they did not competitively localize at Solo accumulation sites, knockdown of either PRG or LARG suppressed Solo-induced actin polymerization to the same extent as double knockdown, indicating that these signaling pathways cooperatively regulate Solo-induced actin polymerization.

It has been believed that platelet integrin IIb{beta}3 recognizes fibrinogen and several ECM proteins, and we recently showed that IIb{beta}3 binds to several inflammatory cytokines (e.g., CCL5, and CXCL12), which are stored in platelet granules. These ligands bind to the classical ligand (RGD)-binding site (site 1) of integrin IIb{beta}3. Also, they bind to the allosteric site (site 2) of IIb{beta}3, which is distinct from site 1, and allosterically activate IIb{beta}3. Site 2 is known to be involved in allosteric integrin activation and inflammatory signaling. FGF2 is also stored in platelet granules and known to be pro-thrombotic, but it is unclear if FGF2 binds to IIb{beta}3. We studied if FGF2 and its homologue FGF1 bind to IIb{beta}3 and induce allosteric activation. FGF1 (not stored in platelet granules) is known to be anti-thrombotic. Mechanism of FGF1s anti-thrombotic action is unknown. Here we describe that FGF1 and FGF2 bound to site 1 of IIb{beta}3, indicating that IIb{beta}3 is a new receptor for FGF1/2. Notably, FGF2 bound to site 2 and allosterically activated IIb{beta}3. Point mutations in the site 2-binding interface of FGF2 suppressed this activation, indicating that FGF2 binding to site 2 is required for activation (FGF2 is an agonist to site 2). In contrast, FGF1 bound to site 2 but did not activate IIb{beta}3, and instead suppressed integrin activation induced by FGF2, indicating that FGF1 acts as an antagonist of site 2. A non-mitogenic FGF1 mutant (R50E), which is defective in binding to site 1 of v{beta}3, suppressed IIb{beta}3 activation by FGF2 as effectively as WT FGF1. We propose that FGF1 R50E has therapeutic potential for anti-thrombosis.

Dystrophin is an essential muscle protein that contributes to cell membrane stability by linking the actin cytoskeleton to the extracellular matrix. The absence or impaired function of dystrophin causes muscular dystrophy. Focal adhesions are mechanosensitive adhesion complexes that also connect the cytoskeleton to the extracellular matrix. However, the interplay between dystrophin and focal adhesion force transmission has not been investigated. Using a bioluminescent tension sensor, we measured focal adhesion tension in transgenic C2C12 myoblasts expressing wild type (WT) dystrophin, a non-pathogenic SNP (I232M), or two missense mutations associated with Duchenne (L54R), or Becker muscular dystrophy (L172H). We found that myoblasts expressing WT or nonpathogenic I232M dystrophin showed increased focal adhesion tension compared to non-transgenic myoblasts, while myoblasts expressing L54R or L172H dystrophin presented with decreased focal adhesion tension. Moreover, myoblasts expressing L54R or L172H dystrophin showed decreased YAP activation and exhibited slower and less directional migration compared to cells expressing WT or I232M dystrophin. Our results suggest that disease-causing missense mutations in dystrophin may disrupt a cellular tension sensing pathway in dystrophic skeletal muscle.

Transforming growth factor beta (TGF{beta}) 1 regulates cell differentiation and proliferation in different physiological settings, but is also involved in fibrotic progression and protects tumors from the immune system. Integrin V{beta}6 has been shown to activate latent TGF{beta}1 by applying mechanical forces onto the latency-associated peptide (LAP). While the extracellular binding between V{beta}6 and LAP1 is well characterized, less is known about the cytoplasmic adaptations that enable V{beta}6 to apply such forces. Here, we generated new tools to facilitate the analysis of this interaction. We combined the integrin-binding part of LAP1 with a GFP and the Fc chain of human IgG. This chimeric protein, sLAP1, revealed a mechanical rearrangement of immobilized sLAP1 by V{beta}6 integrin. This unique interaction was not observed between sLAP1 and other integrins. We also analyzed V{beta}6 integrin binding to LAP2 and LAP3 by creating respective sLAPs. Compared to sLAP1, integrin V{beta}6 showed less binding to sLAP3 and no rearrangement. These observations indicate differences in the binding of V{beta}6 to LAP1 and LAP3 that have not been appreciated so far. Finally, V{beta}6-sLAP1 interaction was maintained even at strongly reduced cellular contractility, highlighting the special mechanical connection between V{beta}6 integrin and latent TGF{beta}1.

Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study was aimed at identifying Solo-interacting proteins using the BioID, a proximal-dependent labeling method and elucidating the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG co-localized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling and RhoA activation. Moreover, overexpression of the binding domains of Solo and PRG had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.