Cytoskeleton Complex Research Articles

SWR1-Independent Association of H2A.Z to the LINC Complex Promotes Meiotic Chromosome Motion.

The H2A.Z histone variant is deposited into the chromatin by the SWR1 complex, affecting multiple aspects of meiosis. We describe here a SWR1-independent localization of H2A.Z at meiotic telomeres and the centrosome. We demonstrate that H2A.Z colocalizes and interacts with Mps3, the SUN component of the linker of nucleoskeleton, and cytoskeleton (LINC) complex that spans the nuclear envelope and links meiotic telomeres to the cytoskeleton, promoting meiotic chromosome movement. H2A.Z also interacts with the meiosis-specific Ndj1 protein that anchors telomeres to the nuclear periphery via Mps3. Telomeric localization of H2A.Z depends on Ndj1 and the N-terminal domain of Mps3. Although telomeric attachment to the nuclear envelope is maintained in the absence of H2A.Z, the distribution of Mps3 is altered. The velocity of chromosome movement during the meiotic prophase is reduced in the htz1Δ mutant lacking H2A.Z, but it is unaffected in swr1Δ cells. We reveal that H2A.Z is an additional LINC-associated factor that contributes to promote telomere-driven chromosome motion critical for error-free gametogenesis.

Genetic loss of importin α4 causes abnormal sperm morphology and impacts on male fertility in mouse.

Importin α proteins play a central role in the transport of cargo from the cytoplasm to the nucleus. In this study, we observed that male knock-out mice for importin α4, which is encoded by the Kpna4 gene (Kpna4-/- ), were subfertile and yielded smaller litter sizes than those of wild-type (WT) males. In contrast, mice lacking the closely related importin α3 (Kpna3-/- ) were fertile. In vitro fertilization and sperm motility assays demonstrated that sperm from Kpna4-/- mice had significantly reduced quality and motility. In addition, acrosome reaction was also impaired in Kpna4-/- mice. Transmission electron microscopy revealed striking defects, including abnormal head morphology and multiple axoneme structures in the flagella of Kpna4-/- mice. A five-fold increase in the frequency of abnormalities in Kpna4-/- mice compared to WT mice indicates the functional importance of importin α4 in normal sperm development. Moreover, Nesprin-2, which is a component of the linker of nucleus and cytoskeleton complex, was expressed at lower levels in sperm from Kpna4-/- mice and was localized with abnormal axonemes, suggesting incorrect formation of the nuclear membrane-cytoskeleton structure during spermiogenesis. Proteomics analysis of Kpna4-/- testis showed significantly altered expression of proteins related to sperm formation, which provided evidence that genetic loss of importin α4 perturbed chromatin status. Collectively, these findings indicate that importin α4 is critical for establishing normal sperm morphology in mice, providing new insights into male germ cell development by highlighting the requirement of importin α4 for normal fertility.

The SUN1 splicing variants SUN1_888 and SUN1_916 differentially regulate nucleolar structure.

The nucleolar structure is highly dynamic and strictly regulated in response to internal cues, such as metabolic rates, and to external cues, such as mechanical forces applied to cells. Although the multilayered nucleolar structure is largely determined by the liquid-like properties of RNA and proteins, the mechanisms regulating the morphology and number of nucleoli remain elusive. The linker of the nucleoskeleton and cytoskeleton (LINC) complex comprises inner nuclear membrane Sad1/UNC-84 (SUN) proteins and outer nuclear membrane-localized nesprins. We previously showed that the depletion of SUN1 proteins affects nucleolar morphologies. This study focuses on the function of SUN1 splicing variants in determining nucleolar morphology. An RNA interference strategy showed that the predominantly expressed variants, SUN1_888 and SUN1_916, were crucial for nucleolar morphology but functionally distinct. In addition, the depletion of either SUN1_888 or SUN1_916 altered the chromatin structure and affected the distribution of histone modifications. Based on these results, we propose a model in which the LINC complex plays a role in modulating nucleolar morphology and numbers via chromatin.

Structural Analysis of Different LINC Complexes Reveals Distinct Binding Modes

Linker of nucleoskeleton and cytoskeleton (LINC) complexes are molecular tethers that span the nuclear envelope (NE) and physically connect the nucleus to the cytoskeleton. They transmit mechanical force across the NE in processes such as nuclear anchorage, nuclear migration, and homologous chromosome pairing during meiosis. LINC complexes are composed of KASH proteins traversing the outer nuclear membrane, and SUN proteins crossing the inner nuclear membrane. Humans have several SUN- and KASH-containing proteins, yet what governs their proper engagement is poorly understood. To investigate this question, we solved high resolution crystal structures of human SUN2 in complex with the KASH-peptides of Nesprin3, Nesprin4, and KASH5. In comparison to the published structures of SUN2-KASH1/2 we observe alternative binding modes for these KASH peptides. While the core interactions between SUN and the C-terminal residues of the KASH peptide are similar in all five complexes, the extended KASH-peptide adopts at least two different conformations. The much-improved resolution allows for a more detailed analysis of other elements critical for KASH interaction, including the KASH-lid and the cation loop, and a possible self-locked state for unbound SUN. In summary, we observe distinct differences between the examined SUN-KASH complexes. These differences may have an important role in regulating the SUN-KASH network.

FLN-1/filamin is required to anchor the actomyosin cytoskeleton and for global organization of sub-cellular organelles in a contractile tissue.

Actomyosin networks are organized in space, direction, size, and connectivity to produce coordinated contractions across cells. We use the C. elegans spermatheca, a tube composed of contractile myoepithelial cells, to study how actomyosin structures are organized. FLN-1/filamin is required for the formation and stabilization of a regular array of parallel, contractile, actomyosin fibers in this tissue. Loss of fln-1 results in the detachment of actin fibers from the basal surface, which then accumulate along the cell junctions and are stabilized by spectrin. In addition, actin and myosin are captured at the nucleus by the linker of nucleoskeleton and cytoskeleton complex (LINC) complex, where they form large foci. Nuclear positioning and morphology, distribution of the endoplasmic reticulum and the mitochondrial network are also disrupted. These results demonstrate that filamin is required to prevent large actin bundle formation and detachment, to prevent excess nuclear localization of actin and myosin, and to ensure correct positioning of organelles.

Mps2 links Csm4 and Mps3 to form a telomere-associated LINC complex in budding yeast.

The linker of the nucleoskeleton and cytoskeleton (LINC) complex is composed of two transmembrane proteins: the KASH domain protein localized to the outer nuclear membrane and the SUN domain protein to the inner nuclear membrane. In budding yeast, the sole SUN domain protein, Mps3, is thought to pair with either Csm4 or Mps2, two KASH-like proteins, to form two separate LINC complexes. Here, we show that Mps2 mediates the interaction between Csm4 and Mps3 to form a heterotrimeric telomere-associated LINC (t-LINC) complex in budding yeast meiosis. Mps2 binds to Csm4 and Mps3, and all three are localized to the telomere. Telomeric localization of Csm4 depends on both Mps2 and Mps3; in contrast, Mps2's localization depends on Mps3 but not Csm4. Mps2-mediated t-LINC complex regulates telomere movement and meiotic recombination. By ectopically expressing CSM4 in vegetative yeast cells, we reconstitute the heterotrimeric t-LINC complex and demonstrate its ability to tether telomeres. Our findings therefore reveal the heterotrimeric composition of the t-LINC complex in budding yeast and have implications for understanding variant LINC complex formation.

Apoptotic stress induces Bax-dependent, caspase-independent redistribution of LINC complex nesprins

The canonical function of Bcl-2 family proteins is to regulate mitochondrial membrane integrity. In response to apoptotic signals the multi-domain pro-apoptotic proteins Bax and Bak are activated and perforate the mitochondrial outer membrane by a mechanism which is inhibited by their interaction with pro-survival members of the family. However, other studies have shown that Bax and Bak may have additional, non-canonical functions, which include stress-induced nuclear envelope rupture and discharge of nuclear proteins into the cytosol. We show here that the apoptotic stimuli cisplatin and staurosporine induce a Bax/Bak-dependent degradation and subcellular redistribution of nesprin-1 and nesprin-2 but not nesprin-3, of the linker of nucleoskeleton and cytoskeleton (LINC) complex. The degradation and redistribution were caspase-independent and did not occur in Bax/Bak double knockout (DKO) mouse embryo fibroblasts (MEFs). Re-expression of Bax in Bax/Bak DKO MEFs restored stress-induced redistribution of nesprin-2 by a mechanism which requires Bax membrane localization and integrity of the α helices 5/6, and the Bcl-2 homology 3 (BH3) domain. We found that nesprin-2 interacts with Bax in close proximity to perinuclear mitochondria in mouse and human cells. This interaction requires the mitochondrial targeting and N-terminal region but not the BH3 domain of Bax. Our results identify nesprin-2 as a Bax binding partner and also a new function of Bax in impairing the integrity of the LINC complex.

Changes in Nuclear Shape and Gene Expression in Response to Simulated Microgravity Are LINC Complex-Dependent.

Microgravity is known to affect the organization of the cytoskeleton, cell and nuclear morphology and to elicit differential expression of genes associated with the cytoskeleton, focal adhesions and the extracellular matrix. Although the nucleus is mechanically connected to the cytoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, the role of this group of proteins in these responses to microgravity has yet to be defined. In our study, we used a simulated microgravity device, a 3-D clinostat (Gravite), to investigate whether the LINC complex mediates cellular responses to the simulated microgravity environment. We show that nuclear shape and differential gene expression are both responsive to simulated microgravity in a LINC-dependent manner and that this response changes with the duration of exposure to simulated microgravity. These LINC-dependent genes likely represent elements normally regulated by the mechanical forces imposed by gravity on Earth.

Isolated nuclei stiffen in response to low intensity vibration

The nucleus, central to all cellular activity, relies on both direct mechanical input and its molecular transducers to sense and respond to external stimuli. While it has been shown that isolated nuclei can adapt to applied force ex vivo, the mechanisms governing nuclear mechanoadaptation in response to physiologic forces in vivo remain unclear. To investigate nuclear mechanoadaptation in cells, we developed an atomic force microscopy (AFM) based procedure to probe live nuclei isolated from mesenchymal stem cells (MSCs) following the application of low intensity vibration (LIV) to determine whether nuclear stiffness increases as a result of LIV. Results indicated that isolated nuclei were, on average, 30% softer than nuclei tested within intact MSCs prior to LIV. When the nucleus was isolated following LIV (0.7 g, 90 Hz, 20 min) applied four times (4×) separated by 1 h intervals, stiffness of isolated nuclei increased 75% compared to non-LIV controls. LIV-induced nuclear stiffening required functional Linker of Nucleoskeleton and Cytoskeleton (LINC) complex, but was not accompanied by increased levels of the nuclear envelope proteins LaminA/C or Sun-2. While depleting LaminA/C or Sun-1&2 resulted in either a 47% or 39% increased heterochromatin to nuclear area ratio in isolated nuclei, the heterochromatin to nuclear area ratio was decreased by 25% in LIV-treated nuclei compared to controls, indicating LIV-induced changes in the heterochromatin structure. Overall, our findings indicate that increased apparent cell stiffness in response to exogenous mechanical challenge of MSCs in the form of LIV is in part retained by increased nuclear stiffness and changes in heterochromatin structure.

Regulation of myonuclear positioning and muscle function by the skeletal muscle-specific CIP protein

The appropriate arrangement of myonuclei within skeletal muscle myofibers is of critical importance for normal muscle function, and improper myonuclear localization has been linked to a variety of skeletal muscle diseases, such as centronuclear myopathy and muscular dystrophies. However, the molecules that govern myonuclear positioning remain elusive. Here, we report that skeletal muscle-specific CIP (sk-CIP) is a regulator of nuclear positioning. Genetic deletion of sk-CIP in mice results in misalignment of myonuclei along the myofibers and at specialized structures such as neuromuscular junctions (NMJs) and myotendinous junctions (MTJs) in vivo, impairing myonuclear positioning after muscle regeneration, leading to severe muscle dystrophy in mdx mice, a mouse model of Duchenne muscular dystrophy. sk-CIP is localized to the centrosome in myoblasts and relocates to the outer nuclear envelope in myotubes upon differentiation. Mechanistically, we found that sk-CIP interacts with the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex and the centriole Microtubule Organizing Center (MTOC) proteins to coordinately modulate myonuclear positioning and alignment. These findings indicate that sk-CIP may function as a muscle-specific anchoring protein to regulate nuclear position in multinucleated muscle cells.

Insights into eukaryogenesis from the fossil record.

Eukaryogenesis-the process by which the eukaryotic cell emerged-has long puzzled scientists. It has been assumed that the fossil record has little to say about this process, in part because important characters such as the nucleus and mitochondria are rarely preserved, and in part because the prevailing model of early eukaryotes implies that eukaryogenesis occurred before the appearance of the first eukaryotes recognized in the fossil record. Here, I propose a different scenario for early eukaryote evolution than is widely assumed. Rather than crown group eukaryotes originating in the late Paleoproterozoic and remaining ecologically minor components for more than half a billion years in a prokaryote-dominated world, I argue for a late Mesoproterozoic origin of the eukaryotic crown group, implying that eukaryogenesis can be studied using the fossil record. I review the proxy records of four crown group characters: the capacity to form cysts as evidenced by the presence of excystment structures; a complex cytoskeleton as evidenced by spines or pylomes; sterol synthesis as evidenced by steranes; and aerobic respiration-and therefore mitochondria-as evidenced by eukaryotes living in oxic environments, and argue that it might be possible to use these proxy records to infer the order in which these characters evolved. The records indicate that both cyst formation and a complex cytoskeleton appeared by late Paleoproterozoic time, and sterol synthesis appeared in the late Mesoproterozioc or early Neoproterozoic. The origin of aerobic respiration cannot as easily be pinned down, but current evidence permits the possibility that it evolved sometime in the Mesoproterozoic.

Mutated lamin A modulates stiffness in muscle cells

The cytoskeleton is a complex network interlinking filaments that extend throughout the cytoplasm from the nucleus to the plasma membrane. Three major types of filaments are found in the cytoskeleton: actin filaments, microtubules, and intermediate filaments. They play a key role in the ability of cells to both resist mechanical stress and generate force. However, the precise involvement of intermediate filament proteins in these processes remains unclear. Here, we focused on nuclear A-type lamins, which are connected to the cytoskeleton via the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Using micro-constriction rheology, we investigated the impact of A-type lamins (p.H222P) mutation on the mechanical properties of muscle cells. We demonstrate that the expression of point mutation of lamin A in muscle cells increases cellular stiffness compared with cells expressing wild type lamin A and that the chemical agent selumetinib, an inhibitor of the ERK1/2 signaling, reversed the mechanical alterations in mutated cells. These results highlight the interplay between A-type lamins and mechano-signaling, which are supported by cell biology measurements.

A Na,K-ATPase-Fodrin-Actin Membrane Cytoskeleton Complex is Required for Endothelial Fenestra Biogenesis.

Fenestrae are transcellular plasma membrane pores that mediate blood–tissue exchange in specialised vascular endothelia. The composition and biogenesis of the fenestra remain enigmatic. We isolated and characterised the protein composition of large patches of fenestrated plasma membrane, termed sieve plates. Loss-of-function experiments demonstrated that two components of the sieve plate, moesin and annexin II, were positive and negative regulators of fenestra formation, respectively. Biochemical analyses showed that moesin is involved in the formation of an actin–fodrin submembrane cytoskeleton that was essential for fenestra formation. The link between the fodrin cytoskeleton and the plasma membrane involved the fenestral pore protein PV-1 and Na,K-ATPase, which is a key regulator of signalling during fenestra formation both in vitro and in vivo. These findings provide a conceptual framework for fenestra biogenesis, linking the dynamic changes in plasma membrane remodelling to the formation of a submembrane cytoskeletal signalling complex.

The effect of endurance training on levels of LINC complex proteins in skeletal muscle fibers of STZ-induced diabetic rats

The changes of the linker of nucleoskeleton and cytoskeleton (LINC) complex have been studied in many muscular abnormality conditions; however, the effects of diabetes and physical activities on it have still remained to be defined. Therefore, the purpose of the this study was to evaluate the impacts of a six-week endurance training on the levels of SUN1 and Nesprin-1 proteins in Soleus and EDL muscles from diabetic wistar rats. A total number of 48 male Wistar rats (10 weeks, 200-250 gr) were randomly divided into healthy control (HC, N = 12), healthy trained (HT, N = 12), diabetic control (DC, N = 12), and diabetic trained (DT, N = 12) groups. Diabetes was also induced by a single intraperitoneally injection of streptozocin (45 mg/kg). The training groups ran a treadmill for five consecutive days within six weeks. The levels of the SUN1 and the Nesprin-1 proteins were further determined via ELISA method. The induction of diabetes had significantly decreased the levels of Nesprin-1 protein in the soleus and EDL muscles but it had no effects on the SUN1 in these muscles. As well, the findings revealed that six weeks of endurance training had significantly increased the levels of Nesprin-1 in DT and HT groups in the soleus as well as the EDL muscles; however, it had no impacts on the SUN1 in these muscles. The muscle fiber cross-sectional area (CSA) and myonuclei also decreased in diabetic control rats in both studied muscles. The training further augmented these parameters in both studied muscles in HT and DT groups. The present study provides new evidence that diabetes changes Nesprin-1 protein levels in skeletal muscle and endurance exercise training can modify it.

Predatory protists.

The vast majority of eukaryotic life is made up of single cells commonly referred to as protists. In this primer, Leander provides an introduction to predatory protists - cells that eat other cells. This lifestyle, in particular the use of phagocytosis, makes endosymbiosis possible and enabled the evolution of complex cells.

The testis-specific LINC component SUN3 is essential for sperm head shaping during mouse spermiogenesis

Sperm head shaping is a key event in spermiogenesis and is tightly controlled via the acrosome-manchette network. Linker of nucleoskeleton and cytoskeleton (LINC) complexes consist of Sad1 and UNC84 domain-containing (SUN) and Klarsicht/ANC-1/Syne-1 homology (KASH) domain proteins and form conserved nuclear envelope bridges implicated in transducing mechanical forces from the manchette to sculpt sperm nuclei into a hook-like shape. However, the role of LINC complexes in sperm head shaping is still poorly understood. Here we assessed the role of SUN3, a testis-specific LINC component harboring a conserved SUN domain, in spermiogenesis. We show that CRISPR/Cas9-generated Sun3 knockout male mice are infertile, displaying drastically reduced sperm counts and a globozoospermia-like phenotype, including a missing, mislocalized, or fragmented acrosome, as well as multiple defects in sperm flagella. Further examination revealed that the sperm head abnormalities are apparent at step 9 and that the sperm nuclei fail to elongate because of the absence of manchette microtubules and perinuclear rings. These observations indicate that Sun3 deletion likely impairs the ability of the LINC complex to transduce the cytoskeletal force to the nuclear envelope, required for sperm head elongation. We also found that SUN3 interacts with SUN4 in mouse testes and that the level of SUN4 proteins is drastically reduced in Sun3-null mice. Altogether, our results indicate that SUN3 is essential for sperm head shaping and male fertility, providing molecular clues regarding the underlying pathology of the globozoospermia-like phenotype.

Prophase-Specific Perinuclear Actin Coordinates Centrosome Separation and Positioning to Ensure Accurate Chromosome Segregation

SummaryCentrosome separation in late G2/ early prophase requires precise spatial coordination that is determined by a balance of forces promoting and antagonizing separation. The major effector of centrosome separation is the kinesin Eg5. However, the identity and regulation of Eg5-antagonizing forces is less well characterized. By manipulating candidate components, we find that centrosome separation is reversible and that separated centrosomes congress toward a central position underneath the flat nucleus. This positioning mechanism requires microtubule polymerization, as well as actin polymerization. We identify perinuclear actin structures that form in late G2/early prophase and interact with microtubules emanating from the centrosomes. Disrupting these structures by breaking the interactions of the linker of nucleoskeleton and cytoskeleton (LINC) complex with perinuclear actin filaments abrogates this centrosome positioning mechanism and causes an increase in subsequent chromosome segregation errors. Our results demonstrate how geometrical cues from the cell nucleus coordinate the orientation of the emanating spindle poles before nuclear envelope breakdown.

Spatiotemporal Mislocalization of Nuclear Membrane-Associated Proteins in γ-Irradiation-Induced Senescent Cells.

Cellular senescence, induced by genotoxic or replication stress, is accompanied by defects in nuclear morphology and nuclear membrane-heterochromatin disruption. In this work, we analyzed cytological and molecular changes in the linker of nucleoskeleton and cytoskeleton (LINC) complex proteins in senescence triggered by γ-irradiation. We used human mammary carcinoma and osteosarcoma cell lines, both original and shRNA knockdown clones targeting lamin B receptor (LBR) and leading to LBR and lamin B (LB1) reduction. The expression status and integrity of LINC complex proteins (nesprin-1, SUN1, SUN2), lamin A/C, and emerin were analyzed by immunodetection using confocal microscopy and Western blot. The results show frequent mislocalization of these proteins from the nuclear membrane to cytoplasm and micronuclei and, in some cases, their fragmentation and amplification. The timing of these changes clearly preceded the onset of senescence. The LBR deficiency triggered neither senescence nor changes in the LINC protein distribution before irradiation. However, the cytological changes following irradiation were more pronounced in shRNA knockdown cells compared to original cell lines. We conclude that mislocalization of LINC complex proteins is a significant characteristic of cellular senescence phenotypes and may influence complex events at the nuclear membrane, including trafficking and heterochromatin attachment.

Differentiating Luminal and Membrane-Associated Nuclear Envelope Proteins

The nuclear envelope (NE) consists of two concentric nuclear membranes separated by the lumen, an ∼40-nm-wide fluid layer. NE proteins are implicated in important cellular processes ranging from gene expression to nuclear positioning. Although recent progress has been achieved in quantifying the assembly states of NE proteins in their native environment with fluorescence fluctuation spectroscopy, these studies raised questions regarding the association of NE proteins with nuclear membranes during the assembly process. Monitoring the interaction of proteins with membranes is important because the binding event is often associated with conformational changes that are critical to cellular signaling pathways. Unfortunately, the close physical proximity of both membranes poses a severe experimental challenge in distinguishing luminal and membrane-associated NE proteins. This study seeks to address this problem by introducing new, to our knowledge, fluorescence-based assays that overcome the restrictions imposed by the NE environment. We found that luminal proteins violate the Stokes-Einstein relation, which eliminates a straightforward use of protein mobility as a marker of membrane association within the NE. However, a surprising anomaly in the temperature-dependent mobility of luminal proteins was observed, which was developed into an assay for distinguishing between soluble and membrane-bound NE proteins. We further introduced a second independent tool for distinguishing both protein populations by harnessing the previously reported undulations of the nuclear membranes. These membrane undulations introduce local volume changes that produce an additional fluorescence fluctuation signal for luminal, but not for membrane-bound, proteins. After testing both methods using simple model systems, we apply the two assays to investigate a previously proposed model of membrane association for the luminal domain of SUN2, a constituent protein of the linker of nucleoskeleton and cytoskeleton complex. Finally, we investigate the effect of C- and N-terminal tagging of the luminal ATPase torsinA on its ability to associate with nuclear membranes.

SUN1/2 Are Essential for RhoA/ROCK-Regulated Actomyosin Activity in Isolated Vascular Smooth Muscle Cells.

Vascular smooth muscle cells (VSMCs) are the predominant cell type in the blood vessel wall. Changes in VSMC actomyosin activity and morphology are prevalent in cardiovascular disease. The actin cytoskeleton actively defines cellular shape and the LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, comprised of nesprin and the Sad1p, UNC-84 (SUN)-domain family members SUN1/2, has emerged as a key regulator of actin cytoskeletal organisation. Although SUN1 and SUN2 function is partially redundant, they possess specific functions and LINC complex composition is tailored for cell-type-specific functions. We investigated the importance of SUN1 and SUN2 in regulating actomyosin activity and cell morphology in VSMCs. We demonstrate that siRNA-mediated depletion of either SUN1 or SUN2 altered VSMC spreading and impaired actomyosin activity and RhoA activity. Importantly, these findings were recapitulated using aortic VSMCs isolated from wild-type and SUN2 knockout (SUN2 KO) mice. Inhibition of actomyosin activity, using the rho-associated, coiled-coil-containing protein kinase1/2 (ROCK1/2) inhibitor Y27632 or blebbistatin, reduced SUN2 mobility in the nuclear envelope and decreased the association between SUN2 and lamin A, confirming that SUN2 dynamics and interactions are influenced by actomyosin activity. We propose that the LINC complex exists in a mechanical feedback circuit with RhoA to regulate VSMC actomyosin activity and morphology.