Bone mineral density and genetic markers involved in three connected pathways (focal adhesion,actin cytoskeleton regulation and cell cycle): the CUMAGAS-BMD information system

The focal adhesion, the actin cytoskeleton and cell-cycle are connected pathways and their genes are implicated in the pathogenesis of low BMD. Data from 211 studies that investigated the association between BMD and gene variants involved in these pathways were catalogued in a web-based information system and analyzed. In individual studies, significant association was found for 16 variants in lumbar spine, 11 in femoral neck and 5 in hip. In meta-analysis, significant results were shown for the variants COL1A1 rs1800012 (in lumbar spine and femoral neck), COL1A1 rs1107946 (in lumbar spine), TGFB1 rs1982073 (in femoral neck and hip) and TGFB1 rs1800469 (in lumbar spine).     

VASP-dependent regulation of actin cytoskeleton rigidity, cell adhesion, and detachment

Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins are established regulators of actin-based motility, platelet aggregation, and growth cone guidance. However, the molecular mechanisms involved essentially remain elusive. Here we report on a novel mechanism of VASP action, namely the regulation of tensile strength, contractility, and rigidity of the actin cytoskeleton. Compared to wild-type cells fibroblasts derived from VASP-deficient mice have thicker and more stable actin stress fibres. Furthermore focal adhesions are enlarged, myosin light chain phosphorylation is increased, and the rigidity of the filament-supported plasma membrane is elevated about three- to fourfold, as is evident from atomic force microscopy. Moreover, fibronectin-coated beads adhere stronger to the surface of VASP-deficient cells. The resistance of these beads to mechanical displacement by laser tweezers is dramatically increased in an F-actin-dependent mode. Cytoskeletal stabilization coincides with slower cell adhesion and detachment, while overall adhesion is increased. Interestingly, many of these effects observed in VASP (−/−) cells are recapitulated in VASP-overexpressing cells, hinting towards a balanced stoichiometry necessary for appropriate VASP function. Taken together, our results suggest that VASP regulates surface protrusion formation and cell adhesion through modulation of the mechanical properties of the actin cytoskeleton.Annette B. Galler, Maísa I. García Arguinzonis these authors contributed equally to this work     

Single cell rigidity sensing: A complex relationship between focal adhesion dynamics and large-scale actin cytoskeleton remodeling

ABSTRACT

Many physiological and pathological processes involve tissue cells sensing the rigidity of their environment. In general, tissue cells have been shown to react to the stiffness of their environment by regulating their level of contractility, and in turn applying traction forces on their environment to probe it. This mechanosensitive process can direct early cell adhesion, cell migration and even cell differentiation. These processes require the integration of signals over time and multiple length scales. Multiple strategies have been developed to understand force- and rigidity-sensing mechanisms and much effort has been concentrated on the study of cell adhesion complexes, such as focal adhesions, and cell cytoskeletons. Here, we review the major biophysical methods used for measuring cell-traction forces as well as the mechanosensitive processes that drive cellular responses to matrix rigidity on 2-dimensional substrates.     

Cadmium affects focal adhesion kinase (FAK) in mesangial cells: Involvement of CaMK‐II and the actin cytoskeleton

The toxic metal ion cadmium (Cd²⁺) induces pleiotropic effects on cell death and survival, in part through effects on cell signaling mechanisms and cytoskeletal dynamics. Linking these phenomena appears to be calmodulin‐dependent activation of the Ca²⁺/calmodulin‐dependent protein kinase II (CaMK‐II). Here we show that interference with the dynamics of the filamentous actin cytoskeleton, either by stabilization or destabilization, results in disruption of focal adhesions at the ends of organized actin structures, and in particular the loss of vinculin and focal adhesion kinase (FAK) from the contacts is a result. Low‐level exposure of renal mesangial cells to CdCl₂ disrupts the actin cytoskeleton and recapitulates the effects of manipulation of cytoskeletal dynamics with biological agents. Specifically, Cd²⁺ treatment causes loss of vinculin and FAK from focal contacts, concomitant with cytoskeletal disruption, and preservation of cytoskeletal integrity with either a calmodulin antagonist or a CaMK‐II inhibitor abrogates these effects of Cd²⁺. Notably, inhibition of CaMK‐II decreases the migration of FAK‐phosphoTyr925 to a membrane‐associated compartment where it is otherwise sequestered from focal adhesions in a Cd²⁺‐dependent manner. These results add further insight into the mechanism of the CaMK‐II‐dependent effects of Cd²⁺ on cellular function. J. Cell. Biochem. 114: 1832–1842, 2013. © 2013 Wiley Periodicals, Inc.     

The pleckstrin homology domain-containing protein CKIP-1 is involved in regulation of cell morphology and the actin cytoskeleton and interaction with actin capping protein

CKIP-1 is a pleckstrin homology domain-containing protein that interacts with protein kinase CK2. To elucidate the functions of CKIP-1, we generated human osteosarcoma cell lines with tetracycline-regulated expression of Flag-CKIP-1. Flag-CKIP-1 expression resulted in distinct changes in cellular morphology. Therefore, we examined the actin profile by immunofluorescence, quantitative measurement of phalloidin binding, and immunoblot analysis. These studies demonstrate that Flag-CKIP-1 expression resulted in increases in F-actin staining and protein levels of beta-actin. To elucidate the mechanisms behind the observed phenotype, we utilized tandem affinity purification to isolate CKIP-1 interacting proteins. Mass spectrometry analysis led to the identification of the actin capping protein subunits, CPalpha and CPbeta, as novel CKIP-1 interaction partners. Interactions were confirmed by coimmunoprecipitation and by colocalization. Furthermore, we demonstrate that Ser9 of CPalpha is phosphorylated by protein kinase CK2 in vitro, that CPalpha is phosphorylated in vivo, and that treatment with a CK2-specific inhibitor results in a decrease in CPalpha phosphorylation. Finally, we demonstrate that CKIP-1 and CK2 inhibit the activity of actin capping protein at the barbed ends of actin filaments. Overall, our results are consistent with CKIP-1 playing a role in the regulation of the actin cytoskeleton through its interactions with actin capping protein.     

Cellular contractility and substrate elasticity: a numerical investigation of the actin cytoskeleton and cell adhesion

Numerous experimental studies have established that cells can sense the stiffness of underlying substrates and have quantified the effect of substrate stiffness on stress fibre formation, focal adhesion area, cell traction, and cell shape. In order to capture such behaviour, the current study couples a mixed mode thermodynamic and mechanical framework that predicts focal adhesion formation and growth with a material model that predicts stress fibre formation, contractility, and dissociation in a fully 3D implementation. Simulations reveal that SF contractility plays a critical role in the substrate-dependent response of cells. Compliant substrates do not provide sufficient tension for stress fibre persistence, causing dissociation of stress fibres and lower focal adhesion formation. In contrast, cells on stiffer substrates are predicted to contain large amounts of dominant stress fibres. Different levels of cellular contractility representative of different cell phenotypes are found to alter the range of substrate stiffness that cause the most significant changes in stress fibre and focal adhesion formation. Furthermore, stress fibre and focal adhesion formation evolve as a cell spreads on a substrate and leading to the formation of bands of fibres leading from the cell periphery over the nucleus. Inhibiting the formation of FAs during cell spreading is found to limit stress fibre formation. The predictions of this mutually dependent material-interface framework are strongly supported by experimental observations of cells adhered to elastic substrates and offer insight into the inter-dependent biomechanical processes regulating stress fibre and focal adhesion formation.     

Classical cadherin adhesion molecules: coordinating cell adhesion, signaling and the cytoskeleton

Classical cadherin adhesion molecules are fundamental determinants of tissue organization in both health and disease. Recent advances in understanding the molecular and cellular basis of cadherin function have revealed that these adhesion molecules serve as molecular couplers, linking cell surface adhesion and recognition to both the actin cytoskeleton and cell signalling pathways. We will review some of these developments, to provide an overview of progress in this rapidly-developing area of cell and developmental biology.     

Small G Rac1 is involved in replication cycle of dengue serotype 2 virus in EAhy926 cells via the regulation of actin cytoskeleton

Bleeding is a clinical characteristic of severe dengue and may be due to increased vascular permeability. However, the pathogenesis of severe dengue remains unclear. In this study, we showed that the Rac1-microfilament signal pathway was involved in the process of DENV serotype 2 (DENV2) infection in EAhy926 cells. DENV2 infection induced dynamic changes in actin organization, and treatment with Cytochalasin D or Jasplakinolide disrupted microfilament dynamics, reduced DENV2 entry, and inhibited DENV2 assembly and maturation. Rac1 activities decreased during the early phase and gradually increased by the late phase of infection. Expression of the dominant-negative form of Rac1 promoted DENV2 entry but inhibited viral assembly, maturation and release. Our findings demonstrated that Rac1 plays an important role in the DENV2 life cycle by regulating actin reorganization in EAhy926 cells. This finding provides further insight into the pathogenesis of severe dengue.     

Stretch, tension and adhesion - adaptive mechanisms of the actin cytoskeleton in podocytes

Podocytes form an epithelial layer on the outer aspect of the basement membrane of glomerular capillaries. The interdigitating pattern of podocyte foot processes (PFPs) generates a unique and extremely long cell-cell contact area - the filtration slit. Thus, the interdigitating PFPs are the morphological basis for the high hydraulic conductivity of the glomerular capillaries. Any disturbance in this interdigitating pattern results in a drop of glomerular filtration rate impairing renal function. PFPs are based on the actin cytoskeleton, consisting of a subplasmalemmal network and a central core of filament bundles. Besides giving PFPs their morphology, the actin cytoskeleton anchors cell-cell contact and cell-matrix proteins in podocytes. Several human genetic diseases as well as transgenic mouse models provide evidence for the crucial role of the actin cytoskeleton in podocytes. Varying flow rates of the filtrate, increased glomerular capillary pressure in glomerular hypertension, and varying activation states of contractile proteins in PFPs impose a mechanical load on the actin cytoskeleton, challenging the intricate arrangement of PFPs and podocyte adhesion. Here we review data about the actin cytoskeleton of podocytes and the response of podocytes to mechanical load. From these data possible mechanisms are emerging how the actin cytoskeleton may allow podocytes to adapt to states of increased mechanical load.     

Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation

Cell-extracellular matrix adhesion is an important determinant of cell morphology. We show here that migfilin, a LIM-containing protein, localizes to cell-matrix adhesions, associates with actin filaments, and is essential for cell shape modulation. Migfilin interacts with the cell-matrix adhesion protein Mig-2 (mitogen inducible gene-2), a mammalian homolog of UNC-112, and the actin binding protein filamin through its C- and N-terminal domains, respectively. Loss of Mig-2 or migfilin impairs cell shape modulation. Mig-2 recruits migfilin to cell-matrix adhesions, while the interaction with filamin mediates the association of migfilin with actin filaments. Migfilin therefore functions as an important scaffold at cell-matrix adhesions. Together, Mig-2, migfilin and filamin define a connection between cell matrix adhesions and the actin cytoskeleton and participate in the orchestration of actin assembly and cell shape modulation.     

Invited review: focal adhesion and small heat shock proteins in the regulation of actin remodeling and contractility in smooth muscle.

Smooth muscle cells are able to adapt rapidly to chemical and mechanical signals impinging on the cell surface. It has been suggested that dynamic changes in the actin cytoskeleton contribute to the processes of contractile activation and mechanical adaptation in smooth muscle. In this review, evidence for functionally important changes in actin polymerization during smooth muscle contraction is summarized. The functions and regulation of proteins associated with "focal adhesion complexes" (membrane-associated dense plaques) in differentiated smooth muscle, including integrins, focal adhesion kinase (FAK), c-Src, paxillin, and the 27-kDa small heat shock protein (HSP27) are described. Integrins in smooth muscles are key elements of mechanotransduction pathways that communicate with and are regulated by focal adhesion proteins that include FAK, c-Src, and paxillin as well as proteins known to mediate cytoskeletal remodeling. Evidence that functions of FAK and c-Src protein kinases are closely intertwined is discussed as well as evidence that focal adhesion proteins mediate key signal transduction events that regulate actin remodeling and contraction. HSP27 is reviewed as a potentially significant effector protein that may regulate actin dynamics and cross-bridge function in response to activation of p21-activated kinase and the p38 mitogen-activated protein kinase signaling pathway by signaling pathways linked to integrin proteins. These signaling pathways are only part of a large number of yet to be defined pathways that mediate acute adaptive responses of the cytoskeleton in smooth muscle to environmental stimuli.     

Actin-binding protein G (AbpG) participates in modulating the actin cytoskeleton and cell migration in Dictyostelium discoideum

Cell migration is involved in various physiological and pathogenic events, and the complex underlying molecular mechanisms have not been fully elucidated. The simple eukaryote Dictyostelium discoideum displays chemotactic locomotion in stages of its life cycle. By characterizing a Dictyostelium mutant defective in chemotactic responses, we identified a novel actin-binding protein serving to modulate cell migration and named it actin-binding protein G (AbpG); this 971–amino acid (aa) protein contains an N-terminal type 2 calponin homology (CH2) domain followed by two large coiled-coil regions. In chemoattractant gradients, abpG⁻ cells display normal directional persistence but migrate significantly more slowly than wild-type cells; expressing Flag-AbpG in mutant cells eliminates the motility defect. AbpG is enriched in cortical/lamellipodial regions and colocalizes well with F-actin; aa 401–600 and aa 501–550 fragments of AbpG show the same distribution as full-length AbpG. The aa 501–550 region of AbpG, which is essential for AbpG to localize to lamellipodia and to rescue the phenotype of abpG⁻ cells, is sufficient for binding to F-actin and represents a novel actin-binding protein domain. Compared with wild-type cells, abpG⁻ cells have significantly higher F-actin levels. Collectively our results suggest that AbpG may participate in modulating actin dynamics to optimize cell locomotion.     

A critical role of tropomyosins in TGF-beta regulation of the actin cytoskeleton and cell motility in epithelial cells

We have investigated transforming growth factor beta (TGF-beta)-mediated induction of actin stress fibers in normal and metastatic epithelial cells. We found that stress fiber formation requires de novo protein synthesis, p38Mapk and Smad signaling. We show that TGF-beta via Smad and p38Mapk up-regulates expression of actin-binding proteins including high-molecular-weight tropomyosins, alpha-actinin and calponin h2. We demonstrate that, among these proteins, tropomyosins are both necessary and sufficient for TGF-beta induction of stress fibers. Silencing of tropomyosins with short interfering RNAs (siRNAs) blocks stress fiber assembly, whereas ectopic expression of tropomyosins results in stress fibers. Ectopic-expression and siRNA experiments show that Smads mediate induction of tropomyosins and stress fibers. Interestingly, TGF-beta induction of stress fibers was not accompanied by changes in the levels of cofilin phosphorylation. TGF-beta induction of tropomyosins and stress fibers are significantly inhibited by Ras-ERK signaling in metastatic breast cancer cells. Inhibition of the Ras-ERK pathway restores TGF-beta induction of tropomyosins and stress fibers and thereby reduces cell motility. These results suggest that induction of tropomyosins and stress fibers play an essential role in TGF-beta control of cell motility, and the loss of this TGF-beta response is a critical step in the acquisition of metastatic phenotype by tumor cells.     

Involvement of the actin cytoskeleton in the regulation of serotonin transporter (SET) activity: possible mechanism underlying SET regulation by protein kinase C

Our previous report has revealed that PKC activation by 12-O-tetradecanoylphorbol 13-acetate (TPA) inhibited the uptake activity of serotonin transporter (SET), via an indirect mechanism unknown, but not likely via direct phosphorylation of SET by PKC (Sakai et al., 1997. J. Neurochem. 68, 2618-2624). To elucidate whether PKC can directly phosphorylate SET in vivo, FLAG-tagged SET (FLAG-SET) was expressed in COS-7 cells and the TPA-induced incorporation of (32)P into immunoprecipitated FLAG-SET was examined. PKC activation with TPA caused no phosphorylation of FLAG-SET expressed in COS-7 cells. On the other hand, morphological change associated with the disruption of filamentous actin (F-actin) was seen in TPA-treated COS-7 cells. Therefore, we studied the effects of cytochalasin D, an inhibitor of actin polymerization, on the uptake activity of the serotonin transporter (SET) to elucidate whether the actin cytoskeleton modulates the SET uptake activity. The treatment with cytochalasin D inhibited the uptake activity of both native and recombinant SET in a concentration-dependent manner. Eadie-Hofstee analysis revealed that cytochalasin D down-regulated the recombinant SET uptake activity by reducing the V(max), but not the K(m), mimicking the result observed in TPA-induced inhibition of SET activity (Sakai et al., 1997. J. Neurochem. 68, 2618-2624). The cytochalasin D-induced inhibition of SET activity was partially, but significantly, reversed by jasplakinolide, a cell permeable stabilizer of F-actin, whereas TPA-induced inhibition of SET activity was not reversed by jasplakinolide. To elucidate whether the subcellular localization of SET was changed in response to cytochalasin D or TPA, we expressed the SET fused with the green fluorescent protein (SET-GFP) in COS-7 cells and observed the subcellular distribution of SET-GFP under a confocal laser scanning fluorescent microscope. Neither cytochalasin D nor TPA markedly changed the SET-GFP cellular localization, although these drugs caused morphological change in the GFP-transfected COS-7 cells. In addition, SET activity was not altered by the treatment with either colchicine, an inhibitor of microtubule polymerization, or taxol, a stabilizer of microtubule polymerization. These results suggest that the SET uptake activity was regulated by the state of the actin cytoskeleton and that TPA exerts its inhibitory action on SET activity, in part, via disruption of F-actin and subsequent morphological change in cells.     

Expression in an arbuscular mycorrhizal fungus of genes putatively involved in metabolism,transport, the cytoskeleton and the cell cycle

Arbuscular mycorrhizal (AM) fungi are multinucleate, coenocytic, obligate symbionts with no known sexual stages and very wide host and habitat ranges. While contributing vitally to the growth of land plants they face unique challenges in metabolism, transport, growth and development. To provide clues to the strategies that AM fungi have adopted, random sequencing of cDNA's from Glomus intraradices was undertaken. Putative genes for enzymes, transporters, structural proteins and cell-cycle regulatory factors were discovered. Among the EST's of particular interest are sequences with homology to known trehalase, arsenite transporter, cysteine synthase, tubulins, actin, dynein, cell cycle regulatory proteins, and three meiosis-related proteins. The significance of these sequences is discussed in the context of what is known about AM metabolism, transport, growth and phylogeny.