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Roland Kaunas - One of the best experts on this subject based on the ideXlab platform.
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Dependence of cyclic stretch-induced Stress Fiber reorientation on stretch waveform.
Journal of biomechanics, 2011Co-Authors: Abhishek Tondon, Hui-ju Hsu, Roland KaunasAbstract:Cyclic uniaxial stretching of adherent nonmuscle cells induces the gradual reorientation of their actin Stress Fibers perpendicular to the stretch direction to an extent dependent on stretch frequency. By subjecting cells to various temporal waveforms of cyclic stretch, we revealed that Stress Fibers are much more sensitive to strain rate than strain frequency. By applying asymmetric waveforms, Stress Fibers were clearly much more responsive to the rate of lengthening than the rate of shortening during the stretch cycle. These observations were interpreted using a theoretical model of networks of Stress Fibers with sarcomeric structure. The model predicts that stretch waveforms with fast lengthening rates generate greater average Stress Fiber tension than that generated by fast shortening. This integrated approach of experiment and theory provides new insight into the mechanisms by which cells respond to matrix stretching to maintain tensional homeostasis.
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Multiple Roles for Myosin II in Tensional Homeostasis Under Mechanical Loading
Cellular and Molecular Bioengineering, 2011Co-Authors: Roland Kaunas, Shinji DeguchiAbstract:Cyclic stretching of adherent cells regulates cell morphology, signal transduction and cell function. It is well established that actin Stress Fibers are mechano-sensitive structural elements that reorganize in response to applied Stress and strain. In this review, we discuss studies revealing the roles of myosin II in Stress Fiber remodeling including Stress Fiber assembly, disassembly, and tension maintenance. The results of these studies are interpreted with mathematical models that describe a mechanism by which Stress Fibers reorganize in response to cyclic stretch and predict how changes in Stress Fiber tension regulate signal transduction dependent on the spatial and temporal patterns of the applied strain.
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cyclic stretch induced Stress Fiber dynamics dependence on strain rate rho kinase and mlck
Biochemical and Biophysical Research Communications, 2010Co-Authors: Chinfu Lee, Candice Haase, Shinji Deguchi, Roland KaunasAbstract:Stress Fiber realignment is an important adaptive response to cyclic stretch for nonmuscle cells, but the mechanism by which such reorganization occurs is not known. By analyzing Stress Fiber dynamics using live cell microscopy, we revealed that Stress Fiber reorientation perpendicular to the direction of cyclic uniaxial stretching at 1 Hz did not involve disassembly of the Stress Fiber distal ends located at focal adhesion sites. Instead, these distal ends were often used to assemble new Stress Fibers oriented progressively further away from the direction of stretch. Stress Fiber disassembly and reorientation were not induced when the frequency of stretch was decreased to 0.01 Hz, however. Treatment with the Rho-kinase inhibitor Y27632 reduced Stress Fibers to thin Fibers located in the cell periphery which bundled together to form thick Fibers oriented parallel to the direction of stretching at 1 Hz. In contrast, these thin Fibers remained diffuse in cells subjected to stretch at 0.01 Hz. Cyclic stretch at 1 Hz also induced actin Fiber formation parallel to the direction of stretch in cells treated with the myosin light chain kinase (MLCK) inhibitor ML-7, but these Fibers were located centrally rather than peripherally. These results shed new light on the mechanism by which Stress Fibers reorient in response to cyclic stretch in different regions of the actin cytoskeleton.
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stretch induced Stress Fiber remodeling and the activations of jnk and erk depend on mechanical strain rate but not fak
PLOS ONE, 2010Co-Authors: Andrea K Locke, Susan Q Vanderzyl, Roland KaunasAbstract:Background Cells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin Stress Fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that Stress Fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized Stress Fiber organization and MAPK activities are altered to an extent dependent on stretch frequency. Principal Findings Bovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in Stress Fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting Stress Fiber organization. Disrupting Stress Fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced Stress Fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts. Conclusions These results indicate that cyclic stretch-induced Stress Fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in Fiber strain. These findings thus shed new light into the roles of Stress Fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment.
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a dynamic stochastic model of frequency dependent Stress Fiber alignment induced by cyclic stretch
PLOS ONE, 2009Co-Authors: Hui-ju Hsu, Chinfu Lee, Roland KaunasAbstract:Background Actin Stress Fibers (SFs) are mechanosensitive structural elements that respond to forces to affect cell morphology, migration, signal transduction and cell function. Cells are internally Stressed so that SFs are extended beyond their unloaded lengths, and SFs tend to self-adjust to an equilibrium level of extension. While there is much evidence that cells reorganize their SFs in response to matrix deformations, it is unclear how cells and their SFs determine their specific response to particular spatiotemporal changes in the matrix. Methodology/Principal Findings Bovine aortic endothelial cells were subjected to cyclic uniaxial stretch over a range of frequencies to quantify the rate and extent of Stress Fiber alignment. At a frequency of 1 Hz, SFs predominantly oriented perpendicular to stretch, while at 0.1 Hz the extent of SF alignment was markedly reduced and at 0.01 Hz there was no alignment at all. The results were interpreted using a simple kinematic model of SF networks in which the dynamic response depended on the rates of matrix stretching, SF turnover, and SF self-adjustment of extension. For these cells, the model predicted a threshold frequency of 0.01 Hz below which SFs no longer respond to matrix stretch, and a saturation frequency of 1 Hz above which no additional SF alignment would occur. The model also accurately described the dependence of SF alignment on matrix stretch magnitude. Conclusions The dynamic stochastic model was capable of describing SF reorganization in response to diverse temporal and spatial patterns of stretch. The model predicted that at high frequencies, SFs preferentially disassembled in the direction of stretch and achieved a new equilibrium by accumulating in the direction of lowest stretch. At low stretch frequencies, SFs self-adjusted to dissipate the effects of matrix stretch. Thus, SF turnover and self-adjustment are each important mechanisms that cells use to maintain mechanical homeostasis.
Pekka Lappalainen - One of the best experts on this subject based on the ideXlab platform.
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Calponin-3 is critical for coordinated contractility of actin Stress Fibers.
Scientific reports, 2018Co-Authors: Katarzyna Ciuba, Sari Tojkander, William Hawkes, Konstantin Kogan, Ulrike Engel, Thomas Iskratsch, Pekka LappalainenAbstract:Contractile actomyosin bundles, Stress Fibers, contribute to morphogenesis, migration, and mechanosensing of non-muscle cells. In addition to actin and non-muscle myosin II (NMII), Stress Fibers contain a large array of proteins that control their assembly, turnover, and contractility. Calponin-3 (Cnn3) is an actin-binding protein that associates with Stress Fibers. However, whether Cnn3 promotes Stress Fiber assembly, or serves as either a positive or negative regulator of their contractility has remained obscure. Here, we applied U2OS osteosarcoma cells as a model system to study the function of Cnn3. We show that Cnn3 localizes to both NMII-containing contractile ventral Stress Fibers and transverse arcs, as well as to non-contractile dorsal Stress Fibers that do not contain NMII. Fluorescence-recovery-after-photobleaching experiments revealed that Cnn3 is a dynamic component of Stress Fibers. Importantly, CRISPR/Cas9 knockout and RNAi knockdown studies demonstrated that Cnn3 is not essential for Stress Fiber assembly. However, Cnn3 depletion resulted in increased and uncoordinated contractility of Stress Fibers that often led to breakage of individual actomyosin bundles within the Stress Fiber network. Collectively these results provide evidence that Cnn3 is dispensable for the assembly of actomyosin bundles, but that it is required for controlling proper contractility of the Stress Fiber network.
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CaMKK2 Regulates Mechanosensitive Assembly of Contractile Actin Stress Fibers.
Cell reports, 2018Co-Authors: Sari Tojkander, Katarzyna Ciuba, Pekka LappalainenAbstract:Stress Fibers are contractile actomyosin bundles that guide cell adhesion, migration, and morphogenesis. Their assembly and alignment are under precise mechanosensitive control. Thus, Stress Fiber networks undergo rapid modification in response to changes in biophysical properties of the cell's surroundings. Stress Fiber maturation requires mechanosensitive activation of 5'AMP-activated protein kinase (AMPK), which phosphorylates vasodilator-stimulated phosphoprotein (VASP) to inhibit actin polymerization at focal adhesions. Here, we identify Ca2+-calmodulin-dependent kinase kinase 2 (CaMKK2) as a critical upstream factor controlling mechanosensitive AMPK activation. CaMKK2 and Ca2+ influxes were enriched around focal adhesions at the ends of contractile Stress Fibers. Inhibition of either CaMKK2 or mechanosensitive Ca2+ channels led to defects in phosphorylation of AMPK and VASP, resulting in a loss of contractile bundles and a decrease in cell-exerted forces. These data provide evidence that Ca2+, CaMKK2, AMPK, and VASP form a mechanosensitive signaling cascade at focal adhesions that is critical for Stress Fiber assembly.
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UNC-45a promotes myosin folding and Stress Fiber assembly.
The Journal of cell biology, 2017Co-Authors: Jaakko Ilmari Lehtimaki, Aidan M. Fenix, Tommi Kotila, Giuseppe Balistreri, Lassi Paavolainen, Markku Varjosalo, Dylan T. Burnette, Pekka LappalainenAbstract:Contractile actomyosin bundles, Stress Fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin Stress Fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in Stress Fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional Stress Fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.
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vimentin intermediate filaments control actin Stress Fiber assembly through gef h1 and rhoa
Journal of Cell Science, 2017Co-Authors: Yaming Jiu, Ramaswamy Krishnan, Johan Peranen, Niccole Schaible, Fang Cheng, John E Eriksson, Pekka LappalainenAbstract:The actin and intermediate filament cytoskeletons contribute to numerous cellular processes, including morphogenesis, cytokinesis and migration. These two cytoskeletal systems associate with each other, but the underlying mechanisms of this interaction are incompletely understood. Here, we show that inactivation of vimentin leads to increased actin Stress Fiber assembly and contractility, and consequent elevation of myosin light chain phosphorylation and stabilization of tropomyosin-4.2 (see Geeves et al., 2015). The vimentin-knockout phenotypes can be rescued by re-expression of wild-type vimentin, but not by the non-filamentous 'unit length form' vimentin, demonstrating that intact vimentin intermediate filaments are required to facilitate the effects on the actin cytoskeleton. Finally, we provide evidence that the effects of vimentin on Stress Fibers are mediated by activation of RhoA through its guanine nucleotide exchange factor GEF-H1 (also known as ARHGEF2). Vimentin depletion induces phosphorylation of the microtubule-associated GEF-H1 on Ser886, and thereby promotes RhoA activity and actin Stress Fiber assembly. Taken together, these data reveal a new mechanism by which intermediate filaments regulate contractile actomyosin bundles, and may explain why elevated vimentin expression levels correlate with increased migration and invasion of cancer cells.
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Palladin promotes assembly of non-contractile dorsal Stress Fibers through VASP recruitment.
Journal of cell science, 2014Co-Authors: Gergana Gateva, Sari Tojkander, Sami Koho, Olli Carpen, Pekka LappalainenAbstract:Stress Fibers are major contractile actin structures in non-muscle cells where they have an important role in adhesion, morphogenesis and mechanotransduction. Palladin is a multidomain protein, which associates with Stress Fibers in a variety of cell types. However, the exact role of palladin in Stress Fiber assembly and maintenance has remained obscure, and whether it functions as an actin filament crosslinker or scaffolding protein was unknown. We demonstrate that palladin is specifically required for the assembly of non-contractile dorsal Stress Fibers, and is, consequently, essential for the generation of Stress Fiber networks and the regulation of cell morphogenesis in osteosarcoma cells migrating in a three-dimensional collagen matrix. Importantly, we reveal that palladin is necessary for the recruitment of vasodilator stimulated phosphoprotein (VASP) to dorsal Stress Fibers and that it promotes Stress Fiber assembly through VASP. Both palladin and VASP display similar rapid dynamics at dorsal Stress Fibers, suggesting that they associate with Stress Fibers as a complex. Thus, palladin functions as a dynamic scaffolding protein that promotes the assembly of dorsal Stress Fibers by recruiting VASP to these structures.
Hui-ju Hsu - One of the best experts on this subject based on the ideXlab platform.
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Dependence of cyclic stretch-induced Stress Fiber reorientation on stretch waveform.
Journal of biomechanics, 2011Co-Authors: Abhishek Tondon, Hui-ju Hsu, Roland KaunasAbstract:Cyclic uniaxial stretching of adherent nonmuscle cells induces the gradual reorientation of their actin Stress Fibers perpendicular to the stretch direction to an extent dependent on stretch frequency. By subjecting cells to various temporal waveforms of cyclic stretch, we revealed that Stress Fibers are much more sensitive to strain rate than strain frequency. By applying asymmetric waveforms, Stress Fibers were clearly much more responsive to the rate of lengthening than the rate of shortening during the stretch cycle. These observations were interpreted using a theoretical model of networks of Stress Fibers with sarcomeric structure. The model predicts that stretch waveforms with fast lengthening rates generate greater average Stress Fiber tension than that generated by fast shortening. This integrated approach of experiment and theory provides new insight into the mechanisms by which cells respond to matrix stretching to maintain tensional homeostasis.
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a dynamic stochastic model of frequency dependent Stress Fiber alignment induced by cyclic stretch
PLOS ONE, 2009Co-Authors: Hui-ju Hsu, Chinfu Lee, Roland KaunasAbstract:Background Actin Stress Fibers (SFs) are mechanosensitive structural elements that respond to forces to affect cell morphology, migration, signal transduction and cell function. Cells are internally Stressed so that SFs are extended beyond their unloaded lengths, and SFs tend to self-adjust to an equilibrium level of extension. While there is much evidence that cells reorganize their SFs in response to matrix deformations, it is unclear how cells and their SFs determine their specific response to particular spatiotemporal changes in the matrix. Methodology/Principal Findings Bovine aortic endothelial cells were subjected to cyclic uniaxial stretch over a range of frequencies to quantify the rate and extent of Stress Fiber alignment. At a frequency of 1 Hz, SFs predominantly oriented perpendicular to stretch, while at 0.1 Hz the extent of SF alignment was markedly reduced and at 0.01 Hz there was no alignment at all. The results were interpreted using a simple kinematic model of SF networks in which the dynamic response depended on the rates of matrix stretching, SF turnover, and SF self-adjustment of extension. For these cells, the model predicted a threshold frequency of 0.01 Hz below which SFs no longer respond to matrix stretch, and a saturation frequency of 1 Hz above which no additional SF alignment would occur. The model also accurately described the dependence of SF alignment on matrix stretch magnitude. Conclusions The dynamic stochastic model was capable of describing SF reorganization in response to diverse temporal and spatial patterns of stretch. The model predicted that at high frequencies, SFs preferentially disassembled in the direction of stretch and achieved a new equilibrium by accumulating in the direction of lowest stretch. At low stretch frequencies, SFs self-adjusted to dissipate the effects of matrix stretch. Thus, SF turnover and self-adjustment are each important mechanisms that cells use to maintain mechanical homeostasis.
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A kinematic model of stretch-induced Stress Fiber turnover and reorientation.
Journal of theoretical biology, 2008Co-Authors: Roland Kaunas, Hui-ju HsuAbstract:A kinetic model based on constrained mixture theory was developed to describe the reorganization of actin Stress Fibers in adherent cells in response to diverse patterns of mechanical stretch. The model was based on reports that Stress Fibers are pre-extended at a "homeostatic" level under normal, non-perturbed conditions, and that perturbations in Stress Fiber length destabilize Stress Fibers. In response to a step change in matrix stretch, the model predicts that Stress Fibers are initially stretched in registry with the matrix, but that these overly stretched Fibers are gradually replaced by new Fibers assembled with the homeostatic level of stretch in the new configuration of the matrix. In contrast, average Fiber stretch is chronically perturbed from the homeostatic level when the cells are subjected to cyclic equibiaxial stretch. The model was able to describe experimentally measured time courses of Stress Fiber reorientation perpendicular to the direction of cyclic uniaxial stretch, as well as the lack of alignment in response to equibiaxial stretch. The model also accurately described the relationship between stretch magnitude and the extent of Stress Fiber alignment in endothelial cells subjected to cyclic uniaxial stretch. Further, in the case of cyclic simple elongation with transverse matrix contraction, Stress Fibers orient in the direction of least perturbation in stretch. In summary, the model predicts that the rate of stretch-induced Stress Fiber disassembly determines the rate of alignment, and that Stress Fibers tend to orient toward the direction of minimum matrix stretch where the rate of Stress Fiber turnover is a minimum.
Shu Chien - One of the best experts on this subject based on the ideXlab platform.
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regulation of stretch induced jnk activation by Stress Fiber orientation
Cellular Signalling, 2006Co-Authors: Roland Kaunas, Shunichi Usami, Shu ChienAbstract:Cyclic mechanical stretch associated with pulsatile blood pressure can modulate cytoskeletal remodeling and intracellular signaling in vascular endothelial cells. The aim of this study was to evaluate the role of stretch-induced actin Stress Fiber orientation in intracellular signaling involving the activation of c-jun N-terminal kinase (JNK) in bovine aortic endothelial cells. A stretch device was designed with the capability of applying cyclic uniaxial and equibiaxial stretches to cultured endothelial cells, as well as changing the direction of cyclic uniaxial stretch. In response to 10% cyclic equibiaxial stretch, which did not result in Stress Fiber orientation, JNK activation was elevated for up to 6 h. In response to 10% cyclic uniaxial stretch, JNK activity was only transiently elevated, followed by a return to basal level as the actin Stress Fibers became oriented perpendicular to the direction of stretch. After the Stress Fibers had aligned perpendicularly and the JNK activity had subsided, a 90° change in the direction of cyclic uniaxial stretch reactivated JNK, and this activation again subsided as Stress Fibers became re-oriented perpendicular to the new direction of stretch. Disrupting actin filaments with cytochalasin D blocked the Stress Fiber orientation in response to cyclic uniaxial stretch and it also caused the uniaxial stretch-induced JNK activation to become sustained. These results suggest that Stress Fiber orientation perpendicular to the direction of stretch provides a mechanism for both structural and biochemical adaptation to cyclic mechanical stretch.
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cooperative effects of rho and mechanical stretch on Stress Fiber organization
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Roland Kaunas, Shunichi Usami, Phu Nguyen, Shu ChienAbstract:The small GTPase Rho regulates the formation of actin Stress Fibers in adherent cells through activation of its effector proteins Rho-kinase and mDia. We found in bovine aortic endothelial cells that inhibitions of Rho, Rho-kinase, and mDia (with C3, Y27632, and F1F2Δ1, respectively) suppressed Stress Fiber formation, but Fibers appeared after 10% cyclic uniaxial stretch (1-Hz frequency). In contrast to the predominately perpendicular alignment of Stress Fibers to the stretch direction in normal cells, the Stress Fibers in cells with Rho pathway inhibition became oriented parallel to the stretch direction. In cells with normal Rho activity, the extent of perpendicular orientation of Stress Fibers depended on the magnitude of stretch. Expressing active RhoV14 plasmid in these cells enhanced the stretch-induced Stress Fiber orientation by an extent equivalent to an additional ≈3% stretch. This augmentation of the stretch-induced perpendicular orientation by RhoV14 was blocked by Y27632 and by F1F2Δ1. Thus, the activity of the Rho pathway plays a critical role in determining both the direction and extent of stretch-induced Stress Fiber orientation in bovine aortic endothelial cells. Our results demonstrate that the stretch-induced Stress Fiber orientation is a function of the interplay between Rho pathway activity and the magnitude of stretching.
Ulla M Wewer - One of the best experts on this subject based on the ideXlab platform.
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adam12 syndecan 4 signaling promotes beta 1 integrin dependent cell spreading through protein kinase calpha and rhoa
Journal of Biological Chemistry, 2003Co-Authors: Charles Kumar Thodeti, Reidar Albrechtsen, Morten Grauslund, Meena Asmar, Yoshikazu Takada, Arthur M Mercurio, John R Couchman, Christer Larsson, Ulla M WewerAbstract:Abstract The ADAMs (a disintegrinand metalloprotease) comprise a large family of multidomain proteins with cell-binding and metalloprotease activities. The ADAM12 cysteine-rich domain (rADAM12-cys) supports cell attachment using syndecan-4 as a primary cell surface receptor that subsequently triggers β1integrin-dependent cell spreading, Stress Fiber assembly, and focal adhesion formation. This process contrasts with cell adhesion on fibronectin, which is integrin-initiated but syndecan-4-dependent. In the present study, we investigated ADAM12/syndecan-4 signaling leading to cell spreading and Stress Fiber formation. We demonstrate that syndecan-4, when present in significant amounts, promotes β1 integrin-dependent cell spreading and Stress Fiber formation in response to rADAM12-cys. A mutant form of syndecan-4 deficient in protein kinase C (PKC)α activation or a different member of the syndecan family, syndecan-2, was unable to promote cell spreading. GF109203X and Go6976, inhibitors of PKC, completely inhibited ADAM12/syndecan-4-induced cell spreading. Expression of syndecan-4, but not syn4ΔI, resulted in the accumulation of activated β1 integrins at the cell periphery in Chinese hamster ovary β1 cells as revealed by 12G10 staining. Further, expression of myristoylated, constitutively active PKCα resulted in β1 integrin-dependent cell spreading, but additional activation of RhoA was required to induce Stress Fiber formation. In summary, these data provide novel insights into syndecan-4 signaling. Syndecan-4 can promote cell spreading in a β1 integrin-dependent fashion through PKCα and RhoA, and PKCα and RhoA likely function in separate pathways.
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adam12 syndecan 4 signaling promotes beta 1 integrin dependent cell spreading through protein kinase calpha and rhoa
Journal of Biological Chemistry, 2003Co-Authors: Charles Kumar Thodeti, Reidar Albrechtsen, Morten Grauslund, Meena Asmar, Yoshikazu Takada, Arthur M Mercurio, John R Couchman, Christer Larsson, Ulla M WewerAbstract:The ADAMs (a disintegrin and metalloprotease) comprise a large family of multidomain proteins with cell-binding and metalloprotease activities. The ADAM12 cysteine-rich domain (rADAM12-cys) supports cell attachment using syndecan-4 as a primary cell surface receptor that subsequently triggers beta(1) integrin-dependent cell spreading, Stress Fiber assembly, and focal adhesion formation. This process contrasts with cell adhesion on fibronectin, which is integrin-initiated but syndecan-4-dependent. In the present study, we investigated ADAM12/syndecan-4 signaling leading to cell spreading and Stress Fiber formation. We demonstrate that syndecan-4, when present in significant amounts, promotes beta(1) integrin-dependent cell spreading and Stress Fiber formation in response to rADAM12-cys. A mutant form of syndecan-4 deficient in protein kinase C (PKC)alpha activation or a different member of the syndecan family, syndecan-2, was unable to promote cell spreading. GF109203X and Go6976, inhibitors of PKC, completely inhibited ADAM12/syndecan-4-induced cell spreading. Expression of syndecan-4, but not syn4DeltaI, resulted in the accumulation of activated beta(1) integrins at the cell periphery in Chinese hamster ovary beta1 cells as revealed by 12G10 staining. Further, expression of myristoylated, constitutively active PKCalpha resulted in beta(1) integrin-dependent cell spreading, but additional activation of RhoA was required to induce Stress Fiber formation. In summary, these data provide novel insights into syndecan-4 signaling. Syndecan-4 can promote cell spreading in a beta(1) integrin-dependent fashion through PKCalpha and RhoA, and PKCalpha and RhoA likely function in separate pathways.
