Sachiko Fujiwara

Rho-guanine nucleotide exchange factors involved in cyclic stretch-induced reorientation of vascular endothelial cells

Cyclic stretch is an artificial model of mechanical force loading, which induces the reorientation of vascular endothelial cells and their stress fibers in a direction perpendicular to the stretch axis. Rho family GTPases are crucial for cyclic‐stretch‐induced endothelial cell reorientation; however, the mechanism underlying stretch‐induced activation of Rho family GTPases is unknown. A screen of short hairpin RNAs targeting 63 Rho guanine nucleotide exchange factors (Rho‐GEFs) revealed that at least 11 Rho‐GEFs – Abr, alsin, ARHGEF10, Bcr, GEF‐H1 (also known as ARHGEF2), LARG (also known as ARHGEF12), p190RhoGEF (also known as ARHGEF28), PLEKHG1, P‐REX2, Solo (also known as ARHGEF40) and &agr;‐PIX (also known as ARHGEF6) – which specifically or broadly target RhoA, Rac1 and/or Cdc42, are involved in cyclic‐stretch‐induced perpendicular reorientation of endothelial cells. Overexpression of Solo induced RhoA activation and F‐actin accumulation at cell–cell and cell–substrate adhesion sites. Knockdown of Solo suppressed cyclic‐stretch‐ or tensile‐force‐induced RhoA activation. Moreover, knockdown of Solo significantly reduced cyclic‐stretch‐induced perpendicular reorientation of endothelial cells when cells were cultured at high density, but not when they were cultured at low density or pretreated with EGTA or VE‐cadherin‐targeting small interfering RNAs. These results suggest that Solo is involved in cell–cell‐adhesion‐mediated mechanical signal transduction during cyclic‐stretch‐induced endothelial cell reorientation.

LIM Kinase Has a Dual Role in Regulating Lamellipodium Extension by Decelerating the Rate of Actin Retrograde Flow and the Rate of Actin Polymerization

Background: LIMK1 regulates actin dynamics by inactivating cofilin. Results: LIMK1 knockdown accelerated actin polymerization and retrograde flow, but the effect on retrograde flow was more efficient. Conclusion: LIMK1 has a dual role in regulating lamellipodium extension by decelerating actin retrograde flow and polymerization. LIMK1 contributes to lamellipodium extension by decelerating actin retrograde flow. Significance: The dual role of LIMK1 in lamellipodium extension was clarified. Lamellipodium extension is crucial for cell migration and spreading. The rate of lamellipodium extension is determined by the balance between the rate of actin polymerization and the rate of actin retrograde flow. LIM kinase 1 (LIMK1) regulates actin dynamics by phosphorylating and inactivating cofilin, an actin-depolymerizing protein. We examined the role of LIMK1 in lamellipodium extension by measuring the rates of actin polymerization, actin retrograde flow, and lamellipodium extension using time-lapse imaging of fluorescence recovery after photobleaching. In the non-extending lamellipodia of active Rac-expressing N1E-115 cells, LIMK1 expression decelerated and LIMK1 knockdown accelerated actin retrograde flow. In the extending lamellipodia of neuregulin-stimulated MCF-7 cells, LIMK1 knockdown accelerated both the rate of actin polymerization and the rate of actin retrograde flow, but the accelerating effect on retrograde flow was greater than the effect on polymerization, thus resulting in a decreased rate of lamellipodium extension. These results indicate that LIMK1 has a dual role in regulating lamellipodium extension by decelerating actin retrograde flow and polymerization, and in MCF-7 cells endogenous LIMK1 contributes to lamellipodium extension by decelerating actin retrograde flow more effectively than decelerating actin polymerization.

Roles of the cytoskeleton, cell adhesion and rho signalling in mechanosensing and mechanotransduction

All cells sense and respond to various mechanical forces in and mechanical properties of their environment. To respond appropriately, cells must be able to sense the location, direction, strength and duration of these forces. Recent progress in mechanobiology has provided a better understanding of the mechanisms of mechanoresponses underlying many cellular and developmental processes. Various roles of mechanoresponses in development and tissue homeostasis have been elucidated, and many molecules involved in mechanotransduction have been identified. However, the whole picture of the functions and molecular mechanisms of mechanotransduction remains to be understood. Recently, novel mechanisms for sensing and transducing mechanical stresses via the cytoskeleton, cell-substrate and cell-cell adhesions and related proteins have been identified. In this review, we outline the roles of the cytoskeleton, cell-substrate and cell-cell adhesions, and related proteins in mechanosensing and mechanotransduction. We also describe the roles and regulation of Rho-family GTPases in mechanoresponses.

Interplay between Solo and keratin filaments is crucial for mechanical force-induced stress fiber reinforcement

Solo (ARHGEF40) is a RhoA-targeting guanine nucleotide exchange factor that serves to promote stress fibers and precisely organize keratin networks. Solo binds to keratin-8/18 filaments. The interplay between Solo and keratin-8/18 filaments is crucial for tensile force–induced RhoA activation and consequent actin stress fiber reinforcement.

Solo, a RhoA-targeting guanine nucleotide exchange factor, is critical for hemidesmosome formation and acinar development in epithelial cells

Cell-substrate adhesions are essential for various physiological processes, including embryonic development and maintenance of organ functions. Hemidesmosomes (HDs) are multiprotein complexes that attach epithelial cells to the basement membrane. Formation and remodeling of HDs are dependent on the surrounding mechanical environment; however, the upstream signaling mechanisms are not well understood. We recently reported that Solo (also known as ARHGEF40), a guanine nucleotide exchange factor targeting RhoA, binds to keratin8/18 (K8/K18) intermediate filaments, and that their interaction is important for force-induced actin and keratin cytoskeletal reorganization. In this study, we show that Solo co-precipitates with an HD protein, β4-integrin. Co-precipitation assays revealed that the central region (amino acids 330–1057) of Solo binds to the C-terminal region (1451–1752) of β4-integrin. Knockdown of Solo significantly suppressed HD formation in MCF10A mammary epithelial cells. Similarly, knockdown of K18 or treatment with Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed HD formation. As Solo knockdown or Y-27632 treatment is known to disorganize K8/K18 filaments, these results suggest that Solo is involved in HD formation by regulating K8/K18 filament organization via the RhoA-ROCK signaling pathway. We also showed that knockdown of Solo impairs acinar formation in MCF10A cells cultured in 3D Matrigel. In addition, Solo accumulated at the site of traction force generation in 2D-cultured MCF10A cells. Taken together, these results suggest that Solo plays a crucial role in HD formation and acinar development in epithelial cells by regulating mechanical force-induced RhoA activation and keratin filament organization.

Solo and keratin filaments regulate epithelial tubule morphology

Epithelial tubules, consisting of the epithelial cell sheet with a central lumen, are the basic structure of many organs. Mechanical forces play an important role in epithelial tubulogenesis; however, little is known about the mechanisms controlling the mechanical forces during epithelial tubule morphogenesis. Solo (also known as ARHGEF40) is a RhoA-targeting guanine-nucleotide exchange factor that is involved in mechanical force-induced RhoA activation and stress fiber formation. Solo binds to keratin-8/keratin-18 (K8/K18) filaments, and this interaction plays a crucial role in mechanotransduction. In this study, we examined the roles of Solo and K8/K18 filaments in epithelial tubulogenesis using MDCK cells cultured in 3D collagen gels. Knockdown of either Solo or K18 resulted in rounder tubules with increased lumen size, indicating that Solo and K8/K18 filaments play critical roles in forming the elongated morphology of epithelial tubules. Moreover, knockdown of Solo or K18 decreased the level of diphosphorylated myosin light chain (a marker of contractile force) at the luminal and outer surfaces of tubules, suggesting that Solo and K8/K18 filaments are involved in the generation of the myosin II-mediated contractile force during epithelial tubule morphogenesis. In addition, K18 filaments were normally oriented along the long axis of the tubule, but knockdown of Solo perturbed their orientation. These results suggest that Solo plays crucial roles in forming the elongated morphology of epithelial tubules and in regulating myosin II activity and K18 filament organization during epithelial tubule formation.Key words: epithelial tubulogenesis, Solo, keratin, Rho-GEF, myosin.