Abstract
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.
Highlights
Hemidesmosomes (HDs) are epithelial cell-specific adhesion complexes that regulate a wide range of biological processes, including cell migration, proliferation, differentiation, and apoptosis [1,2,3]
HDs are formed at cell-substrate adhesion sites, where α6β4-integrin binds to the extracellular matrix (ECM) on the outside of the cell, and to keratin intermediate filaments through hemidesmosomal proteins on the inside of the cell. β4-integrin interacts with plectin, which anchors keratin filaments to the hemidesmosomal adhesions [4,5]
yellow fluorescent protein (YFP)-tagged Solo was expressed in MCF10A human mammary epithelial cells, and the binding ability of Solo to endogenous β4 was analyzed by immunoprecipitation with an anti-GFP antibody, followed by immunoblotting with an anti-β4 antibody
Summary
Hemidesmosomes (HDs) are epithelial cell-specific adhesion complexes that regulate a wide range of biological processes, including cell migration, proliferation, differentiation, and apoptosis [1,2,3]. Epithelial cells continuously perceive and respond to mechanical forces, derived from the inside and outside of the cells, which leads to reorganization of the cytoskeleton and adhesion structures to adapt to the mechanical environment [7,8]. While the stability of HDs is important for epithelial integrity, the dynamic reorganization of HDs in response to its surrounding environment is critical for maintaining cell and tissue homeostasis. MCF10A mammary epithelial cells cultured in 3D gels develop acinar structures, and this process depends on ECM stiffness and requires α6β4-integrin clustering and HD formation [1,11], indicating that HDs play a crucial role in sensing ECM stiffness during acinar formation in epithelial cells. The molecular mechanisms underlying the mechanical force-dependent control of HD organization and acinar formation remain largely unknown
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