In response to external mechanical stimuli, cells remodel their actin cytoskeleton. Solo, a Rho guanine nucleotide exchange factor (RhoGEF), is involved in mechanical stress responses via cell-substrate adhesions. Using BioID, we identified PDZ-RhoGEF (PRG), a member of the RGS-RhoGEF (regulator of G protein signaling domain-containing RhoGEFs) family, as a Solo-interacting protein. Moreover, we found that Solo regulates PRG during the mechanical stress response. Furthermore, we identified leukemia-associated RhoGEF (LARG), another RGS-RhoGEF member, as a Solo-interacting protein; however, the functional role of this interaction remains unknown. Therefore, in this study, we investigated the interaction between Solo and LARG and found that LARG localizes to Solo accumulation sites at the basal plane and that LARG is required for Solo-induced actin polymerization. Additionally, Solo is required to maintain LARG activity in cells, and this interaction is related to actin regulation in response to substrate stiffness. We further investigated the relationship between LARG and PRG as a function of Solo. We noted that the double knockdown of PRG and LARG suppressed Solo-induced actin polymerization to the same extent or more than every single knockdown, indicating that these signaling pathways cooperatively regulate Solo-induced actin polymerization.

Abstract Desmosomes mechanically couple neighboring epithelial cells to the intermediate filament cytoskeleton. However, the nanoscale organization of opposing cytoplasmic plaques and their dynamic remodeling during epithelial maturation remain poorly understood owing to the resolution limits of conventional optical microscopy. In this study, we applied expansion microscopy (ExM) to Madin–Darby canine kidney epithelial monolayers to resolve the spatial distribution of the core desmosomal proteins within opposing plaques for quantitative mapping. A comparative study of subconfluent and confluent cultures revealed a density-dependent transition of desmoplakin (DSP) from a compact “closed” state to an extended “open” conformation. Specifically, the spacing between opposing DSP C-terminal regions increased significantly with cell density. In contrast, desmoglein 2, plakoglobin and plakophilin 2 remained spatially stable, while the DSP N-terminus exhibited a comparatively subtle cytoplasmic shift. Notably, keratin filament–deficient cells adopt a constitutively open conformation regardless of cell density, demonstrating that keratin anchoring is indispensable for structural remodeling. Our findings reveal keratin-dependent conformational remodeling of DSP as a hallmark of junctional maturation and establish ExM as a robust, accessible approach for deciphering the functional nanoscale architecture of cell–cell junctions.

In response to external mechanical stimuli, cells remodel their actin cytoskeleton. Solo, a Rho guanine nucleotide exchange factor (RhoGEF), is involved in mechanical stress responses. Using BioID, we identified PDZ-RhoGEF (PRG), a member of the RGS-RhoGEF family (regulator of G protein signaling domain-containing RhoGEFs, as a Solo-interacting protein. Moreover, we found that Solo regulates PRG during the mechanical stress response. Furthermore, we identified leukemia-associated RhoGEF (LARG), another RGS-RhoGEF member, as a Solo-interacting protein; however, the functional role of this interaction remains unknown. Therefore, in this study, we investigated the interaction between Solo and LARG and found that LARG localizes to Solo accumulation sites at the basal plane and that LARG is required for Solo-induced actin polymerization. Additionally, Solo is required to maintain LARG activity in cells, and this interaction is related to actin regulation in response to substrate stiffness. We further investigated the relationship between LARG and PRG as a function of Solo. We noted that although they did not competitively localize at Solo accumulation sites, knockdown of either PRG or LARG suppressed Solo-induced actin polymerization to the same extent as double knockdown, indicating that these signaling pathways cooperatively regulate Solo-induced actin polymerization. ### Competing Interest Statement The authors have declared no competing interest. * ### ABBREVIATIONS BioID : proximity-dependent biotin identification; DH : Dbl homology; GST : glutathione-S- transferase; LARG : leukemia-associated RhoGEF; mant-GTP : N-methylanthraniloyl-GTP; PA : polyacrylamide; PH : pleckstrin homology; PLEKHG4B : pleckstrin homology and RhoGEF domain containing 4 B, PRG : PDZ-RhoGEF; RGS : regulator of G protein signaling domain; RhoGEF : Rho-guanine nucleotide exchange factor

OBJECTIVES:Butyrate is one of major metabolites of periodontitis-associated bacteria and often detected in periodontal pockets. Butyrate has been considered to affect human gingival fibroblasts (HGFs); however, there was no information on its long-term effect as occurs in periodontitis. Therefore, this study aimed to evaluate the time-dependent effects of butyrate on HGFs. MATERIAL AND METHODS:The effects of butyrate on HGF proliferation, apoptosis, cell morphology, glucose metabolic activity, butyrate metabolic activity, and cell migration ability were evaluated by cell counting, DNA electrophoresis, cell staining, pH-stat system, HPLC, and scratch test, respectively. RESULTS:HGF proliferation was temporarily inhibited by 5-10 mM butyrate (p < 0.05); however, it resumed at 24 h with morphological changes from spindle to slightly widened (p < 0.05). HGFs cultured with 10 mM butyrate for 12-24 h shifted the glucose metabolic pathway from oxidative phosphorylation to glycolysis (p < 0.05), and increased butyrate consumption, which returned to control levels over 24 h. HGF migration ability tended to decrease at 72 h. CONCLUSIONS:HGF cell proliferation and glucose/butyrate metabolism were temporarily inhibited by butyrate and then recovered in a time-dependent manner, accompanied by changes in cell morphology. These time-dependent effects may help to understand the role of butyrate in the pathology of periodontitis.

Rho guanine nucleotide exchange factors (RhoGEFs) comprise a wide range of proteins with a common domain responsible for the activation of the Rho family of small GTPases and various domains in other regions. The evolutionary divergence of RhoGEFs enables actin cytoskeletal reorganization, leading to complex cellular responses in higher organisms. In this review, we address the involvement of RhoGEFs in the mechanical stress response of mammalian cells. The cellular mechanical stress response is essential for the proper and orderly regulation of cell populations, including the maintenance of homeostasis, tissue morphogenesis, and adaptation to the mechanical environment. In particular, this review focuses on the recent findings regarding the Dbl family of RhoGEFs involved in mechanical stress responses at the cell-cell and cell-substrate adhesion sites, and their molecular mechanisms underlying actin cytoskeleton remodeling and signal transduction.

Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study aimed to identify Solo-interacting proteins using the BioID, a proximal-dependent labeling method, and elucidate the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG colocalized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at the Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling. Furthermore, the purified Solo itself had little or negligible GEF activity, even its GEF-inactive mutant directly activated the GEF activity of PRG through interaction. Moreover, overexpression of the Solo and PRG binding domains, respectively, had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.

ABSTRACT Recent findings indicate that Solo, a RhoGEF, is involved in cellular mechanical stress responses. However, the mechanism of actin cytoskeletal remodeling via Solo remains unclear. Therefore, this study was aimed at identifying Solo-interacting proteins using the BioID, a proximal-dependent labeling method and elucidating the molecular mechanisms of function of Solo. We identified PDZ-RhoGEF (PRG) as a Solo-interacting protein. PRG co-localized with Solo in the basal area of cells, depending on Solo localization, and enhanced actin polymerization at Solo accumulation sites. Additionally, Solo and PRG interaction was necessary for actin cytoskeletal remodeling and RhoA activation. Moreover, overexpression of the binding domains of Solo and PRG had a dominant-negative effect on actin polymerization and actin stress fiber formation in response to substrate stiffness. Therefore, Solo restricts the localization of PRG and regulates actin cytoskeletal remodeling in synergy with PRG in response to the surrounding mechanical environment.

PLEKHG4B is a Cdc42-targeting guanine-nucleotide exchange factor implicated in forming epithelial cell-cell junctions. Here we explored the mechanism regulating PLEKHG4B localization. PLEKHG4B localized to the basal membrane in normal Ca2+ medium but accumulated at cell-cell junctions upon ionomycin treatment. Ionomycin-induced junctional localization of PLEKHG4B was suppressed upon disrupting its annexin-A2 (ANXA2)-binding ability. Thus, Ca2+ influx and ANXA2 binding are crucial for PLEKHG4B localization to cell-cell junctions. Treatments with low Ca2+ or BAPTA-AM (an intracellular Ca2+ chelator) suppressed PLEKHG4B localization to the basal membrane. Mutations of the phosphoinositide-binding motif in the pleckstrin homology (PH) domain of PLEKHG4B or masking of membrane phosphatidylinositol-4,5-biphosphate [PI(4,5)P2] suppressed PLEKHG4B localization to the basal membrane, indicating that basal membrane localization of PLEKHG4B requires suitable intracellular Ca2+ levels and PI(4,5)P2 binding of the PH domain. Activation of mechanosensitive ion channels (MSCs) promoted PLEKHG4B localization to cell-cell junctions, and their inhibition suppressed it. Moreover, similar to the PLEKHG4B knockdown phenotypes, inhibition of MSCs or treatment with BAPTA-AM disturbed the integrity of actin filaments at cell-cell junctions. Taken together, our results suggest that Ca2+ influx plays crucial roles in PLEKHG4B localization to cell-cell junctions and the integrity of junctional actin organization, with MSCs contributing to this process.

ABSTRACT Cofilin acts as a key regulator of actin cytoskeletal remodeling via stimulating actin filament disassembly. Cofilin is inactivated by Ser-3 phosphorylation and reactivated by cofilin-phosphatase Slingshot-1 (SSH1). SSH1 is activated upon binding to F-actin, and this activation is inhibited by its phosphorylation at Ser-937 and Ser-978 and the subsequent binding of 14-3-3 proteins. In this study, we identified MARK3 (also named Par-1a and C-TAK1) as a kinase responsible for Ser-937/Ser-978 phosphorylation of SSH1. MARK3-mediated phosphorylation promoted SSH1 binding to 14-3-3 proteins and suppressed its F-actin-assisted cofilin-phosphatase activity. When Jurkat cells were stimulated with SDF-1α, actin filaments formed multidirectional F-actin-rich lamellipodia around the cells in the initial stage, and thereafter, they were rearranged as a single polarized lamellipodium to the direction of cell migration. Upon SDF-1α stimulation, SSH1 was translocated into F-actin-rich lamellipodia, but its Ser-937/Ser-978 non-phosphorylatable mutant SSH1(2SA) was retained at the location of the original cortical F-actin. Knockdown of MARK3 or overexpression of SSH1(2SA), similar to SSH1 knockdown, impaired the conversion of multiple lamellipodia to a single polarized lamellipodium. These results indicate that MARK3-mediated Ser-937/Ser-978 phosphorylation is required for SSH1 liberation from F-actin and translocation to lamellipodia, and hence, facilitates the formation of a single polarized lamellipodium for directional cell migration. Our results suggest that the phosphorylation-dephosphorylation cycle of SSH1 is crucial for its localization to lamellipodia via promoting the dissociation-reassociation cycle of SSH1 to F-actin, and thereby the stimulus-induced lamellipodium formation to the direction of cell movement.

Cell-cell junction formation requires actin cytoskeletal remodeling. Here we show that PLEKHG4B, a Rho-guanine nucleotide exchange factor (Rho-GEF), plays a crucial role in epithelial cell-cell junction formation. Knockdown of PLEKHG4B decreased Cdc42 activity and tended to increase RhoA activity in A549 cells. A549 monolayer cells showed 'closed junctions' with closely packed actin bundles along the cell-cell contacts, but PLEKHG4B knockdown suppressed closed junction formation and exhibited 'open junctions' with split actin bundles located away from the cell-cell boundary. In calcium-switch assays, PLEKHG4B knockdown delayed the conversion of open junctions to closed junctions and β-catenin accumulation at cell-cell junctions. Further, PLEKHG4B knockdown abrogated the reduction in myosin activity normally seen in the later stage of junction formation. The aberrant myosin activation and impairments in closed junction formation in PLEKHG4B-knockdown cells were reverted by ROCK inhibition or LARG/PDZ-RhoGEF knockdown. These results suggest that PLEKHG4B enables actin remodeling during epithelial cell-cell junction maturation, probably by reducing myosin activity in the later stage of junction formation, through suppressing LARG/PDZ-RhoGEF and RhoA-ROCK activities. We also showed that annexin-A2 participates in PLEKHG4B localization to cell-cell junctions.

Collective cell migration plays crucial roles in tissue remodeling, wound healing, and cancer cell invasion. However, its underlying mechanism remains unknown. Previously, we showed that the RhoA-targeting guanine nucleotide exchange factor Solo (ARHGEF40) is required for tensile force-induced RhoA activation and proper organization of keratin-8/keratin-18 (K8/K18) networks. Here, we demonstrate that Solo knockdown significantly increases the rate at which Madin-Darby canine kidney cells collectively migrate on collagen gels. However, it has no apparent effect on the migratory speed of solitary cultured cells. Therefore, Solo decelerates collective cell migration. Moreover, Solo localized to the anteroposterior regions of cell-cell contact sites in collectively migrating cells and was required for the local accumulation of K8/K18 filaments in the forward areas of the cells. Partial Rho-associated protein kinase (ROCK) inhibition or K18 or plakoglobin knockdown also increased collective cell migration velocity. These results suggest that Solo acts as a brake for collective cell migration by generating pullback force at cell-cell contact sites via the RhoA-ROCK pathway. It may also promote the formation of desmosomal cell-cell junctions related to K8/K18 filaments and plakoglobin.

Metals and alloys are used widely in bone prosthetic materials, stents and dental tissue reconstructions. The most common materials are stainless steels and cobalt-chromium-nickel and titanium alloys. These alloys can be easily deformed but are hard to break. However, their affinity for cells and tissues is very low. In addition, they can sometimes provoke unexpected metal allergies. Iron is an abundant trace element essential for humans. However, excess amounts in particular of Fe2+ ions are toxic. We previously succeeded in obtaining 99.9996% ultra-high-purity iron (ABIKO iron). The chemical properties of ABIKO iron are completely different from that of conventional pure iron. For example, the reaction rate in hydrochloric acid is very slow and there is barely any corrosion. Here, we found that, in the absence of any type of coating, mammalian cells could easily attach to, and normally proliferate and differentiate on, ABIKO iron. On the other hand, cell densities and proliferation rate of the surfaces of plates made from Co-Cr-Mo or Ti-6Al-4V were significantly reduced. In addition, several stress and iron response genes, HSP70, SOD1, ATM and IRP2 did not change in the cells on ABIKO iron, while these genes were induced with exogenous application of FeSO4. Cells also secreted and fastened some organics on ABIKO iron. In vitro collagen binding assay showed that ABIKO iron binds higher amount of collagens. These findings highlight ABIKO iron as a novel biocompatible prosthetic material.

Solo (ARHGEF40) is a RhoA-targeting guanine nucleotide exchange factor that regulates tensional force-induced cytoskeletal reorganization. Solo binds to keratin 8/keratin 18 (K8/K18) filaments through multiple sites, but the roles of these interactions in the localization and mechanotransduction-regulating function of Solo remain unclear. Here, we constructed two Solo mutants (L14R/L17R and L49R/L52R) with leucine-to-arginine replacements in the N-terminal conserved region (which we termed the Solo domain) and analyzed their K18-binding activities. These mutations markedly decreased the K18-binding ability of the N-terminal fragment (residues 1-329) of Solo but had no apparent effect on the K18-binding ability of full-length (FL) Solo. When expressed in cultured cells, wild-type Solo-FL showed a unique punctate localization near the ventral surface of cells and caused the reinforcement of actin filaments. In contrast, despite retaining the K18-binding ability, the L14R/L17R and L49R/L52R mutants of Solo-FL were diffusely distributed in the cytoplasm and barely induced actin cytoskeletal reinforcement. Furthermore, wild-type Solo-FL promoted traction force generation against extracellular matrices and tensional force-induced stress fiber reinforcement, but its L14R/L17R and L49R/L52R mutants did not. These results suggest that the K18-binding ability of the N-terminal Solo domain is critical for the ventral localization of Solo and its function in regulating mechanotransduction.

BACKGROUND:Neurochemicals like serotonin and dopamine play crucial roles in human cognitive and emotional functions. Vesicular monoamine transporter 1 (VMAT1) transports monoamine neurotransmitters, and its variant (136Thr) is associated with various psychopathological symptoms and reduced monoamine uptake relative to 136Ile. We previously showed that two human-specific amino acid substitutions (Glu130Gly and Asn136Thr/Ile) of VMAT1 were subject to positive natural selection. However, the potential functional alterations caused by these substitutions (Glu130Gly and Asn136Thr) remain unclear. To assess functional changes in VMAT1 from an evolutionary perspective, we reconstructed ancestral residues and examined the role of these substitutions in monoamine uptake in vitro using fluorescent false neurotransmitters (FFN), which are newly developed substances used to quantitatively assay VMATs.RESULTS:Immunoblotting confirmed that all the transfected YFP-VMAT1 variants are properly expressed in HEK293T cells at comparable levels, and no significant difference was seen in the density and the size of vesicles among them. Our fluorescent assays revealed a significant difference in FFN206 uptake among VMAT1 variants: 130Glu/136Asn, 130Glu/136Thr, and 130Gly/136Ile showed significantly higher levels of FFN206 uptake than 130Gly/136Asn and 130Gly/136Thr, indicating that both 130Glu and 136Ile led to increased neurotransmitter uptake, for which 136Thr and 136Asn were comparable by contrast.CONCLUSIONS:These findings suggest that monoamine uptake by VMAT1 initially declined (from 130Glu/136Asn to 130Gly/136Thr) in human evolution, possibly resulting in higher susceptibility to the external environment of our ancestors.

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.

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.

The discrepancy between observed flower visitors and those predicted based on floral phenotype has often cast doubt on the pollination syndrome concept. Here we show that this paradox may be alleviated by gaining better knowledge of the contributions of different flower visitors to pollination and the effects of floral traits that cannot be readily perceived by humans in Adenophora triphylla var. japonica. The blue, bell-shaped and pendant flowers of A. triphylla appear to fit a bee pollination syndrome. In contrast to this expectation, recent studies show that these flowers are frequented by nocturnal moths. We compared the flower visitor fauna, their visitation frequency and their relative contributions to seed set between day and night in two field populations of A. triphylla in Japan. We also determined the floral traits associated with temporal changes in the visitor assemblage, i.e. the timing of anthesis, the timing of changes in the sexual phase and the diel pattern of nectar production. While A. triphylla flowers were visited by both diurnal and nocturnal insects, the results from pollination experiments demonstrate that their primary pollinators are nocturnal settling-moths. Moreover, the flowers opened just after sunset, changed from staminate to pistillate phase in successive evenings and produced nectar only during the night, which all conform to the activity of nocturnal/crepuscular moths. Our study illustrates that the tradition of stereotyping the pollinators of a flower based on its appearance can be misleading and that it should be improved with empirical evidence of pollination performance and sufficient trait matching.