Kensaku Mizuno

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 PLEKHG4B-knockdown cells exhibited ‘open junctions’ with split actin bundles located away from the cell–cell boundary. In Ca2+-switch assays, PLEKHG4B knockdown delayed the conversion of open junctions to closed junctions and β-catenin accumulation at cell–cell junctions. Furthermore, 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 pathway activities. We also showed that annexin A2 participates in PLEKHG4B localization to cell–cell junctions. This article has an associated First Person interview with the first author of the paper.

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.

Primary cilia are antenna-like sensory organelles that transmit various extracellular signals. Ciliogenesis requires the removal of CP110 and its interactor CEP97 from the mother centriole for initiating ciliary axoneme extension, but the underlying mechanism remains unknown. Here we show that, upon serum starvation, CEP97 is partially degraded by the ubiquitin-proteasome system. CEP97 was polyubiquitylated in serum-starved cells, and overexpression of a non-ubiquitylatable CEP97 mutant effectively blocked CP110 removal and ciliogenesis induced by serum-starvation. Through several screening steps, we identified the cullin-3–RBX1–KCTD10 complex as the E3 ligase that mediates CEP97 degradation and removal from the mother centriole. Depletion of each component of this E3 complex caused aberrant accumulation of CEP97 on the centrosome, suppressed the removal of CEP97 and CP110 from the mother centriole, and impaired ciliogenesis. Moreover, KCTD10 was specifically localized to the mother centriole. These results suggest that CEP97 degradation by the cullin-3–RBX1–KCTD10 complex plays a crucial role in serum-starvation-induced CP110 removal and ciliogenesis.

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.

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Primary cilia are antenna-like sensory organelles extending from the surface of many cell types that play critical roles in tissue development and homeostasis. Here, we examined the effect of nutrient status on primary cilium formation. Glucose deprivation significantly increased the number of ciliated cells under both serum-fed and -starved conditions. Glucose deprivation-induced ciliogenesis was suppressed by overexpression of Rheb, an activator of the mammalian target of rapamycin complex-1 (mTORC1). Inactivating mTORC1 by rapamycin treatment or Raptor knockdown significantly promoted ciliogenesis. These results indicate that glucose deprivation promotes primary cilium formation through mTORC1 inactivation. Rapamycin treatment did not promote autophagy or degradation of OFD1, a negative regulator of ciliogenesis. In contrast, rapamycin treatment increased the level of the p27^KIP1 (also known as CDKN1B) cyclin-dependent kinase inhibitor, and rapamycin-induced ciliogenesis was abrogated in p27^KIP1-depleted cells. These results indicate that mTORC1 inactivation induces ciliogenesis through p27^KIP1 upregulation, but not through autophagy. By contrast, glucose deprivation or rapamycin treatment shortened the cilium length. Thus, glucose deprivation and subsequent inactivation of mTORC1 play dual roles in ciliogenesis: triggering primary cilium formation and shortening cilium length. This article has an associated First Person interview with the first author of the paper.

Primary cilia are non-motile cilia that serve as cellular antennae for sensing and transducing extracellular signals. In general, primary cilia are generated by cell quiescence signals. Recent studies have shown that manipulations to increase actin assembly suppress quiescence-induced ciliogenesis. To further examine the role of actin dynamics in ciliogenesis, we analyzed the effect of jasplakinolide (Jasp), a potent inducer of actin polymerization, on ciliogenesis. Unexpectedly, Jasp treatment induced ciliogenesis in serum-fed cells cultured at low density. In contrast, Jasp had no apparent effect on ciliogenesis in cells cultured at higher densities. Jasp-induced ciliogenesis was correlated with a change in cell morphology from a flat and adherent shape to a round and weakly adherent one. Jasp treatment also induced the phosphorylation and cytoplasmic localization of the YAP transcriptional co-activator and suppressed cell proliferation in low density-cultured cells. Overexpression of an active form of YAP suppressed Jasp-induced ciliogenesis. These results suggest that Jasp induces ciliogenesis through cell rounding and cytoplasmic localization and inactivation of YAP. Knockdown of LATS1/2 only faintly suppressed Jasp-induced YAP phosphorylation, indicating that LATS1/2 are not primarily responsible for Jasp-induced YAP phosphorylation. Furthermore, overexpression of active Src kinase suppressed Jasp-induced cytoplasmic localization of YAP and ciliogenesis, suggesting that down-regulation of Src activity is involved in Jasp-induced YAP inactivation and ciliogenesis. Our data suggest that actin polymerization does not suppress ciliogenesis per se but rather that cell rounding and reduced cell adhesion are more crucially involved in Jasp-induced ciliogenesis.