PLEKHG4B enables actin cytoskeletal remodeling during epithelial cell-cell junction formation.

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 ß-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.

Furry protein suppresses nuclear localization of yes-associated protein (YAP) by activating NDR kinase and binding to YAP.

The Hippo signaling pathway suppresses cell proliferation and tumorigenesis. In the canonical Hippo pathway, large tumor suppressor kinases 1/2 (LATS1/2) phosphorylate the transcriptional coactivator yes-associated protein (YAP) and thereby suppress its nuclear localization and co-transcriptional activity. Nuclear Dbf2-related kinases 1/2 (NDR1/2), which are closely related to LATS1/2, also phosphorylate and inactivate YAP by suppressing its nuclear localization. Furry (FRY) is a cytoplasmic protein that associates with NDR1/2 and activates them, but its role in the nuclear/cytoplasmic localization of YAP remains unknown. Here, we constructed FRY-knockout cell lines to examine the role of FRY in YAP's cytoplasmic localization. FRY depletion markedly increased YAP nuclear localization and decreased NDR1/2 kinase activity and YAP phosphorylation levels, but did not affect LATS1/2 kinase activity. This indicated that FRY suppresses YAP's nuclear localization by promoting its phosphorylation via NDR1/2 activation. NDR1/2 depletion also promoted YAP nuclear localization, but depletion of both FRY and NDR1/2 increased the number of cells with YAP nuclear localization more strongly than did depletion of NDR1/2 alone, suggesting that FRY suppresses YAP nuclear localization by a mechanism in addition to NDR1/2 activation. Co-precipitation assays revealed that Fry uses its N-terminal 1-2400-amino-acid-long region to bind to YAP. Expression of full-length FRY or its 1-2400 N-terminal fragment restored YAP cytoplasmic localization in FRY-knockout cells. Taken together, these results suggest that FRY plays a crucial role in YAP cytoplasmic retention by promoting YAP phosphorylation via NDR1/2 kinase activation and by binding to YAP, leading to its cytoplasmic sequestration.

Roles of TOG and jelly-roll domains of centrosomal protein CEP104 in its functions in cilium elongation and Hedgehog signaling.

Primary cilia are generated through the extension of the microtubule-based axoneme. Centrosomal protein 104 (CEP104) localizes to the tip of the elongating axoneme, and CEP104 mutations are linked to a ciliopathy, Joubert syndrome. Thus, CEP104 has been implicated in ciliogenesis. However, the mechanism by which CEP104 regulates ciliogenesis remains elusive. We report here that CEP104 is critical for cilium elongation but not for initiating ciliogenesis. We also demonstrated that the tumor-overexpressed gene (TOG) domain of CEP104 exhibits microtubule-polymerizing activity and that this activity is essential for the cilium-elongating activity of CEP104. Knockdown/rescue experiments showed that the N-terminal jelly-roll (JR) fold partially contributes to cilium-elongating activity of CEP104, but neither the zinc-finger region nor the SXIP motif is required for this activity. CEP104 binds to a centriole-capping protein, CP110, through the zinc-finger region and to a microtubule plus-end-binding protein, EB1, through the SXIP motif, indicating that the binding of CP110 and EB1 is dispensable for the cilium-elongating activity of CEP104. Moreover, CEP104 depletion does not affect CP110 removal from the mother centriole, which suggests that CEP104 functions after the removal of CP110. Last, we also showed that CEP104 is required for the ciliary entry of Smoothened and export of GPR161 upon Hedgehog signal activation and that the TOG domain plays a critical role in this activity. Our results define the roles of the individual domains of CEP104 in its functions in cilium elongation and Hedgehog signaling and should enhance our understanding of the mechanism underlying CEP104 mutation-associated ciliopathies.

Glucose deprivation induces primary cilium formation through mTORC1 inactivation.

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 p27KIP1 (also known as CDKN1B) cyclin-dependent kinase inhibitor, and rapamycin-induced ciliogenesis was abrogated in p27KIP1-depleted cells. These results indicate that mTORC1 inactivation induces ciliogenesis through p27KIP1 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.

Cullin-3-KCTD10-mediated CEP97 degradation promotes primary cilium formation.

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.

Keratin-binding ability of the N-terminal Solo domain of Solo is critical for its function in cellular mechanotransduction.

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.

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.

Localization of Protein Kinase NDR2 to Peroxisomes and Its Role in Ciliogenesis.

Nuclear Dbf2-related (NDR) kinases, comprising NDR1 and NDR2, are serine/threonine kinases that play crucial roles in the control of cell proliferation, apoptosis, and morphogenesis. We recently showed that NDR2, but not NDR1, is involved in primary cilium formation; however, the mechanism underlying their functional difference in ciliogenesis is unknown. To address this issue, we examined their subcellular localization. Despite their close sequence similarity, NDR2 exhibited punctate localization in the cytoplasm, whereas NDR1 was diffusely distributed within the cell. Notably, NDR2 puncta mostly co-localized with the peroxisome marker proteins, catalase and CFP-SKL (cyan fluorescent protein carrying the C-terminal typical peroxisome-targeting signal type-1 (PTS1) sequence, Ser-Lys-Leu). NDR2 contains the PTS1-like sequence, Gly-Lys-Leu, at the C-terminal end, whereas the C-terminal end of NDR1 is Ala-Lys. An NDR2 mutant lacking the C-terminal Leu, NDR2(ΔL), exhibited almost diffuse distribution in cells. Additionally, NDR2, but neither NDR1 nor NDR2(ΔL), bound to the PTS1 receptor Pex5p. Together, these findings indicate that NDR2 localizes to the peroxisome by using the C-terminal GKL sequence. Intriguingly, topology analysis of NDR2 suggests that NDR2 is exposed to the cytosolic surface of the peroxisome. The expression of wild-type NDR2, but not NDR2(ΔL), recovered the suppressive effect of NDR2 knockdown on ciliogenesis. Furthermore, knockdown of peroxisome biogenesis factor genes (PEX1 or PEX3) partially suppressed ciliogenesis. These results suggest that the peroxisomal localization of NDR2 is implicated in its function to promote primary cilium formation.

NDR2-mediated Rabin8 phosphorylation is crucial for ciliogenesis by switching binding specificity from phosphatidylserine to Sec15.

Primary cilia are antenna-like sensory organelles protruding from the plasma membrane. Defects in ciliogenesis cause diverse genetic disorders. NDR2 was identified as the causal gene for a canine ciliopathy, early retinal degeneration, but its role in ciliogenesis remains unknown. Ciliary membranes are generated by transport and fusion of Golgi-derived vesicles to the pericentrosome, a process requiring Rab11-mediated recruitment of Rabin8, a GDP-GTP exchange factor (GEF) for Rab8, and subsequent Rab8 activation and Rabin8 binding to Sec15, a component of the exocyst that mediates vesicle tethering. This study shows that NDR2 phosphorylates Rabin8 at Ser-272 and defects in this phosphorylation impair preciliary membrane assembly and ciliogenesis, resulting in accumulation of Rabin8-/Rab11-containing vesicles at the pericentrosome. Rabin8 binds to and colocalizes with GTP-bound Rab11 and phosphatidylserine (PS) on pericentrosomal vesicles. The phospho-mimetic S272E mutation of Rabin8 decreases affinity for PS but increases affinity for Sec15. These results suggest that NDR2-mediated Rabin8 phosphorylation is crucial for ciliogenesis by triggering the switch in binding specificity of Rabin8 from PS to Sec15, thereby promoting local activation of Rab8 and ciliary membrane formation.

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 α-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.

A pleckstrin homology-like domain is critical for F-actin binding and cofilin-phosphatase activity of Slingshot-1.

Slingshot-1 (SSH1) is a protein phosphatase that specifically dephosphorylates and activates cofilin, an F-actin-severing protein. SSH1 binds to and co-localizes with F-actin, and the cofilin-phosphatase activity of SSH1 is markedly increased by binding to F-actin. In this study, we performed a secondary structure analysis of SSH1, which predicted the existence of a pleckstrin homology (PH)-like domain in the N-terminal region of SSH1. SSH1 also contains a DEK-C domain in the N-terminal region. The N-terminal fragment of SSH1 bound to and co-localized with F-actin, but mutation at Arg-96 or a Leu-His-Lys (LHK) motif in the PH-like domain reduced this activity. Furthermore, mutation at Arg-96 abrogated the cofilin-phosphatase activity of SSH1 in the presence of F-actin. These results suggest that the N-terminal PH-like domain plays a critical role in F-actin binding and F-actin-mediated activation of the cofilin-phosphatase activity of SSH1.

Insulin receptor substrate-4 binds to Slingshot-1 phosphatase and promotes cofilin dephosphorylation.

Cofilin plays an essential role in cell migration and morphogenesis by enhancing actin filament dynamics via its actin filament-severing activity. Slingshot-1 (SSH1) is a protein phosphatase that plays a crucial role in regulating actin dynamics by dephosphorylating and reactivating cofilin. In this study, we identified insulin receptor substrate (IRS)-4 as a novel SSH1-binding protein. Co-precipitation assays revealed the direct endogenous binding of IRS4 to SSH1. IRS4, but not IRS1 or IRS2, was bound to SSH1. IRS4 was bound to SSH1 mainly through the unique region (amino acids 335-400) adjacent to the C terminus of the phosphotyrosine-binding domain of IRS4. The N-terminal A, B, and phosphatase domains of SSH1 were bound to IRS4 independently. Whereas in vitro phosphatase assays revealed that IRS4 does not directly affect the cofilin phosphatase activity of SSH1, knockdown of IRS4 increased cofilin phosphorylation in cultured cells. Knockdown of IRS4 decreased phosphatidylinositol 3-kinase (PI3K) activity, and treatment with an inhibitor of PI3K increased cofilin phosphorylation. Akt preferentially phosphorylated SSH1 at Thr-826, but expression of a non-phosphorylatable T826A mutant of SSH1 did not affect insulin-induced cofilin dephosphorylation, and an inhibitor of Akt did not increase cofilin phosphorylation. These results suggest that IRS4 promotes cofilin dephosphorylation through sequential activation of PI3K and SSH1 but not through Akt. In addition, IRS4 co-localized with SSH1 in F-actin-rich membrane protrusions in insulin-stimulated cells, which suggests that the association of IRS4 with SSH1 contributes to localized activation of cofilin in membrane protrusions.

CAMP (C13orf8, ZNF828) is a novel regulator of kinetochore-microtubule attachment.

Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore-microtubule attachment, chromosome alignment-maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc-finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1-dependent phosphorylation at multiple sites during mitosis. CAMP-depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K-fibres to the tension exerted upon establishment of sister kinetochore bi-orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C-terminal region containing the zinc-finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP-E and CENP-F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP-E and CENP-F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore-microtubule attachment during bi-orientation.

Furry promotes acetylation of microtubules in the mitotic spindle by inhibition of SIRT2 tubulin deacetylase.

The structure and function of microtubules (MTs) are regulated by post-translational modifications of tubulin subunits, such as acetylation of the Lys40 residue of α-tubulin. Regulation of the organization and dynamics of MTs is essential for the precise formation of the mitotic spindle. Spindle MTs are highly acetylated, but the mechanism regulating this acetylation is largely unknown. Furry (Fry) is an evolutionarily conserved protein that binds to MTs and colocalizes with acetylated MTs in the mitotic spindle. In this study, we examined the role of Fry in the acetylation of MTs in the mitotic spindle. Depletion of Fry significantly reduced the level of MT acetylation in the mitotic spindle. Expression of the N-terminal fragment of Fry induced hyperacetylation of MTs in both mitotic and interphase cells. These results indicate that Fry promotes MT acetylation in the mitotic spindle. We also found that Fry binds to the tubulin deacetylase SIRT2, preferentially in mitotic cells. Cell-free experiments revealed that the N-terminal region of Fry is the domain responsible for binding to and inhibiting the tubulin-deacetylase activity of SIRT2. AGK2, a specific inhibitor of SIRT2, increased the level of MT acetylation in the mitotic spindle, indicating that SIRT2 is involved in the deacetylation of spindle MTs. Furthermore, AGK2 reversed the decrease in MT acetylation induced by Fry depletion. In summary, these results suggest that Fry plays a crucial role in promoting the level of MT acetylation in the mitotic spindle by inhibiting the tubulin-deacetylase activity of SIRT2.

Binding to Cep164, but not EB1, is essential for centriolar localization of TTBK2 and its function in ciliogenesis.

Primary cilia are formed by extending the microtubule-based axoneme from the mother centriole-derived basal body. Recruitment of Tau tubulin kinase-2 (TTBK2) to the mother centriole and subsequent removal of CP110 and its interactor Cep97 are crucial for the initiation of ciliogenesis. We analyzed the roles of two TTBK2-binding proteins, EB1 and Cep164, in centriolar localization of TTBK2. TTBK2 bound EB1 and Cep164 through its SxIP motifs and a proline-rich motif, respectively. Using TTBK2 variants that contained mutations in the SxIP or proline-rich motifs, we obtained evidence that Cep164, but not EB1, is essential for centriolar localization of TTBK2. Depletion of TTBK2 inhibited CP110 removal and ciliogenesis, whereas expression of wild-type TTBK2, but not non-Cep164-binding mutants, rescued CP110 removal and ciliogenesis in TTBK2-depleted cells. Therefore, Cep164 binding is essential for the function of TTBK2 in promoting CP110 removal and ciliogenesis. We also provide evidence that TTBK2 has the potential to effectively phosphorylate Cep164 and Cep97 and inhibits the interaction between Cep164 and its binding partner Dishevelled-3 (an important regulator of ciliogenesis) in a kinase activity-dependent manner.

Coactosin accelerates cell dynamism by promoting actin polymerization.

During development, cells dynamically move or extend their processes, which are achieved by actin dynamics. In the present study, we paid attention to Coactosin, an actin binding protein, and studied its role in actin dynamics. Coactosin was associated with actin and Capping protein in neural crest cells and N1E-115 neuroblastoma cells. Accumulation of Coactosin to cellular processes and its association with actin filaments prompted us to reveal the effect of Coactosin on cell migration. Coactosin overexpression induced cellular processes in cultured neural crest cells. In contrast, knock-down of Coactosin resulted in disruption of actin polymerization and of neural crest cell migration. Importantly, Coactosin was recruited to lamellipodia and filopodia in response to Rac signaling, and mutated Coactosin that cannot bind to F-actin did not react to Rac signaling, nor support neural crest cell migration. It was also shown that deprivation of Rac signaling from neural crest cells by dominant negative Rac1 (DN-Rac1) interfered with neural crest cell migration, and that co-transfection of DN-Rac1 and Coactosin restored neural crest cell migration. From these results we have concluded that Coactosin functions downstream of Rac signaling and that it is involved in neurite extension and neural crest cell migration by actively participating in actin polymerization.

Activation of cytosolic Slingshot-1 phosphatase by gelsolin-generated soluble actin filaments.

Slingshot-1 (SSH1) is a protein phosphatase that dephosphorylates and activates cofilin, an actin-severing and -disassembling protein. SSH1 is bound to and activated by F-actin, but not G-actin. SSH1 is accumulated in the F-actin-rich lamellipodium but is also diffusely distributed in the cytoplasm. It remains unknown whether SSH1 is activated by soluble (low-level polymerized) actin filaments in the cytoplasm. In this study, we show that SSH1 binds to gelsolin via actin filaments in the cytosolic fraction. Gelsolin promoted solubilization of actin filaments and SSH1 in cell-free assays and in cultured cells. SSH1 was activated by gelsolin-generated soluble actin filaments. Furthermore, gelsolin enhanced cofilin dephosphorylation in neuregulin-stimulated cells. Our results suggest that cytosolic SSH1 forms a complex with gelsolin via soluble actin filaments and is activated by gelsolin-generated soluble actin filaments and that gelsolin promotes stimulus-induced cofilin dephosphorylation through increasing soluble actin filaments, which support SSH1 activation in the cytoplasm.

CaMKIIß-mediated LIM-kinase activation plays a crucial role in BDNF-induced neuritogenesis.

LIM-kinase 1 (LIMK1) regulates actin cytoskeletal reorganization by phosphorylating and inactivating actin-depolymerizing factor and cofilin. We examined the role of LIMK1 in brain-derived neurotrophic factor (BDNF)-induced neuritogenesis in primary-cultured rat cortical neurons. Knockdown of LIMK1 or expression of a kinase-dead LIMK1 mutant suppressed BDNF-induced enhancement of primary neurite formation. By contrast, expression of an active form of LIMK1 promoted primary neuritogenesis in the absence of BDNF. BDNF-induced neuritogenesis was inhibited by KN-93, an inhibitor of Ca(2+) /calmodulin-dependent protein kinases (CaMKs), but not by STO-609, an inhibitor of CaMK-kinase (CaMKK). CaMKK activity is required for the activation of CaMKI and CaMKIV, but not CaMKII, which suggests that CaMKII is principally involved in BDNF-induced enhancement of neuritogenesis. Knockdown of CaMKIIß, but not CaMKIIα, suppressed BDNF-induced neuritogenesis. Active CaMKIIß promoted neuritogenesis, and this promotion was inhibited by knockdown of LIMK1, indicating that CaMKIIß is involved in BDNF-induced neuritogenesis via activation of LIMK1. Furthermore, in vitro kinase assays revealed that CaMKIIß phosphorylates LIMK1 at Thr-508 in the kinase domain and activates the cofilin-phosphorylating activity of LIMK1. In summary, these results suggest that CaMKIIß-mediated activation of LIMK1 plays a crucial role in BDNF-induced enhancement of primary neurite formation.

Calcium influx promotes PLEKHG4B localization to cell-cell junctions and regulates the integrity of junctional actin filaments.

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.

Solo regulates the localization and activity of PDZ-RhoGEF for actin cytoskeletal remodeling in response to substrate stiffness.

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.