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SummaryGut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism that ensure optimal energy balance. Yet, the interplay between diet, microbes, and host factors that sustain intestinal oscillations is complex and poorly understood. Here, we report the host C-type lectin antimicrobial peptideReg3γworks with key ileal microbes to orchestrate these interactions in a bi-directional manner, independent from the intestinal core circadian clock. High fat diet diminishes physiologically relevant microbial oscillators essential for host metabolic homeostasis, resulting in arrhythmic hostReg3γexpression and increased abundance and oscillation ofReg3γ-independent gut microbes. This illustrates a transkingdom co-evolved biological rhythm involving reciprocating, sensor-effector signals between key host and microbial components that ultimately drive metabolism, but are also heavily influenced by diet. Restoring the gut microbiota’s capacity to sense and transduce dietary signals mediated by specific host factors such asReg3γcould be harnessed to improve metabolic dysfunction.

Gut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism that ensure optimal energy balance. Yet, the interplay between diet, microbes, and host factors that sustain intestinal oscillations is complex and poorly understood. We report the host C-type lectin antimicrobial peptide REG3γ works with key ileal microbes to orchestrate these interactions in a bi-directional manner, independent from the intestinal core circadian clock. High fat diet diminishes physiologically relevant microbial oscillators essential for host metabolic homeostasis, resulting in arrhythmic host REG3γ expression and increased abundance and oscillation of REG3γ-independent gut microbes. This illustrates a transkingdom co-evolved biological rhythm involving reciprocating, sensor-effector signals between key host and microbial components that ultimately drive metabolism, but are also heavily influenced by diet. Restoring the gut microbiota’s capacity to sense and transduce dietary signals mediated by specific host factors such as REG3γ could be harnessed to improve metabolic dysfunction.

Adhesive intercellular connections at cardiomyocyte intercalated disks (IDs) support contractile force and maintain structural integrity of the heart muscle. Disturbances of the proteins at IDs deteriorate cardiac function and morphology. An adaptor protein afadin, one of the components of adherens junctions, is expressed ubiquitously including IDs. At present, the precise role of afadin in cardiac physiology or disease is unknown. To explore this, we generated conditional knockout (cKO) mice with cardiomyocyte-targeted deletion of afadin. Afadin cKO mice were born according to the expected Mendelian ratio and have no detectable changes in cardiac phenotype. On the other hand, chronic pressure overload induced by transverse aortic constriction (TAC) caused systolic dysfunction, enhanced fibrogenesis and apoptosis in afadin cKO mice. Afadin deletion increased macrophage infiltration and monocyte chemoattractant protein-1 expression, and suppressed transforming growth factor (TGF) β receptor signaling early after TAC procedure. Afadin also associated with TGFβ receptor I at IDs. Pharmacological antagonist of TGFβ receptor I (SB431542) augmented mononuclear infiltration and fibrosis in the hearts of TAC-operated control mice. In conclusion, afadin is a critical molecule for cardiac protection against chronic pressure overload. The beneficial effects are likely to be a result from modulation of TGFβ receptor signaling pathways by afadin.

In palatogenesis, bilateral palatal shelves grow and fuse with each other to establish mesenchyme continuity across the horizontal palate. The palatal shelves are covered with the medial edge epithelium (MEE) in which most apical cells are periderm cells. We investigated localization and roles of tight junction (TJ) and adherens junction (AJ) components and an apical membrane marker in the MEE in palatogenesis. Immunofluorescence and immunoelectron microscopy analyses revealed that TJs were located at the boundary between neighboring periderm cells, whereas AJ components were localized at the boundary between all epithelial cells in the MEE. Specifically, typical AJs were observed at the boundaries between neighboring periderm cells and between periderm cells and underlying epithelial cells where the signal for nectin-1 was observed. The TGF-β-induced desquamation of periderm cells reduced the polarity of remaining epithelial cells as estimated by changes of epithelial cell morphology and the staining of the polarity marker and the AJ components. These less polarized epithelial cells then intermingled and finally disappeared at least partly by apoptosis. These results indicate that periderm cells covering growing palatal shelves have bona fide TJs and their desquamation reduces the polarity of palatal shelf epithelial cells in palatogenesis.

In the auditory epithelium of the cochlea, the sensory hair cells and supporting cells are arranged in a checkerboard-like fashion, but the mechanism underlying this cellular patterning is unclear. We found that mouse hair cells and supporting cells express the immunoglobulin-like adhesion molecules nectin-1 and -3, respectively, and that their interaction mediates the heterotypic adhesion between these two cell types. Genetic removal of nectin-1 or -3 disrupted the checkerboard-like pattern, inducing aberrant attachment between hair cells. When cells expressing either nectin-1 or -3 were cocultured, they arranged themselves into a mosaic pattern. Thus, nectin-1 and -3 promote the formation of the checkerboard-like pattern of the auditory epithelia.

Afadin interacts with the cytoplasmic region of nectins, which are immunoglobulin-like cell adhesion molecules at adherens junctions, and links them to the actin cytoskeleton. Afadin regulates activities of cells in culture such as directional motility, proliferation and survival. We used Cre-loxP technology to generate mice conditionally lacking afadin specifically in the intestinal epithelia after birth. The loss of afadin caused increased paracellular permeability in the intestinal mucosa and enhanced susceptibility to the tissue destruction induced by dextran sulfate sodium. The junctional architecture of the intestinal epithelia appeared to be preserved, whereas the deficiency of afadin caused the mislocalization of nectin-2 and nectin-3 from adherens junctions to basolateral membrane domains but not that of other components of apical junctions. By contrast, such phenotypic changes were undetected in mice lacking nectin-2, nectin-3 or both. These findings suggest that afadin plays crucial roles, independently of the role as the nectin–afadin module, in barrier function and homeostasis of the intestinal epithelia once the epithelial structure has been established.

Nectins are immunoglobulin-like cell adhesion proteins and their interactions recruit various cell-cell junctions. Mutations in human NECTIN-1 cause an ectodermal dysplasia syndrome, but Nectin-1 null mice have only slight defects in teeth, suggesting compensation by other nectin(s). We observed overlapping expression of nectin-3 with nectin-1 and enamel abnormality in the nectin-3 mutant. We, therefore, generated nectin-1;nectin-3 compound mutants. However, all teeth developed and no significant dental abnormalities were observed before birth. At postnatal day 10, the upper molars of compound mutants exhibited conical crown shape and retarded enamel maturation. Nectin-1 was expressed in ameloblasts whereas nectin-3 was expressed in neighboring stratum intermedium cells at this stage. The immunohistochemical localization and electron microscopical observations indicated that the desmosomal junctions between stratum intermedium and ameloblasts were significantly reduced. These results suggest that heterophilic interaction between nectin-1 and nectin-3 recruits desmosomal junctions, and that these are required for proper enamel formation.

Differential expression of cell adhesion molecules regulates stem cell location, self-renewal and lineage selection under steady state conditions and during tissue repair. We show that the intercellular adhesion protein nectin-like molecule 2 (Necl2) is highly expressed in bulge stem cells of adult human and mouse hair follicles. Overexpression of Necl2 in cultured human keratinocytes led to upregulation of calcium/calmodulin-associated Ser/Thr kinase (CASK), increased calcium-independent intercellular adhesion, and inhibition of cell motility and in vitro wound healing. Although the rate of cell proliferation was reduced, terminal differentiation was unaffected. To assess the role of Necl2 in vivo, we examined the epidermis of Necl2-null mice and developed transgenic mice that expressed Necl2 in the basal layer of murine epidermis. Necl2 overexpression led to a reduction in S-phase cells and an increase in quiescent cells retaining DNA label in the bulge. Although epidermal homeostasis appeared normal in both transgenic and knockout mice, wound healing was markedly delayed. Necl2 overexpression resulted in reduced proliferation and increased levels of CASK and E-cadherin at the leading edge of healing wounds, consistent with its effects in culture. Our results demonstrate that Necl2 is involved in regulating epidermal stem cell quiescence and location.

In vivo immune response is triggered in the lymph node, where lymphocytes for entry into, retention at, and migration to effector sites are dynamically regulated. The molecular mechanism underlying retention regulation is the key to elucidating in vivo regulation of immune response. In this study, we describe the function of the adhesion molecule class I-restricted T cell-associated molecule (CRTAM) in regulating CD8+ T cell retention within the lymph node and eventually effector function. We previously identified CRTAM as a receptor predominantly expressed on activated CD8+ T cells, and nectin-like molecule-2 (Necl2) as its ligand. In vivo function of CRTAM-Necl2 interaction was analyzed by generating CRTAM−/− mice. CRTAM−/− mice exhibited reduced protective immunity against viral infection and impaired autoimmune diabetes induction in vivo. Although Ag-specific CRTAM−/− CD8+ T cells showed normal CTL functions in vitro, their number in the draining lymph node was reduced. Because CRTAM+ T cells bound efficiently to Necl2-expressing CD8+ dendritic cells (DCs) that reside in T cell area of lymph node, CRTAM may induce retention by binding to CD8+ DCs at the late stage of activation before proliferation. The CRTAM-mediated late interaction with DCs induced retention of activated CD8+ T cells in an Ag-independent fashion, and this possibly resulted in effective CTL development in the draining lymph node.

In the hippocampus, synapses are formed between mossy fiber terminals and CA3 pyramidal cell dendrites and comprise highly developed synaptic junctions (SJs) and puncta adherentia junctions (PAJs). Dynamic remodeling of synapses in the hippocampus is implicated in learning and memory. Components of both the nectin–afadin and cadherin–catenin cell adhesion systems exclusively accumulate at PAJs. We investigated the role of afadin at synapses in mice in which the afadin gene was conditionally inactivated in hippocampal neurons. In these mutant mice, the signals for not only nectins, but also N-cadherin and β-catenin, were hardly detected in the CA3 area, in addition to loss of the signal for afadin, resulting in disruption of PAJs. Ultrastructural analysis revealed an increase in the number of perforated synapses, suggesting the instability of SJs. These results indicate that afadin is involved not only in the assembly of nectins and cadherins at synapses, but also in synaptic remodeling.

The Doc2 family comprises the brain-specific Doc2alpha and the ubiquitous Doc2beta and Doc2gamma. With the exception of Doc2gamma, these proteins exhibit Ca(2+)-dependent phospholipid-binding activity in their Ca(2+)-binding C2A domain and are thought to be important for Ca(2+)-dependent regulated exocytosis. In excitatory neurons, Doc2alpha interacts with Munc13-1, a member of the Munc13 family, through its N-terminal Munc13-1-interacting domain and the Doc2alpha-Munc13-1 system is implicated in Ca(2+)-dependent synaptic vesicle exocytosis. The Munc13 family comprises the brain-specific Munc13-1, Munc13-2, and Munc13-3, and the non-neuronal Munc13-4. We previously showed that Munc13-4 is involved in Ca(2+)-dependent secretory lysosome exocytosis in mast cells, but the involvement of Doc2 in this process is not determined. In the present study, we found that Doc2alpha but not Doc2beta was endogenously expressed in the RBL-2H3 mast cell line. Doc2alpha colocalized with Munc13-4 on secretory lysosomes, and interacted with Munc13-4 through its two regions, the N terminus containing the Munc13-1-interacting domain and the C terminus containing the Ca(2+)-binding C2B domain. In RBL-2H3 cells, Ca(2+)-dependent secretory lysosome exocytosis was inhibited by expression of the Doc2alpha mutant lacking either of the Munc13-4-binding regions and the inhibition was suppressed by coexpression of Munc13-4. Knockdown of endogenous Doc2alpha also reduced Ca(2+)-dependent secretory lysosome exocytosis, which was rescued by re-expression of human Doc2alpha but not by its mutant that could not bind to Munc13-4. Moreover, Ca(2+)-dependent secretory lysosome exocytosis was severely reduced in bone marrow-derived mast cells from Doc2alpha knockout mice. These results suggest that the Doc2alpha-Muunc13-4 system regulates Ca(2+)-dependent secretory lysosome exocytosis in mast cells.

Actin dynamics play multiple roles in promoting cell movement, changing cell shapes, and establishing intercellular adhesion. Cell contact events are involved in tissue morphogenesis, immune responses, and cancer cell invasion. In epithelial cells, cell–cell contacts mature to form apical junctions with which the actin cytoskeleton physically associates. Living cell imaging shows, however, that the apical junctional complex is less dynamically regulated than the actin cytoskeleton, indicating that their interaction does not remain stable. Given that several cell adhesion modules are clustered at apical junctions, the sum of weak or transient interactions may create linkages that can be strong yet easily remodeled. Here we describe how subcellular protein interactions are coordinated to induce changes in actin organization and dynamics, in response to the status of apical junctions.

Afadin directly links nectin, an immunoglobulin-like cell–cell adhesion molecule, to actin filaments (F-actin) at adherens junctions (AJs). The nectin–afadin complex is important for the formation of not only AJs but also tight junctions (TJs) in epithelial cells. Studies using afadin-knockout mice have revealed that afadin is indispensable for embryonic development by organizing the formation of cell–cell junctions. However, the molecular mechanism of cell–cell junction disorganization during embryonic development in afadin-knockout mice is poorly understood. To address this, we took advantage of embryoid bodies (EBs) as a model system. The formation of cell–cell junctions including AJs and TJs was impaired in afadin-null EBs. The proper accumulation of the Par complex and the activation of Cdc42 and atypical PKC (aPKC), which are crucial for the formation of cell polarity, were also inhibited by knockout of afadin. In addition, the disruption of afadin caused the abnormal deposition of laminin and the dislocalization of its receptors integrin α6 and integrin β1. These results indicate that afadin organizes the formation of cell–cell junctions by regulating cell polarization in early embryonic development.

Rho family GTPases have been implicated in the regulation of endothelial permeability via their actions on actin cytoskeletal organization and integrity of interendothelial junctions. In cell culture studies, activation of RhoA disrupts interendothelial junctions and increases endothelial permeability, whereas activation of Rac1 and Cdc42 enhances endothelial barrier function by promoting the formation of restrictive junctions. The primary regulators of Rho proteins, guanine nucleotide dissociation inhibitors (GDIs), form a complex with the GDP-bound form of the Rho family of monomeric G proteins, and thus may serve as a nodal point regulating the activation state of RhoGTPases. In the present study, we addressed the in vivo role of RhoGDI-1 in regulating pulmonary microvascular permeability using RhoGDI-1 −/− mice. We observed that basal endothelial permeability in lungs of RhoGDI-1 −/− mice was 2-fold greater than wild-type mice. This was the result of opening of interendothelial junctions in lung microvessels which are normally sealed. The activity of RhoA (but not of Rac1 or Cdc42) was significantly increased in RhoGDI-1 −/− lungs as well as in cultured endothelial cells on downregulation of RhoGDI-1 with siRNA, consistent with RhoGDI-1–mediated modulation RhoA activity. Thus, RhoGDI-1 by repressing RhoA activity regulates lung microvessel endothelial barrier function in vivo. In this regard, therapies augmenting endothelial RhoGDI-1 function may be beneficial in reestablishing the endothelial barrier and lung fluid balance in lung inflammatory diseases such as acute respiratory distress syndrome.

The tumor suppressor p53 is a key transcriptional factor regulating the induction of cellular senescence by oncogenic signals. The activity of p53 is regulated by recruitment into promyelocytic leukemia (PML)-nuclear bodies (NBs) as well as by stabilization through posttranslational modifications such as phosphorylation and acetylation. Here we found that MORC3 (microrchidia3)-ATPase activated p53 and induced cellular senescence in normal human and mouse fibroblasts but not p53−/− fibroblasts. Conversely, genotoxic stress–induced phosphorylation and stabilization of p53 but barely increased its transcriptional activity in Morc3−/− fibroblasts. MORC3 localized on PML-NBs in presence of PML and mediated recruitment of p53 and CREB-binding protein (CBP) into PML-NBs. In contrast, expression of ATPase activity-deficient mutant MORC3-E35A or siRNA repression of MORC3 impaired the localization of p53 and Sp100 but not CBP on PML-NBs. These results suggest that MORC3 regulates p53 activity and localization into PML-NBs. We identified a new molecular mechanism that regulates the activity of nuclear proteins by localization to a nuclear subdomain.

Myeloid dendritic cells (mDCs) recognize and respond to polyI:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyI:C to mice and in vivo are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor NK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyI:C led to the retardation of tumor growth, an effect relied on by NK activation. This NK-dependent tumor regression did not occur in TICAM-1 −/− or IFNAR −/− mice, whereas a normal NK antitumor response was induced in PKR −/− , MyD88 −/− , IFN-β −/− , and wild-type mice. IFNAR was a prerequisite for the induction of IFN-α/β and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyI:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vivo transwell analysis. Implanted tumor growth in IFNAR −/− mice was retarded by adoptively transferring polyI:C-treated TICACM-1-positive mDCs but not TICAM-1 −/− mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor NK, resulting in the regression of low MHC-expressing tumors.

Nectins and nectin-like molecules (Necls) are structurally related transmembrane proteins primarily involved in cell adhesion. Nectins and afadin, the adaptor or anchoring protein, stabilize the epithelium and endothelium and establish apical-basal polarity of epithelial cells, independently or in cooperation with other cell adhesion molecules. Necls facilitate cell–cell communication implicated in cell movement and proliferation, immune responses, and cancer cell phenotypes. Necls interact with nectins and specific ligands at cell–cell contacts, whereas Necls associate with integrin αvβ3 and growth factor receptors on the same cell surface. Besides their roles in cell adhesion, nectins regulate the activities of Rho family small G proteins which play critical roles in maintaining the apical junctions of epithelial cells through reorganization of the actin cytoskeleton. Since mice lacking the Rho GDP-dissociation inhibitor (GDI)α show massive proteinuria and degeneration of renal epithelial cells, nectins and other cell adhesion molecules may play roles in the structural and functional aspects of renal diseases. Here we summarize our knowledge of nectins and Necls and discuss cell adhesion biology in the kidney.

Rho family small GTP-binding proteins, including Rho, Rac, and Cdc42, are key determinants of cell movement and actin-dependent cytoskeletal morphogenesis. Rho GDP-dissociation inhibitor (GDI) α and Rho GDIβ (or D4/Ly-GDI), closely related regulators for Rho proteins, are both expressed in hemopoietic cell lineages. Nevertheless, the functional contributions of Rho GDIs remain poorly understood in vivo. In this study, we report that combined disruption of both the Rho GDIα and Rho GDIβ genes in mice resulted in reduction of marginal zone B cells in the spleen, retention of mature T cells in the thymic medulla, and a marked increase in eosinophil numbers. Furthermore, these mice showed lower CD3 expression and impaired CD3-mediated proliferation of T cells. While B cells showed slightly enhanced chemotactic migration in response to CXCL12, peripheral T cells showed markedly reduced chemotactic migration in response to CCL21 and CCL19 associated with decreased receptor levels of CCR7. Overall, Rho protein levels were reduced in the bone marrow, spleen, and thymus but sustained activation of the residual part of RhoA, Rac1, and Cdc42 was detected mainly in the bone marrow and spleen. Rho GDIα and Rho GDIβ thus play synergistic roles in lymphocyte migration and development by modulating activation cycle of the Rho proteins in a lymphoid organ-specific manner.

Rab3A small G protein is a member of the Rab family and is most abundant in the brain, where it is localized on synaptic vesicles. Evidence is accumulating that Rab3A plays a key role in neurotransmitter release and synaptic plasticity. Rab3A cycles between the GDP-bound inactive and GTP-bound active forms, and this change in activity is associated with the trafficking cycle of synaptic vesicles at nerve terminals. Rab3 GTPase-activating protein (GAP) stimulates the GTPase activity of Rab3A and is expected to determine the timing of the dissociation of Rab3A from synaptic vesicles, which may be coupled with synaptic vesicle exocytosis. Rab3 GAP consists of two subunits: the catalytic subunit p130 and the noncatalytic subunit p150. Recently, mutations in p130 were found to cause Warburg Micro syndrome with severe mental retardation. Here, we generated p130-deficient mice and found that the GTP-bound form of Rab3A accumulated in the brain. Loss of p130 in mice resulted in inhibition of Ca 2+ -dependent glutamate release from cerebrocortical synaptosomes and altered short-term plasticity in the hippocampal CA1 region. Thus, Rab3 GAP regulates synaptic transmission and plasticity by limiting the amount of the GTP-bound form of Rab3A.

Neurites recognize their specific partners during the formation of interneuronal connections. In hippocampal pyramidal neurons, axons attach to dendrites for their synaptogenesis, but the dendrites do not form stable contacts with each other, suggesting the presence of a mechanism to allow their selective associations. Nectin-1 (N1), an immunoglobulin domain adhesive protein, is preferentially localized in axons, and its heterophilic partner, N3, is present in both axons and dendrites; we tested their potential roles in interneurite recognition. The overexpression of N1, causing its mislocalization to dendrites, induced atypical dendrodendritic as well as excessive axodendritic associations. On the contrary, the genetic deletion of N1 loosened the contacts between axons and dendritic spines. Those actions of nectins required cadherin–catenin activities, but the overexpression of cadherin itself could not accelerate neurite attachment. These results suggest that the axon-biased localization of N1 and its trans-interaction with N3 in cooperation with the cadherin machinery is critical for the ordered association of axons and dendrites.

Synapses are specialized intercellular junctions whose specificity and plasticity are mediated by synaptic cell adhesion molecules. In hippocampus, the mossy fibers form synapses on the apical dendrites of the CA3 pyramidal cells where synaptic and puncta adherentia junctions (PAJs) are highly developed. Synaptic junctions are the sites of neurotransmission, while PAJs are regarded as mechanical adhesion sites. Cell–cell adhesion molecules nectin-1 and nectin-3 asymmetrically localize at the pre- and post-synaptic sides of PAJs, respectively. To reveal the definitive role of nectins, we analyzed nectin-1−/− and nectin-3−/− mice. In both the mutant mice, the number of PAJs at the synapses between the mossy fiber terminals and the dendrites of the CA3 pyramidal cells was reduced. In addition, the abnormal mossy fiber trajectory was observed. These results indicate that nectins are involved in the formation of PAJs, which maintain the proper mossy fiber trajectory.

Cdc42 is a member of the Rho family of GTPases and plays an important role in the regulation of actin cytoskeletal organization. Activation of Cdc42 and associated signal transduction cascades are dependent upon proper localization of this GTPase. The studies described herein address the hypothesis that Rho GDP-dissociation inhibitor, RhoGDI, plays an essential role in the translocation of Cdc42 to signaling complexes at the plasma membrane and is essential for Cdc42-mediated actin cytoskeletal rearrangements. An activating mutant of Cdc42 that is RhoGDI-binding defective (Cdc42(G12V/R66E)) is characterized and used as a tool to study the functional importance of the Cdc42-RhoGDI interaction. Overexpression of mycCdc42(G12V/R66E) in COS-7 cells results in actin cytoskeletal rearrangements that are indistinguishable from those stimulated by overexpression of mycCdc42(G12V). In addition, the G12V activating mutant of Cdc42 was overexpressed in mesangial cells that are null for RhoGDI expression. MycCdc42(G12V) stimulation of filopodia formation in these cells was indistinguishable from that observed in wild-type mesangial cells. Taken together, the results presented herein indicate that although RhoGDI is a critical regulator of guanine nucleotide binding, cycling of Cdc42 between membranes and the cytosol and cellular transformation, it is not essential for Cdc42-mediated organization of the actin cytoskeleton.

Afadin is an actin filament-binding protein that acts cooperatively in cell adhesion with the cell adhesion molecule nectin, and in directional cell movement with the small G protein Rap1 in a nectin-independent manner. We studied the role of afadin in the organization of the small intestinal epithelium using afadin conditional gene knockout (cKO) mice. Afadin was localized at adherens junctions of all types of epithelial cells throughout the crypt-villus axis. Paneth cells were localized at the base of the crypt in control mice, but not confined there, and migrated into the villi in afadin-cKO mice. The distribution of other types of epithelial cells did not change significantly in the mutant mice. The Paneth cells remaining in the crypt exhibited abnormal shapes, were buried between adjacent cells, and did not face the lumen. In these cells, the formation of adherens junctions and tight junctions was impaired. Rap1 and EphB3 were highly expressed in control Paneth cells but markedly down-regulated in the afadin-deficient Paneth cells. Taken together, the results indicate that afadin plays a role in the restricted localization of Paneth cells at the base of the crypt by maintaining their adhesion to adjacent crypt cells and inhibiting their movement toward the top of villi.

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca2+-dependent neurotransmitter release. These small G proteins are activated by Rab3 GDP/GTP exchange protein (Rab3 GEP). To determine the function of Rab3 GEP during neurotransmitter release, we have knocked out Rab3 GEP in mice. Rab3 GEP−/− mice developed normally but died immediately after birth. Embryos at E18.5 showed no evoked action potentials of the diaphragm and gastrocnemius muscles in response to electrical stimulation of the phrenic and sciatic nerves, respectively. In contrast, axonal conduction of the spinal cord and the phrenic nerve was not impaired. Total numbers of synaptic vesicles, especially those docked at the presynaptic plasma membrane, were reduced at the neuromuscular junction ∼10-fold compared with controls, whereas postsynaptic structures and functions appeared normal. Thus, Rab3 GEP is essential for neurotransmitter release and probably for formation and trafficking of the synaptic vesicles.

Rab GDP dissociation inhibitor α (Rab GDIα) is a regulator of the Rab small G proteins implicated in neurotransmission, and mutations of Rab GDIα cause human X-linked mental retardation associated with epileptic seizures. In Rab GDIα-deficient mice, synaptic potentials in the CA1 region of the hippocampus displayed larger enhancement during repetitive stimulation, which was apparently opposite to the phenotype of Rab3A-deficient mice. Furthermore, the Rab GDIα-deficient mice showed hypersensitivity to bicuculline, an inducer of epileptic seizures. These results suggest that Rab GDIα plays a specialized role in Rab3A recycling to suppress hyperexcitability via modulation of presynaptic forms of plasticity.

Afadin is an actin filament–binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell–cell adherens junctions (AJs). To explore the function of afadin in cell–cell adhesion during embryogenesis, we generated afadin−/− mice and embryonic stem cells. In wild-type mice at embryonic days 6.5–8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin−/− mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin−/− embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell–cell AJs and tight junctions were improperly organized in the ectoderm of afadin−/− mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.

Mouse embryonic stem cells (ESCs) have the potential to differentiate into germ cells (GCs) in vivo and in vitro. Interestingly, XY ESCs can give rise to both male and female GCs in culture, irrespective of the genetic sex. Recent studies showed that ESC-derived primordial GCs contributed to functional gametogenesis in vivo; however, in vitro differentiation techniques have never succeeded in generating mature oocytes from ESCs due to cryptogenic growth arrest during the preantral follicle stages of development. To address this issue, a mouse ESC line, capable of producing follicle-like structures (FLSs) efficiently, was established to investigate their properties using conventional molecular biological methods. The results revealed that the ESC-derived FLSs were morphologically similar to ovarian primary-to-secondary follicles but never formed an antrum; instead, the FLSs eventually underwent abnormal development or cell death in culture, or formed teratomas when transplanted under the kidney capsule in mice. Gene expression analyses demonstrated that the FLSs lacked transcripts for genes essential to late folliculogenesis, including gonadotropin receptors and steroidogenic enzymes, whereas some other genes were overexpressed in FLSs compared to the adult ovary. The E-Cadherin protein, which is involved in cell-to-cell interactions, was also expressed ectopically. Remarkably, it was seen that oocyte-like cells in the FLSs exhibited androgenetic genomic imprinting, which is ordinarily indicative of male GCs. Although the FLSs did not express male GC marker genes, the DNA methyltransferase, Dnmt3L, was expressed at an abnormally high level. Furthermore, the expression of sex determination factors was ambiguous in FLSs as both male and female determinants were expressed weakly. These data suggest that the developmental dysfunction of the ESC-derived FLSs may be attributable to aberrant gene expression and genomic imprinting, possibly associated with uncertain sex determination in culture.

Polycomb-group (PcG) complex 1 acts as an E3 ubiquitin ligase both for histone H2A to silence transcription and for geminin to regulate its stability. Scmh1 is a substoichiometric component of PcG complex 1 that provides the complex with an interaction domain for geminin. Scmh1 is unstable and regulated through the ubiquitin-proteasome system, but its molecular roles are unknown, so we generated Scmh1-deficient mice to elucidate its function. Loss of Scmh1 caused derepression of Hoxb4 and Hoxa9, direct targets of PcG complex 1-mediated transcriptional silencing in hematopoietic cells. Double knockdown of Hoxb4 and Hoxa9 or transduction of a dominant-negative Hoxb4N→A mutant caused geminin accumulation. Age-related transcriptional downregulation of derepressed Hoxa9 also leads to geminin accumulation. Transduction of Scmh1 lacking a geminin-binding domain restored derepressed expression of Hoxb4 and Hoxa9 but did not downregulate geminin like full-length Scmh1. Each of Hoxb4 and Hoxa9 can form a complex with Roc1-Ddb1-Cul4a to act as an E3 ubiquitin ligase for geminin. We suggest that geminin dysregulation may be restored by derepressed Hoxb4 and Hoxa9 in Scmh1-deficient mice. These findings suggest that PcG and a subset of Hox genes compose a homeostatic regulatory system for determining expression level of geminin.

Osteoclasts are multinucleated giant cells that reside in osseous tissues and resorb bone. Signaling mediated by receptor activator of nuclear factor (NF)-κB (RANK) and its ligand leads to the nuclear factor of activated T cells 2/c1 (NFAT2 or NFATc1) expression, a critical step in the formation of functional osteoclasts. In addition, adaptor proteins harboring immunoreceptor tyrosine-based activation motifs, such as DNAX-activating protein of 12 kDa (DAP12), play essential roles. In this study, we identified the gene encoding the lectin Siglec-15 as NFAT2-inducible, and we found that the protein product links RANK ligand-RANK-NFAT2 and DAP12 signaling in mouse osteoclasts. Both the recognition of sialylated glycans by the Siglec-15 V-set domain and the association with DAP12 through its Lys-272 are essential for its function. When Siglec-15 expression was knocked down, fewer multinucleated cells developed, and those that did were morphologically contracted with disordered actin-ring structures. These changes were accompanied by significantly reduced bone resorption. Siglec-15 formed complexes with Syk through DAP12 in response to vitronectin. Furthermore, chimeric molecules consisting of the extracellular and transmembrane regions of Siglec-15 with a K272A mutation and the cytoplasmic region of DAP12 significantly restored bone resorption in cells with knocked down Siglec-15 expression. Together, these results suggested that the Siglec-15-DAP12-Syk-signaling cascade plays a critical role in functional osteoclast formation.

Rationale: Vascular endothelial growth factor (VEGF), a major proangiogenic agent, exerts its proangiogenic action by binding to VEGF receptor 2 (VEGFR2), the activity of which is regulated by direct interactions with other cell surface proteins, including integrin α V β 3 . However, how the interaction between VEGFR2 and integrin α V β 3 is regulated is not clear. Objective: To investigate whether Necl-5/poliovirus receptor, an immunoglobulin-like molecule that is known to bind integrin α V β 3 , regulates the interaction between VEGFR2 and integrin α V β 3 , and to clarify the role of Necl-5 in the VEGF-induced angiogenesis. Methods and Results: Necl-5-knockout mice displayed no obvious defect in vascular development; however, recovery of blood flow after hindlimb ischemia and the VEGF-induced neovascularization in implanted Matrigel plugs were impaired in Necl-5-knockout mice. To clarify the mechanism of the regulation of angiogenesis by Necl-5, we investigated the roles of Necl-5 in the VEGF-induced angiogenic responses in vitro. Knockdown of Necl-5 by siRNAs in human umbilical vein endothelial cells (HUVECs) inhibited the VEGF-induced capillary-like network formation on Matrigel, migration, and proliferation, and conversely, enhanced apoptosis. Coimmunoprecipitation assays showed the interaction of Necl-5 with VEGFR2, and knockdown of Necl-5 prevented the VEGF-induced interaction of integrin α V β 3 with VEGFR2. Knockdown of Necl-5 suppressed the VEGFR2-mediated activation of downstream proangiogenic and survival signals, including Rap1, Akt, and endothelial nitric oxide synthase. Conclusions: These results demonstrate the critical role of Necl-5 in angiogenesis and suggest that Necl-5 may regulate the VEGF-induced angiogenesis by controlling the interaction of VEGFR2 with integrin α v β 3 , and the VEGFR2-mediated Rap1-Akt signaling pathway.

Nectins have recently been identified as new cell adhesion molecules (CAMs) consisting of four members. They show immunoglobulin-like structures and exclusively localize at adherens junctions (AJs) between two neighboring cells. During the formation of cell-cell junctions, nectins function in cooperation with or independently of cadherins, major CAMs at AJs. Similar to cadherins, which are linked to the actin cytoskeleton by binding to catenins, nectins also bind to afadin through their C-terminal region and are linked to the actin cytoskeleton. In addition to nectins, there are nectin-like molecules (Necls), which resemble nectins in their structures and consist of five members. Nectins and Necls are involved in the formation of various kinds of cell-cell adhesion, and also play key roles in diverse cellular functions including cell movement, proliferation, survival, and differentiation. Thus, nectins and Necls are crucial for physiology and pathology of multicellular organisms.(Communicated by Shigetada NAKANISHI, M.J.A.).

The intercalated disc, a cell-cell contact site between neighboring cardiac myocytes, plays an important role in maintaining the homeostasis of the heart by transmitting electric and mechanical signals. Changes in the architecture of the intercalated disc have been observed in dilated cardiomyopathy. Among cell-cell junctions in the intercalated disc, adherens junctions are involved in anchoring myofibrils and transmitting force. Nectins are Ca 2+ -independent, immunoglobulin-like cell-cell adhesion molecules that exist in adherens junctions. However, the role of nectins in cardiac homeostasis and integrity of the intercalated disc are unknown. Among the isoforms of nectins, nectin-2 and -4 were expressed at the intercalated disc in the heart. Nectin-2–knockout mice showed normal cardiac structure and function under physiological conditions. Four weeks after banding of the ascending aorta, cardiac function was significantly impaired in nectin-2–knockout mice compared with wild-type mice, although both nectin-2–knockout and wild-type mice developed similar degrees of cardiac hypertrophy. Banded nectin-2–knockout mice displayed cardiac fibrosis more evidently than banded wild-type mice. The disruption of the intercalated discs and disorganized myofibrils were observed in banded nectin-2–knockout mice. Furthermore, the number of apoptotic cardiac myocytes was increased in banded nectin-2–knockout mice. In the hearts of banded nectin-2–knockout mice, Akt remained at lower phosphorylation levels until 2 weeks after banding, whereas c-Jun N-terminal kinase and p38 mitogen-activated protein kinase were highly phosphorylated compared with those of wild-type mice. These results indicate that nectin-2 is required to maintain structure and function of the intercalated disc and protects the heart from pressure-overload–induced cardiac dysfunction.

A new transforming gene has been molecularly cloned from hamster SHOK cells transformed with DNA extracted from a human thyroid carcinoma cell line and named the cot (cancer Osaka thyroid) oncogene. cDNA sequencing disclosed that this oncogene codes for a protein with 415 amino acid residues, and computer matching showed 42 to 48% similarity matches with serine protein kinases. Its gene product was identified as a 52-kDa protein by transcription and translation in vitro. Expression of cot cDNA under transcriptional control by a retroviral long terminal repeat induced morphological transformation of NIH 3T3 cells as well as SHOK cells. Protein kinase activity associated with constructed p60gag-cot was detected by immune complex kinase assay with anti-gag antiserum. The cot oncogene was overexpressed in transformed SHOK cells and found to have a rearranged 3' end in the last coding exon, which probably resulted in a deletion and an altered C' terminus in the transforming protein. This DNA rearrangement appeared to have occurred during transfection of the tumor DNA into hamster SHOK cells and not in the original thyroid tumor.

Nectins and nectin-like molecules (Necls) are immunoglobulin-like transmembrane cell adhesion molecules that are expressed in various cell types. Homophilic and heterophilic engagements between family members provide cells with molecular tools for intercellular communications. Nectins primarily regulate cell-cell adhesions, whereas Necls are involved in a greater variety of cellular functions. Recent studies have revealed that nectins and NECL-5, in cooperation with integrin alphavbeta3 and platelet-derived growth factor receptor, are crucial for the mechanisms that underlie contact inhibition of cell movement and proliferation; this has important implications for the development and tissue regeneration of multicellular organisms and the phenotypes of cancer cells.

Evidence is accumulating that Rab3A plays a key role in neurotransmitter release and synaptic plasticity. Recently mutations in the catalytic subunit p130 and the noncatalytic subunit p150 of Rab3 GTPase–activating protein were found to cause Warburg Micro syndrome and Martsolf syndrome, respectively, both of which exhibit mental retardation. We have found that loss of p130 in mice results in inhibition of Ca 2+ ‐dependent glutamate release from cerebrocortical synaptosomes and alters short‐term plasticity in the hippocampal CA1 region, probably through the accumulation of the GTP‐bound form of Rab3A. Here, we describe the procedures for the measurement of the GTP‐bound pool of Rab3A with pull‐down assay using mouse brains and the biochemical method for the measurement of glutamate release from mouse synaptosomes.

Nectin is an immunoglobulin-like cell-cell adhesion molecule, which plays essential roles in the initial step of formation of adherens junctions and tight junctions. We demonstrate here the role of nectin-1 in the epidermis using nectin-1–/– mice. Newborn nectin-1–/– pups showed shiny and slightly reddish skin; the amount of loricrin, one of the differentiation markers and also a major component of cornified cell envelopes, was markedly reduced in the epidermis of nectin-1–/– mice. The amounts of repetin and SPRRP, other components of cornified cell envelopes, were markedly elevated probably due to a compensatory mechanism to overcome the impaired expression of loricrin. However, cornified cells from nectin-1–/– mice were sensitive to mechanical stress. Moreover, Ca2+-induced activation of ERK through Rap1 and expression of loricrin were reduced in primary cultured nectin-1–/– keratinocytes; in turn, the inhibition of ERK activation reduced the amount of loricrin in wild-type keratinocytes. These results indicate that nectin-1 plays a key role in the expression of loricrin in the epidermis.

Synaptic scaffolding molecule (S-SCAM) interacts with a wide variety of molecules at excitatory and inhibitory synapses. It comprises three alternative splicing variants, S-SCAMalpha, -beta, and -gamma. We generated mutant mice lacking specifically S-SCAMalpha. S-SCAMalpha-deficient mice breathe and feed normally but die within 24 h after birth. Primary cultured hippocampal neurons from mutant mice have abnormally elongated dendritic spines. Exogenously expressed S-SCAMalpha corrects this abnormal morphology, while S-SCAMbeta and -gamma have no effect. Active RhoA decreases in cortical neurons from mutant mice. Constitutively active RhoA and ROCKII shift the length of dendritic spines toward the normal level, whereas ROCK inhibitor (Y27632) blocks the effect by S-SCAMalpha. S-SCAMalpha fails to correct the abnormal spine morphology under the treatment of N-methyl-d-aspartate (NMDA) receptor inhibitor (AP-5), Ca(2+)/calmodulin kinase inhibitor (KN-62), or tyrosine kinase inhibitor (PP2). NMDA treatment increases active RhoA in dendrites in wild-type hippocampal neurons, but not in mutant neurons. The ectopic expression of S-SCAMalpha, but not -beta, recovers the NMDA-responsive accumulation of active RhoA in dendrites. Phosphorylation of extracellular signal-regulated kinase 1/2 and Akt and calcium influx in response to NMDA are not impaired in mutant neurons. These data indicate that S-SCAMalpha is a scaffold required to activate RhoA protein in response to NMDA receptor signaling in dendrites.

Seminiferous epithelia of the testes contain two types of intercellular junctions: Sertoli-Sertoli junctions and Sertoli-spermatid junctions. The former junctions are equipped with tight and adherens junctions while the latter junctions are not. Ca2+ -independent immunoglobulin-like cell-cell adhesion molecules, nectin-2 and nectin-3, asymmetrically localize at the Sertoli cell side and at the spermatid side of Sertoli-spermatid junctions, respectively. They heterophilically trans-interact to make contact between the two cells. Nectin-2(-/-) mice have shown male-specific infertility, disrupted Sertoli-spermatid junctions and morphologically impaired spermatid development. Here we report testicular phenotypes of nectin-3(-/-) mice exhibiting male-specific infertility. Nectin-3(-/-) mice had defects in the later steps of sperm morphogenesis including distorted nuclei and abnormal distribution of mitochondria, as well as in localization of nectin-2 at the Sertoli-spermatid junctions. Transplantation of wild-type spermatogenic stem cells into the nectin-3(-/-) testes partially rescued these defects in sperm morphogenesis. These results indicate that the heterophilic trans-interaction between nectin-2 and nectin-3 is essential for the formation and maintenance of Sertoli-spermatid junctions that plays a critical role in spermatid development.

Nectins are Ca2+-independent immunoglobulin-like cell-cell-adhesion molecules consisting of four members. Nectins homophilically and heterophilically trans-interact to form a variety of cell-cell junctions, including cadherin-based adherens junctions in epithelial cells and fibroblasts in culture, synaptic junctions in neurons, and Sertoli cell-spermatid junctions in the testis, in cooperation with, or independently of, cadherins. To further explore the function of nectins, we generated nectin 1–/– and nectin 3–/– mice. Both nectin 1–/– and nectin 3–/– mice showed a virtually identical ocular phenotype, microphthalmia, accompanied by a separation of the apex-apex contact between the pigment and non-pigment cell layers of the ciliary epithelia. Immunofluorescence and immunoelectron microscopy revealed that nectin 1 and nectin 3, but not nectin 2, localized at the apex-apex junctions between the pigment and non-pigment cell layers of the ciliary epithelia. However, nectin 1–/– and nectin 3–/– mice showed no impairment of the apicolateral junctions between the pigment epithelia where nectin 1, nectin 2 and nectin 3 localized, or of the apicolateral junctions between the non-pigment epithelia where nectin 2 and nectin 3, but not nectin 1, localized. These results indicate that the heterophilic trans-interaction between nectin 1 and nectin 3 plays a sentinel role in establishing the apex-apex adhesion between the pigment and non-pigment cell layers of the ciliary epithelia that is essential for the morphogenesis of the ciliary body.

DENN/MADD is a component of a signalling protein complex that is localized to the cytosol and exerts multiple functions by using different binding partners. Human DENN/MADD is physically the same death-domain protein as rat Rab3 GDP/GTP exchange protein (Rab3GEP). DENN/MADD regulates the recycling of Rab3 small G proteins under normal conditions and has an essential role in Ca(2+)-dependent neurotransmitter release and exocytosis. It is also involved in blocking the apoptosis of neuronal cells under conditions of cytotoxic stress. Recent research supports an important role for DENN/MADD in neuroprotection: reduced endogenous DENN/MADD expression and enhanced pro-apoptotic signalling has been found in brains affected by Alzheimer's disease.

Apical–basal polarity and a highly organized actin cytoskeleton are main characteristics of epithelial cells that support exchange of ions and nutrients from one body compartment to another. The junctional complexes, localized to the apical end of the basolateral domain of the plasma membrane, are not simply epithelial barriers in paracellular transport or fences preventing diffusion of integral proteins in the plasma membrane, but also contain proteins involved in the maintenance of the physiologic epithelial cell state and signal transduction. Claudin-based tight junctions and E-cadherin-based adherens junctions have been extensively studied. Nectins, along with a unique scaffolding protein, afadin, form homophilic and heterophilic trans-dimers and play a key role in identifying cell partners in the primordial cell–cell adhesion. Nectin-based cell–cell adhesion participates in the epithelial morphogenesis, both independently and cooperatively with claudin-based tight junctions and cadherin-based adherens junctions. This review discusses how these adhesion systems interact with each other to form apical junctional complexes, and how they reorganize the actin cytoskeleton in a multistage process of cell adhesion, migration, and polarization.

Rac1, a member of the Rho family of small GTP-binding proteins, is involved in the regulation of the actin cytoskeleton via activation of lamellipodia and membrane ruffle formation. RhoGDI (Rho-family-specific GDP-dissociation inhibitor) forms a complex with Rho proteins in the cytosol of mammalian cells. It not only regulates guanine nucleotide binding to Rho proteins, but may also function as a molecular shuttle to carry Rho proteins from an inactive cytosolic pool to the membrane for activation. These studies tested if RhoGDI is necessary for the translocation of Rac1 from the cytosol to the plasma membrane for the formation of membrane ruffles. We describe a novel mutant of Rac1, R66E (Arg66→Glu), that fails to bind RhoGDI. This RhoGDI-binding-defective mutation is combined with a Rac1-activating mutation G12V, resulting in a double-mutant [Rac1(G12V/R66E)] that fails to interact with RhoGDI in COS-7 cells, but remains constitutively activated. This double mutant stimulates membrane ruffling to a similar extent as that observed after epidermal growth factor treatment of non-transfected cells. To confirm that Rac1 can signal ruffle formation in the absence of interaction with RhoGDI, Rac1(G12V) was overexpressed in cultured mesangial cells derived from a RhoGDI knockout mouse. Rac1-mediated membrane ruffling was indistinguishable between the RhoGDI(−/−) and RhoGDI(+/+) cell lines. In both the COS-7 and cultured mesangial cells, Rac1(G12V) and Rac1(G12V/R66E) co-localize with membrane ruffles. These findings suggest that interaction with RhoGDI is not essential in the mechanism by which Rac1 translocates to the plasma membrane to stimulate ruffle formation.

Lipopolysaccharide (LPS) is a pathogenic factor that increases bone resorption in periodontal diseases. LPS treatment of osteoblasts was shown to induce the receptor activator of NF-κB ligand (RANKL), an essential secretory or membrane-bound factor for osteoclast function, in a manner dependent on extracellular signal-regulated kinase (ERK) activation. However, the mechanisms regulating this process remained unknown. Here, we show that RANKL mRNA induction and ERK activation, when treated with synthetic lipid A (an active center of LPS), were markedly reduced in mouse osteoblasts lacking Cot/Tpl2, which was recently recognized as an essential kinase for the induction of TNF-α by LPS in macrophages. In contrast, c-Jun N-terminal kinase (JNK), p38 kinase, Raf-1, and NF-κB were normally activated in cot/tpl2-/- osteoblasts. These findings indicate that Cot/Tpl2 is essential for LPS-induced ERK activation and RANKL induction in osteoblasts.

The Rab3 GDP/GTP exchange protein (Rab3 GEP) activates the Rab3 small GTP-binding protein (G protein) family, including Rab3A that is an important member controlling synaptic vesicle trafficking. Here, we examined the role of Rab3 GEP in regulating neurotransmitter release in autapses of mouse hippocampal neurons in culture. The release probability was markedly reduced in Rab3 GEP−/− neurons, whereas the readily releasable pool size was not different between WT and Rab3 GEP−/− neurons, indicating that Rab3 GEP up-regulates a postdocking step of synaptic exocytosis. Because Rab3A reportedly down-regulates Ca 2+ -triggered fusion of synaptic vesicles, these results provide evidence for a role of Rab3 GEP in the postdocking process distinct from Rab3A activation.

Rab3A is a member of the Rab3 small GTP-binding protein (G protein) subfamily along with Rab3B., 3C., and 3D, and the Rab3 proteins belong to the Rab family (Takai et al., 2001). The Rab family members are involved in vesicle trafficking, such as exocytosis and endocytosis (Takai et al., 2001). Evidence is accumulating that Rab3A is involved in Ca2+-dependent exocytosis, particularly neurotransmitter release from nerve terminals (Takai et al., 2001). The Ca2+-dependent neurotransmitter release is regulated by several steps: translocation of synaptic vesicles from the reserve pool to the active zone of the presynaptic plasm membrane where Ca2+ channels localize, docking of the vesicles to the active zone, transition from the docking to the priming of the vesicles in the readily releasable pool, and fusion of the vesicles with the plasma membrane induced by Ca2+ influx (Fig. 1) (Takai et al., 2001). Studies on Rab3A knockout mice have revealed that Rab3A is not essential for basal neurotransmitter release but modulates synaptic plasticity (Geppert et al.,1994; 1997; Castillo et al., 1997). In Rab3A knockout mice, synaptic depression is increased during repetitive stimulation in the CA1 region of the hippocampus (Geppert et al., 1994), and mossy fiber long-term potentiation (LTP) in the CA3 region is abolished (Castillo et al., 1997). In cultured hippocampal neurons derived from Rab3A knockout mice, release probability is increased, while the size of the readily releasable pool measured by hypertonic solution is normal (Geppert et al., 1997). Rab3A appears to up-regulate the steps of the translocation and docking as well as to down-regulate the step of the fusion.

Small GTP-binding protein GDP dissociation stimulator (Smg GDS) regulates GDP/GTP exchange reaction of Ki-Ras and the Rho and Rap1 family members and inhibits their binding to membranes. In fibroblasts, Smg GDS shows mitogenic and transforming activities in cooperation with Ki-Ras. However, the physiological function of Smg GDS remains unknown. Here we show that mice lacking Smg GDS died of heart failure shortly after birth, not resulting from developmental heart defects but from enhanced apoptosis of cardiomyocytes triggered by cardiovascular overload. Furthermore, neonatal thymocytes and developing neuronal cells underwent apoptotic cell death. Smg GDS−/− thymocytes were susceptible to apoptotic inducers, such as etoposide and UV irradiation. Smg GDS−/− thymocytes were protected from etoposide-induced cell death by ex vivo transduction of the Smg GDS cDNA. These phenotypes partly coincide with those observed in Ki-Ras-deficient mice, suggesting that Smg GDS is involved in antiapoptotic cell survival signaling through Ki-Ras.

The Rho small G protein family members regulate various actin cytoskeleton-dependent cell functions. The Rho GDI (GDP dissociation inhibitor) family, consisting of Rho GDIalpha, -beta, and -gamma, is a regulator that keeps the Rho family members in the cytosol as the GDP-bound inactive form and translocates the GDP-bound form from the membranes to the cytosol after the GTP-bound form accomplishes their functions. Rho GDIalpha is ubiquitously expressed in mouse tissues and shows GDI activity on all the Rho family members in vitro. We have generated mice lacking Rho GDIalpha by homologous recombination to clarify its in vivo function. Rho GDIalpha -/- mice showed several abnormal phenotypes. Firstly, Rho GDIalpha -/- mice were initially viable but developed massive proteinuria mimicking nephrotic syndrome, leading to death due to renal failure within a year. Histologically, degeneration of tubular epithelial cells and dilatation of distal and collecting tubules were readily detected in the kidneys. Secondly, Rho GDIalpha -/- male mice were infertile and showed impaired spermatogenesis with vacuolar degeneration of seminiferous tubules in their testes. Thirdly, Rho GDIalpha -/- embryos derived from Rho GDIalpha -/- female mice were defective in the postimplantation development. In addition, these morphological and functional abnormalities showed age-dependent progression. These results suggest that the signaling pathways of the Rho family members regulated by Rho GDIalpha play important roles in maintaining the structure and physiological function of at least kidneys and reproductive systems in adult mice.

Afadin is an intracellular binding partner of nectins, cell-cell adhesion molecules, and plays important roles in the formation of cell-cell junctions. Afadin-knockout mice show early embryonic lethality, therefore little is known about the function of afadin during organ development. In this study, we generated mice lacking afadin expression in endothelial cells, and found that the majority of these mice were embryonically lethal as a result of severe subcutaneous edema. Defects in the lymphatic vessels of the skin were observed, although the morphology in the blood vessels was almost normal. Severe disruption of VE-cadherin-mediated cell-cell junctions occurred only in lymphatic endothelial cells, but not in blood endothelial cells. Knockout of afadin did not affect the differentiation and proliferation of lymphatic endothelial cells. Using in vitro assays with blood and lymphatic microvascular endothelial cells (BMVECs and LMVECs, respectively), knockdown of afadin caused elongated cell shapes and disruption of cell-cell junctions among LMVECs, but not BMVECs. In afadin-knockdown LMVECs, enhanced F-actin bundles at the cell periphery and reduced VE-cadherin immunostaining were found, and activation of RhoA was strongly increased compared with that in afadin-knockdown BMVECs. Conversely, inhibition of RhoA activation in afadin-knockdown LMVECs restored the cell morphology. These results indicate that afadin has different effects on blood and lymphatic endothelial cells by controlling the levels of RhoA activation, which may critically regulate the lymphangiogenesis of mouse embryos.

We investigated N-ras activation in childhood acute lymphoblastic leukemia (dALL) by the polymerase chain reaction (PCR) and the oligonucleotide hybridization method. The frequency of point-mutation of the N-ras gene was not high (2 of 15), and one positive case who relapsed was analyzed in detail. Although N-ras gene activation was detected at both onset and relapse, the mutation sites were different. At onset, Gly (GGT) was changed to Ser (AGT) at codon 12, and at relapse, Gly (GGT) to Asp (GAT) was observed at the same codon. In addition, the DNA at relapse showed a remarkably higher transforming activity than the DNA at onset on two independent recipient cell lines. The identical cell surface phenotype and the same rearrangement patterns of both the immunoglobulin (Ig) heavy chain and T-cell receptor (TCR) gamma chain genes indicated that the leukemic cells at onset and those at relapse were derived from the same precursor cell. Therefore, this case supports the concept that ras activation is not the event initiating leukemogenesis, but may be involved in leukemic progression.

A cDNA clone encoding the mouse counterpart to adult hamster liver purified growth inhibitory factor (PGIF) was isolated from a mouse liver cDNA library by using antibodies raised against PGIF and sequenced. It contained a single open reading frame with a coding capacity for a 323 amino acid protein. Sequence analysis showed that it shared high homology with rat- and human liver arginases: the cDNA clone was 92% identical for rat arginase at the nucleotide level and was 93% identical to it at the deduced amino acid level. These results suggest that PGIF derived from adult hamster liver was identical or closely related to an isoform of hamster liver arginases.