地方国立大学の定員増における新教育組織の設置に伴う入口戦略としての入試制度設計と実施――徳島大学 MPEプログラムを例として――

The Journal of University Admissions Research


Rapid, high-sensitivity detection of biomolecules using dual-comb biosensing

Rapid, sensitive detection of biomolecules is important for biosensing of infectious pathogens as well as biomarkers and pollutants. For example, biosensing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still strongly required for the fight against coronavirus disease 2019 (COVID-19) pandemic. Here, we aim to achieve the rapid and sensitive detection of SARS-CoV-2 nucleocapsid protein antigen by enhancing the performance of optical biosensing based on optical frequency combs (OFC). The virus-concentration-dependent optical spectrum shift produced by antigen-antibody interactions is transformed into a photonic radio-frequency (RF) shift by a frequency conversion between the optical and RF regions in the OFC, facilitating rapid and sensitive detection with well-established electrical frequency measurements. Furthermore, active-dummy temperature-drift compensation with a dual-comb configuration enables the very small change in the virus-concentration-dependent signal to be extracted from the large, variable background signal caused by temperature disturbance. The achieved performance of dual-comb biosensing will greatly enhance the applicability of biosensors to viruses, biomarkers, environmental hormones, and so on.


s-Afadin binds to MAGUIN/Cnksr2 and regulates the localization of the AMPA receptor and glutamatergic synaptic response in hippocampal neurons.

A hippocampal mossy fiber synapse implicated in learning and memory is a complex structure in which a presynaptic bouton attaches to the dendritic trunk by puncta adherentia junctions (PAJs) and wraps multiply branched spines. The postsynaptic densities (PSDs) are localized at the heads of each of these spines and faces to the presynaptic active zones. We previously showed that the scaffolding protein afadin regulates the formation of the PAJs, PSDs, and active zones in the mossy fiber synapse. Afadin has two splice variants: l-afadin and s-afadin. l-Afadin, but not s-afadin, regulates the formation of the PAJs but the roles of s-afadin in synaptogenesis remain unknown. We found here that s-afadin more preferentially bound to MAGUIN (a product of the Cnksr2 gene) than l-afadin in vivo and in vitro. MAGUIN/CNKSR2 is one of the causative genes for nonsyndromic X-linked intellectual disability accompanied by epilepsy and aphasia. Genetic ablation of MAGUIN impaired PSD-95 localization and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor surface accumulation in cultured hippocampal neurons. Our electrophysiological analysis revealed that the postsynaptic response to glutamate, but not its release from the presynapse, was impaired in the MAGUIN-deficient cultured hippocampal neurons. Furthermore, disruption of MAGUIN did not increase the seizure susceptibility to flurothyl, a GABAA receptor antagonist. These results indicate that s-afadin binds to MAGUIN and regulates the PSD-95-dependent cell surface localization of the AMPA receptor and glutamatergic synaptic responses in the hippocampal neurons and that MAGUIN is not involved in the induction of epileptic seizure by flurothyl in our mouse model.


Highly sensitive detection of nucleocapsid protein from SARS-CoV-2 using a near-infrared surface plasmon resonance sensing system


Ultrasensitive detection of SARS-CoV-2 nucleocapsid protein using large gold nanoparticle-enhanced surface plasmon resonance.

The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein.


JRAB/MICAL-L2 undergoes liquid-liquid phase separation to form tubular recycling endosomes.

Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid-liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.


Dynamin 1 is important for microtubule organization and stabilization in glomerular podocytes.

Dynamin 1 is a neuronal endocytic protein that participates in vesicle formation by scission of invaginated membranes. Dynamin 1 is also expressed in the kidney; however, its physiological significance to this organ remains unknown. Here, we show that dynamin 1 is crucial for microtubule organization and stabilization in glomerular podocytes. By immunofluorescence and immunoelectron microscopy, dynamin 1 was concentrated at microtubules at primary processes in rat podocytes. By immunofluorescence of differentiated mouse podocytes (MPCs), dynamin 1 was often colocalized with microtubule bundles, which radially arranged toward periphery of expanded podocyte. In dynamin 1-depleted MPCs by RNAi, α-tubulin showed a dispersed linear filament-like localization, and microtubule bundles were rarely observed. Furthermore, dynamin 1 depletion resulted in the formation of discontinuous, short acetylated α-tubulin fragments, and the decrease of microtubule-rich protrusions. Dynamins 1 and 2 double-knockout podocytes showed dispersed acetylated α-tubulin and rare protrusions. In vitro, dynamin 1 polymerized around microtubules and cross-linked them into bundles, and increased their resistance to the disassembly-inducing reagents Ca and podophyllotoxin. In addition, overexpression and depletion of dynamin 1 in MPCs increased and decreased the nocodazole resistance of microtubules, respectively. These results suggest that dynamin 1 supports the microtubule bundle formation and participates in the stabilization of microtubules.


Afadin regulates actomyosin organization through αE-catenin at adherens junctions.

Actomyosin-undercoated adherens junctions are critical for epithelial cell integrity and remodeling. Actomyosin associates with adherens junctions through αE-catenin complexed with β-catenin and E-cadherin in vivo; however, in vitro biochemical studies in solution showed that αE-catenin complexed with β-catenin binds to F-actin less efficiently than αE-catenin that is not complexed with β-catenin. Although a "catch-bond model" partly explains this inconsistency, the mechanism for this inconsistency between the in vivo and in vitro results remains elusive. We herein demonstrate that afadin binds to αE-catenin complexed with β-catenin and enhances its F-actin-binding activity in a novel mechanism, eventually inducing the proper actomyosin organization through αE-catenin complexed with β-catenin and E-cadherin at adherens junctions.


Actin Cytoskeletal Reorganization Function of JRAB/MICAL-L2 Is Fine-tuned by Intramolecular Interaction between First LIM Zinc Finger and C-terminal Coiled-coil Domains

JRAB/MICAL-L2 is an effector protein of Rab13, a member of the Rab family of small GTPase. JRAB/MICAL-L2 consists of a calponin homology domain, a LIM domain, and a coiled-coil domain. JRAB/MICAL-L2 engages in intramolecular interaction between the N-terminal LIM domain and the C-terminal coiled-coil domain, and changes its conformation from closed to open under the effect of Rab13. Open-form JRAB/MICAL-L2 induces the formation of peripheral ruffles via an interaction between its calponin homology domain and filamin. Here, we report that the LIM domain, independent of the C-terminus, is also necessary for the function of open-form JRAB/MICAL-L2. In mechanistic terms, two zinc finger domains within the LIM domain bind the first and second molecules of actin at the minus end, potentially inhibiting the depolymerization of actin filaments (F-actin). The first zinc finger domain also contributes to the intramolecular interaction of JRAB/MICAL-L2. Moreover, the residues of the first zinc finger domain that are responsible for the intramolecular interaction are also involved in the association with F-actin. Together, our findings show that the function of open-form JRAB/MICAL-L2 mediated by the LIM domain is fine-tuned by the intramolecular interaction between the first zinc finger domain and the C-terminal domain.


Protein kinase C iota facilitates insulin-induced glucose transport by phosphorylation of soluble nSF attachment protein receptor regulator (SNARE) double C2 domain protein b.

Double C2 domain protein b (DOC2b), one of the synaptotagmins, has been shown to translocate to the plasma membrane, and to initiate membrane-fusion processes of vesicles containing glucose transporter 4 proteins on insulin stimulation. However, the mechanism by which DOC2b is regulated remains unclear. Herein, we identified the upstream regulatory factors of DOC2b in insulin signal transduction. We also examined the role of DOC2b on systemic homeostasis using DOC2b knockout (KO) mice. We first identified DOC2b binding proteins by immunoprecipitation and mutagenesis experiments. Then, DOC2b KO mice were generated by disrupting the first exon of the DOC2b gene. In addition to the histological examination, glucose metabolism was assessed by measuring parameters on glucose/insulin tolerance tests. Insulin-stimulated glucose uptake was also measured using isolated soleus muscle and epididymal adipose tissue. We identified an isoform of atypical protein kinase C (protein kinase C iota) that can bind to DOC2b and phosphorylates one of the serine residues of DOC2b (S34). This phosphorylation is essential for DOC2b translocation. DOC2b KO mice showed insulin resistance and impaired oral glucose tolerance on insulin and glucose tolerance tests, respectively. Insulin-stimulated glucose uptake was impaired in isolated soleus muscle and epididymal adipose tissues from DOC2b KO mice. We propose a novel insulin signaling mechanism by which protein kinase C iota phosphorylates DOC2b, leading to glucose transporter 4 vesicle translocation, fusion and facilitation of glucose uptake in response to insulin. The present results also showed DOC2b to play important roles in systemic glucose homeostasis.


Involvement of l-afadin, but not s-afadin, in the formation of puncta adherentia junctions of hippocampal synapses.

A hippocampal mossy fiber synapse has a complex structure in which presynaptic boutons attach to the dendritic trunk by puncta adherentia junctions (PAJs) and wrap multiply-branched spines, forming synaptic junctions. It was previously shown that afadin regulates the formation of the PAJs cooperatively with nectin-1, nectin-3, and N-cadherin. Afadin is a nectin-binding protein with two splice variants, l-afadin and s-afadin: l-afadin has an actin filament-binding domain, whereas s-afadin lacks it. It remains unknown which variant is involved in the formation of the PAJs or how afadin regulates it. We showed here that re-expression of l-afadin, but not s-afadin, in the afadin-deficient cultured hippocampal neurons in which the PAJ-like structure was disrupted, restored this structure as estimated by the accumulation of N-cadherin and αΝ-catenin. The l-afadin mutant, in which the actin filament-binding domain was deleted, or the l-afadin mutant, in which the αΝ-catenin-binding domain was deleted, did not restore the PAJ-like structure. These results indicate that l-afadin, but not s-afadin, regulates the formation of the hippocampal synapse PAJ-like structure through the binding to actin filaments and αN-catenin. We further found here that l-afadin bound αN-catenin, but not γ-catenin, whereas s-afadin bound γ-catenin, but hardly αN-catenin. These results suggest that the inability of s-afadin to form the hippocampal synapse PAJ-like structure is due to its inability to efficiently bind αN-catenin.


Dancing Styles of Collective Cell Migration: Image-Based Computational Analysis of JRAB/MICAL-L2.

Collective cell migration is observed during morphogenesis, angiogenesis, and wound healing, and this type of cell migration also contributes to efficient metastasis in some kinds of cancers. Because collectively migrating cells are much better organized than a random assemblage of individual cells, there seems to be a kind of order in migrating clusters. Extensive research has identified a large number of molecules involved in collective cell migration, and these factors have been analyzed using dramatic advances in imaging technology. To date, however, it remains unclear how myriad cells are integrated as a single unit. Recently, we observed unbalanced collective cell migrations that can be likened to either precision dancing or , Japanese traditional dancing similar to the style at Rio Carnival, caused by the impairment of the conformational change of JRAB/MICAL-L2. This review begins with a brief history of image-based computational analyses on cell migration, explains why quantitative analysis of the stylization of collective cell behavior is difficult, and finally introduces our recent work on JRAB/MICAL-L2 as a successful example of the multidisciplinary approach combining cell biology, live imaging, and computational biology. In combination, these methods have enabled quantitative evaluations of the "dancing style" of collective cell migration.

Frontiers in Cell and Developmental Biology


Conformational plasticity of JRAB/MICAL-L2 provides "law and order" in collective cell migration.

In fundamental biological processes, cells often move in groups, a process termed collective cell migration. Collectively migrating cells are much better organized than a random assemblage of individual cells. Many molecules have been identified as factors involved in collective cell migration, and no one molecule is adequate to explain the whole picture. Here we show that JRAB/MICAL-L2, an effector protein of Rab13 GTPase, provides the "law and order" allowing myriad cells to behave as a single unit just by changing its conformation. First, we generated a structural model of JRAB/MICAL-L2 by a combination of bioinformatic and biochemical analyses and showed how JRAB/MICAL-L2 interacts with Rab13 and how its conformational change occurs. We combined cell biology, live imaging, computational biology, and biomechanics to show that impairment of conformational plasticity in JRAB/MICAL-L2 causes excessive rigidity and loss of directionality, leading to imbalance in cell group behavior. This multidisciplinary approach supports the concept that the conformational plasticity of a single molecule provides "law and order" in collective cell migration.


Junctional Rab13-binding protein (JRAB) regulates cell spreading via filamins.

We previously showed that Rab13 and its effector protein, junctional Rab13-binding protein (JRAB)/molecules interacting with CasL-like 2 (MICAL-L2), regulate junctional development by modulating cell adhesion molecule transport and actin cytoskeletal reorganization in epithelial cells. Here, we investigated how JRAB regulates reorganization of the actin cytoskeleton in NIH3T3 fibroblasts, in an attempt to obtain novel insights into the mechanism of JRAB action. To this end, we expressed mutant proteins that adopt a constitutively open or closed state and then examined effect on cellular morphology of the resulting actin cytoskeletal reorganization. Expression of the JRABΔCT mutant (constitutively 'closed' state) induced stress fibers, whereas expression of the JRABΔCC mutant (constitutively 'open' state) caused cell spreading with membrane ruffles. Next, we identified the proteins involved in JRAB-induced rearrangement of actin cytoskeleton leading to morphological changes. In NIH3T3 cells expressing HA-JRABΔCC, filamin, an actin cross-linking protein, coimmunoprecipitated with HA-JRABΔCC. Expression of ASB2 induced degradation of all three filamin isoforms and inhibited the JRABΔCC-induced cell spreading. Consistent with our previous results, actinin-1/-4 were also immunoprecipitated with HA-JRABΔCC. However, actinin-1/-4 have no effect on the cell spreading regulated by JRABΔCC. These data suggest that JRAB contributes to the rearrangement of the actin cytoskeleton during cell spreading via filamins.


Rab13 small G protein and junctional Rab13-binding protein (JRAB) orchestrate actin cytoskeletal organization during epithelial junctional development.

During epithelial junctional development, both vesicle transport and reorganization of the actin cytoskeleton must be spatiotemporally regulated. Coordination of these cellular functions is especially important, but the precise mechanism remains elusive. Previously, we identified junctional Rab13-binding protein (JRAB)/molecules interacting with CasL-like 2 (MICAL-L2) as an effector of the Rab13 small G protein, and we found that the Rab13-JRAB system may be involved in the formation of cell-cell adhesions via transport of adhesion molecules. Here, we showed that JRAB interacts with two actin-binding proteins, actinin-1 and -4, and filamentous actin via different domains and regulates actin cross-linking and stabilization through these interactions. During epithelial junctional development, JRAB is prominently enriched in the actin bundle at the free border; subsequently, JRAB undergoes a Rab13-dependent conformational change that is required for maturation of cell-cell adhesion sites. These results suggest that Rab13 and JRAB regulate reorganization of the actin cytoskeleton throughout epithelial junctional development from establishment to maturation of cell-cell adhesion.


Expression of RASSF6 in kidney and the implication of RASSF6 and the Hippo pathway in the sorbitol-induced apoptosis in renal proximal tubular epithelial cells.

RASSF6, a member of RASSF tumour suppressor proteins, binds to mammalian Ste20-like kinases (MST1/2), core kinases of the proapoptotic Hippo pathway and cooperates with the Hippo pathway to induce apoptosis. We originally identified RASSF6 as a putative interactor of membrane-associated guanylate kinase inverted (MAGI)-1 by the yeast two-hybrid screening. We used human kidney cDNA library for the screening. MAGI-1 is abundantly expressed in kidney and is a core component of the slit diaphragm. These findings suggest that RASSF6 is expressed in kidney. However, the function of RASSF6 in kidney is not yet studied. We performed this study to confirm the interaction of RASSF6 with MAGI-1, to analyse the expression of RASSF6 in kidney and to gain insight into the function of RASSF6 in kidney. RASSF6 binds to PDZ domains of MAGI-1 through its C-terminal PDZ-binding motif and is coimmunoprecipitated with MAGI-1 from rat liver. RASSF6 is localized in normal kidney glomerulus but disappears when the slit diaphragm is disrupted in nephrotic kidney. RASSF6 is also localized on apical membranes in renal proximal tubular epithelial cells. We demonstrated that RASSF6 as well as the Hippo pathway are involved in the sorbitol-induced apoptosis in immortalized human proximal renal tubular epithelial HK-2 cells.


Rubicon and PLEKHM1 negatively regulate the endocytic/autophagic pathway via a novel Rab7-binding domain.

The endocytic and autophagic pathways are involved in the membrane trafficking of exogenous and endogenous materials to lysosomes. However, the mechanisms that regulate these pathways are largely unknown. We previously reported that Rubicon, a Beclin 1-binding protein, negatively regulates both the autophagic and endocytic pathways by unidentified mechanisms. In this study, we performed database searches to identify potential Rubicon homologues that share the common C-terminal domain, termed the RH domain. One of them, PLEKHM1, the causative gene of osteopetrosis, also suppresses endocytic transport but not autophagosome maturation. Rubicon and PLEKHM1 specifically and directly interact with Rab7 via their RH domain, and this interaction is critical for their function. Furthermore, we show that Rubicon but not PLEKHM1 uniquely regulates membrane trafficking via simultaneously binding both Rab7 and PI3-kinase.


Rab13 regulates neurite outgrowth in PC12 cells through its effector protein, JRAB/MICAL-L2.

Neurite outgrowth is the first step in the processes of neuronal differentiation and regeneration and leads to synaptic polarization and plasticity. Rab13 small G protein shows an increased mRNA expression level during neuronal regeneration; it is therefore thought to be involved in this process. We previously identified JRAB (junctional Rab13-binding protein)/MICAL-L2 (molecules interacting with CasL-like 2) as a novel Rab13 effector protein. Here, we show that Rab13 regulates neurite outgrowth in the rat pheochromocytoma cell line PC12 through an interaction with JRAB/MICAL-L2. The expression of JRAB/MICAL-L2 alone inhibits neurite outgrowth, whereas coexpression of the dominant active form of Rab13 rescues this effect. We also demonstrate an intramolecular interaction between the N-terminal calponin-homology (CH) and LIM domains of JRAB/MICAL-L2 and the C-terminal coiled-coil domain. Finally, we show that the binding of Rab13 to JRAB/MICAL-L2 stimulates the interaction of JRAB/MICAL-L2 with actinin-4, an actin-binding protein, which localizes to the cell body and the tips of the neurites in PC12 cells. These results suggest that Rab13 and JRAB/MICAL-L2 may act to transfer actinin-4 from the cell body to the tips of neurites, where actinin-4 is involved in the reorganization of the actin cytoskeleton which results in neurite outgrowth.


APP Anterograde Transport Requires Rab3A GTPase Activity for Assembly of the Transport Vesicle

The amyloid precursor protein (APP) is anterogradely transported by conventional kinesin in a distinct transport vesicle, but both the biochemical composition of such a vesicle and the specific kinesin-1 motor responsible for transport are poorly defined. APP may be sequentially cleaved by beta- and gamma-secretases leading to accumulation of beta-amyloid (Abeta) peptides in brains of Alzheimer's disease patients, whereas cleavage of APP by alpha-secretases prevents Abeta generation. Here, we demonstrate by time-lapse analysis and immunoisolations that APP is a cargo of a vesicle containing the kinesin heavy chain isoform kinesin-1C, the small GTPase Rab3A, and a specific subset of presynaptic protein components. Moreover, we report that assembly of kinesin-1C and APP in this vesicle type requires Rab3A GTPase activity. Finally, we show cleavage of APP in transport vesicles by alpha-secretase activity, likely mediated by ADAM10. Together, these data indicate that maturation of APP transport vesicles, including recruitment of conventional kinesin, requires Rab3 GTPase activity.


Rab family small G proteins in regulation of epithelial apical junctions.

Tight junctions (TJs) and adherens junctions (AJs) comprise epithelial apical junctions that adhere neighboring epithelial cells and determine tissue organization. They are highly dynamic structures that undergo continuous remodeling during physiological morphogenesis and under pathological conditions. The assembly and disassembly of epithelial apical junctions is regulated by the interplay between a variety of cellular processes, such as the remodeling of actin cytoskeletons and the endocytic recycling of apical junctional proteins, and coordinated by many signaling pathways. Accumulating evidences demonstrate that Rab family small G proteins are crucially involved in the regulation of epithelial apical junctions. Rab proteins localized both at endosomes and apical junctions can influence the assembly and disassembly of epithelial apical junctions. In this review, we summarize how Rab proteins influence epithelial apical junctions and describe the role of Rab8/13-a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) complexes in the regulation of epithelial apical junctions.


Interaction of Rab3B with microtubule-binding protein Gas8 in NIH 3T3 cells.

Rab3 subfamily small G proteins (Rab3A, Rab3B, Rab3C, and Rab3D) control the regulated exocytosis in neuronal/secretory cells. Rab3B is also detected and upregulated in non-neuronal/non-secretory cells, whereas its function remains elusive. In the present study, we identified growth-arrest-specific gene 8 (Gas8), an evolutionally conserved microtubule-binding protein that is upregulated in growth-arrested NIH 3T3 cells and involved in the dynein motor regulation in flagellar/ciliary axoneme, as a novel Rab3B-binding protein using a yeast two-hybrid system. Rab3B as well as Gas8 was upregulated in growth-arrested NIH 3T3 cells and enriched in testis and lung with well-developed flagella/cilia. Gas8 was specifically interacted with the GTP-bound form of Rab3B and co-localized with Rab3B at the Golgi in NIH 3T3 cells. Furthermore, Rab3B was relocated upon expression of the Rab3B-binding domain of Gas8. These results suggest that Gas8 links Rab3B to microtubules in NIH 3T3 cells.


Involvement of actinin-4 in the recruitment of JRAB/MICAL-L2 to cell-cell junctions and the formation of functional tight junctions.

Tight junctions (TJs) are cell-cell adhesive structures that undergo continuous remodeling. We previously demonstrated that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) localized at TJs and mediated the endocytic recycling of the integral TJ protein occludin and the formation of functional TJs. Here, we investigated how JRAB/MICAL-L2 was targeted to TJs. Using a series of deletion mutants, we found the plasma membrane (PM)-targeting domain within JRAB/MICAL-L2. We then identified actinin-4, which was originally isolated as an actin-binding protein associated with cell motility and cancer invasion/metastasis, as a binding protein for the PM-targeting domain of JRAB/MICAL-L2, using a yeast two-hybrid system. Actinin-4 was colocalized with JRAB/MICAL-L2 at cell-cell junctions and linked JRAB/MICAL-L2 to F-actin. Although actinin-4 bound to JRAB/MICAL-L2 without Rab13, the actinin-4-JRAB/MICAL-L2 interaction was enhanced by Rab13 activation. Depletion of actinin-4 by using small interfering RNA inhibited the recruitment of occludin to TJs during the Ca(2+) switch. During the epithelial polarization after replating, JRAB/MICAL-L2 was recruited from the cytosol to cell-cell junctions. This JRAB/MICAL-L2 recruitment as well as the formation of functional TJs was delayed in actinin-4-depleted cells. These results indicate that actinin-4 is involved in recruiting JRAB/MICAL-L2 to cell-cell junctions and forming functional TJs.


Doc2α and Munc13-4 Regulate Ca2+-Dependent Secretory Lysosome Exocytosis in Mast Cells.

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.


Involvement of Rab13 and JRAB/MICAL-L2 in epithelial cell scattering.

Epithelial cell scattering recapitulates the first steps of carcinoma invasion/metastasis. While the balance between cell-cell adhesive activity and cell motility ultimately determines this process, its molecular mechanisms remain unclear. Adherence junctions and tight junctions (TJs) are primarily responsible for cell-cell adhesive activity and subjected to dynamic remodeling. We previously showed that Rab13 and its effector protein JRAB/MICAL-L2 mediate the endocytic recycling of the integral TJ protein occludin and the assembly of functional TJs. In this study, we examined the role of Rab13 and JRAB/MICAL-L2 in the scattering of Madin-Darby canine kidney (MDCK) cells in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). Knockdown of Rab13 in canine MDCK cells suppressed the TPA-induced scattering, and this phenotype was restored by re-expression of human Rab13. During TPA-induced MDCK cell scattering, Rab13 was transiently activated and returned to its basal level, and both Rab13 and JRAB/MICAL-L2 were colocalized with F-actin at cell-cell contact sites and then accumulated at emerging lamellipodial structures. TPA-induced MDCK cell scattering was also inhibited by knockdown of canine JRAB/MICAL-L2 and rescued by re-expression of mouse JRAB/MICAL-L2. These results indicate that Rab13 and JRAB/MICAL-L2 are involved in epithelial cell scattering.


The Interaction of JRAB/MICAL-L2 with Rab8 and Rab13 Coordinates the Assembly of Tight Junctions and Adherens Junctions.

The assembly of tight junctions (TJs) and adherens junctions (AJs) is regulated by the transport of integral TJ and AJ proteins to and/or from the plasma membrane (PM) and it is tightly coordinated in epithelial cells. We previously reported that Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) mediated the endocytic recycling of an integral TJ protein occludin and the formation of functional TJs. Here, we investigated the role of Rab13 and JRAB/MICAL-L2 in the transport of other integral TJ and AJ proteins claudin-1 and E-cadherin to the PM by using a Ca(2+)-switch model. Although knockdown of Rab13 specifically suppressed claudin-1 and occludin but not E-cadherin transport, knockdown of JRAB/MICAL-L2 and expression of its Rab13-binding domain (JRAB/MICAL-L2-C) inhibited claudin-1, occludin, and E-cadherin transport. We then identified Rab8 as another JRAB/MICAL-L2-C-binding protein. Knockdown of Rab8 inhibited the Rab13-independent transport of E-cadherin to the PM. Rab8 and Rab13 competed with each other for the binding to JRAB/MICAL-L2 and functionally associated with JRAB/MICAL-L2 at the perinuclear recycling/storage compartments and PM, respectively. These results suggest that the interaction of JRAB/MICAL-L2 with Rab8 and Rab13 coordinates the assembly of AJs and TJs.


Analysis on the Emerging Role of Rab3 GTPase-Activating Protein in Warburg Micro and Martsolf Syndrome.

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 Ca2+-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.


Identification and Characterization of JRAB/MICAL-L2, a Junctional Rab13-Binding Protein.

The Rab family small G proteins are localized to distinct subsets of intracellular membranes and play a key role in membrane traffic through the interaction with their specific effector protein(s). Rab13 is identified as a plaque protein at tight junctions (TJs) and has been shown to regulate the assembly of functional TJs in epithelial cells. We have demonstrated that Rab13 mediates the endocytic recycling of integral TJ protein occludin, and identified a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2) as a Rab13 effector protein using a yeast two-hybrid system. JRAB/MICAL-L2 has a calponin-homology domain in the N-terminus, a LIM domain in the middle, and a coiled-coil domain at the C-terminus, and specifically binds to the GTP-bound form of Rab13 via its C-terminus. It is localized to TJs in epithelial cells and distributed along stress fibers in fibroblasts. In epithelial cells, JRAB/MICAL-L2 as well as Rab13 mediates the endocytic recycling of occludin, but not transferrin receptor, and the formation of functional TJs. This chapter describes the procedures for the isolation of JRAB/MICAL-L2 and the analysis of its functions.


Regulation of epithelial cell adhesion and repulsion : role of endocytic recycling.

A proper balance between cell adhesion and repulsion is essential for cellular morphogenesis during epithelial-mesenchymal transition and mesenchymal-epithelial transition. A number of ligand-receptor pairs including hepatocyte growth factor/scatter factor-Met and semaphorin-plexin are known to control this balance through the complex intracellular signaling pathways. Cell adhesion to other cells and extracellular matrix (ECM) is mediated by cell adhesion molecules (CAMs) and ECM receptors, respectively, which are associated with cytoskeleton through a variety of plaque proteins strengthening and/or weakening adhesion activities. Cell repulsion requires the downregulation of cell adhesion and the extensive changes in cytoskeletal dynamics. The endocytic recycling of CAMs and ECM receptors has recently emerged as an important mechanism to control the balance between cell adhesion and repulsion. Molecule interacting with CasL (MICAL) family proteins are originally identified as a plaque protein associated with ECM receptors integrins and implicated in semaphorin-plexin dependent repulsive axon guidance. We have recently shown that MICAL family protein JRAB/MICAL-L2 functions as an effector protein for Rab family small G protein Rab13 and regulates the endocytic recycling of tight junctional CAM occludin and controls the adhesion and repulsion of epithelial cells.

The Journal of Medical Investigation : JMI


Cell-Surface Biotinylation to Study Endocytosis and Recycling of Occludin.

The dynamic turnover of adherens junctions (AJs) and tight junctions (TJs) is essential for epithelial morphogenesis during normal development and differentiation. Although the endocytic recycling of E-cadherin is characterized and implicated in AJ turnover, the molecular basis for TJ turnover is poorly understood. Occludin and claudins are distinct transmembrane proteins localized to the TJs. Although claudins are an indispensable structural component of TJ strands, depletion of occludin in mice reveals well-developed TJ strands and complex histological abnormalities. To examine the intracellular transport of transmembrane proteins to and from the cell surface, cell-surface biotinylation is a proven powerful method. Using this method, we successfully demonstrated that occludin was endocytosed and recycled back to the cell surface in both fibroblastic baby hamster kidney (BHK) and epithelial MTD-1A cells. The endocytic recycling of occludin as well as the formation of functional TJs was dependent on Rab13 and a junctional Rab13-binding protein (JRAB)/molecule interacting with CasL-like 2 (MICAL-L2). We describe the method to study the intracellular transport of occludin to and from the cell surface in both fibroblastic and epithelial cells.


Involvement of Rabring7 in EGF receptor degradation as an E3 ligase.

Rab7, a member of the Rab family small G proteins, is involved in the late stage of the endocytic pathway. We previously identified a Rab7 target protein, Rabring7, which contains a RING finger domain at its C termini, but the precise role of Rabring7 remains unknown. In this study, we demonstrate using an in vitro ubiquitination assay with recombinant E1 and E2 proteins that Rabring7 has E3 ligase activity and that it preferentially reacts with Ubc4 and Ubc5 as its E2 proteins. Rabring7 ubiquitinated itself but not Rab7, and a mutation at Cys-229 in the RING finger domain (Rabring7C229S) completely diminished its E3 ligase activity. In the ligand-induced degradation of EGF receptor (EGFR), Rabring7 accelerated the degradation of EGFR, whereas Rabring7C229S inhibited the degradation induced by cCbl, another E3 ligase. These results suggest that Rabring7 is involved in the endocytic trafficking of EGFR through its E3 ligase activity.

Biochemical and Biophysical Research Communications


Rab3 GTPase-activating protein regulates synaptic transmission and plasticity through the inactivation of Rab3.

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.

Proceedings of the National Academy of Sciences of the United States of America


JRAB/MICAL-L2 is a junctional Rab13-binding protein mediating the endocytic recycling of occludin.

The dynamic turnover of tight junctions (TJs) is essential for epithelial-mesenchymal transitions and/or mesenchymal-epithelial transitions during epithelial morphogenesis. We previously demonstrated that Rab13 specifically mediates the endocytic recycling of occludin. Here, we identified MICAL-L2 (molecule interacting with CasL-like 2) as a novel Rab13-binding protein. Immunoprecipitation and immunofluorescence microscopy showed that MICAL-L2 specifically bound to the GTP-bound form of Rab13 via its C terminus, which contained a coiled-coil domain, and localized at TJs in epithelial MTD-1A cells. Recycling assay demonstrated that a MICAL-L2 mutant lacking the Rab13-binding domain (MICAL-L2-N) specifically inhibited the endocytic recycling of occludin but not transferrin receptor. Ca2+ switch assay further revealed that MICAL-L2-N as well as Rab13 Q67L inhibited the recruitment of occludin to the plasma membrane, the development of transepithelial electrical resistance, and the formation of a paracellular diffusion barrier. MICAL-L2 was displaced from TJs upon actin depolymerization and was distributed along radiating actin cables and stress fibers in Ca2+-depleted MTD-1A and fibroblastic NIH3T3 cells, respectively. These results suggest that MICAL-L2 mediates the endocytic recycling of occludin and the formation of functional TJs by linking Rab13 to actin cytoskeleton. We rename MICAL-L2 as JRAB (junctional Rab13-binding protein).


Rabring7: A target protein for Rab7 small G protein.

Rab7, a member of the Rab family of small G proteins, has been shown to regulate late endocytic traffic and lysosome biogenesis, but the exact roles and the mode of actions of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins and works together with them to coordinate the individual step of vesicle traffic. Rabring7 (Rab7-interacting ring finger protein) is a Rab7 target protein that has been isolated using a CytoTrap system. This protein shows no homology with RILP, which has been reported as another Rab7 target protein. Rabring7 is recruited efficiently to late endosome/lysosome by the GTP-bound form of Rab7. Exogenous expression of Rabring7 not only affects epidermal growth factor degradation but also induces the perinuclear aggregation of lysosomes and the increased acidity in the lysosomes. This chapter describes the procedures for the isolation of Rabring7 with a CytoTrap system, the analysis of the Rab7-Rabring7 interactions, and the properties of Rabring7.


Rab13 Mediates the Continuous Endocytic Recycling of Occludin to the Cell Surface

During epithelial morphogenesis, adherens junctions (AJs) and tight junctions (TJs) undergo dynamic reorganization, whereas epithelial polarity is transiently lost and reestablished. Although ARF6-mediated endocytic recycling of E-cadherin has been characterized and implicated in the rapid remodeling of AJs, the molecular basis for the dynamic rearrangement of TJs remains elusive. Occludin and claudins are integral membrane proteins comprising TJ strands and are thought to be responsible for establishing and maintaining epithelial polarity. Here we investigated the intracellular transport of occludin and claudins to and from the cell surface. Using cell surface biotinylation and immunofluorescence, we found that a pool of occludin was continuously endocytosed and recycled back to the cell surface in both fibroblastic baby hamster kidney cells and epithelial MTD-1A cells. Biochemical endocytosis and recycling assays revealed that a Rab13 dominant active mutant (Rab13 Q67L) inhibited the postendocytic recycling of occludin, but not that of transferrin receptor and polymeric immunoglobulin receptor in MTD-1A cells. Double immunolabelings showed that a fraction of endocytosed occludin was colocalized with Rab13 in MTD-1A cells. These results suggest that Rab13 specifically mediates the continuous endocytic recycling of occludin to the cell surface in both fibroblastic and epithelial cells.


Involvement of Rab27 in antigen-induced histamine release from rat basophilic leukemia 2H3 cells

The Rab family small G proteins regulate discrete steps in vesicular transport pathways. Recent studies indicate that one member of the Rab family, Rab27A, regulates the transport of lysosome-related organelles, such as melanosome distribution in melanocytes, lytic granule release in cytotoxic T cells, and dense granule release in platelets. Here, we have examined the involvement of Rab27A in the exocytic transport of another lysosome-related organelle, the basophilic secretory granule, in basophils. We have found that Rab27A locates on basophilic secretory granules containing histamine in rat basophilic leukemia (RBL) 2H3 cells. In addition, exogenous expression of dominant active Rab27A reduces antigen-induced histamine release from the cells. We have moreover identified Munc13-4 as a Rab27A target using a CytoTrap system and found that exogenous expression of Munc13-4 affects antigen-induced histamine release from RBL-2H3 cells. These results demonstrate that Rab27A plays a crucial role in antigen-induced histamine release from RBL-2H3 cells.


Identification and characterization of Noc2 as a potential Rab3B effector protein in epithelial cells

The Rab3 family small G proteins (Rab3A-D) are involved in the regulated secretory pathway of brain and secretory tissues. Among Rab3-interacting proteins, Rabphilin-3, Rim, and Noc2, all of which contain a conserved Rab3-binding domain (RBD3), are generally recognized Rab3 effector proteins in neurons and secretory cells. Although Rab3B was also detected in epithelial cells, its function remained unknown. We isolated cDNA sequences from human epithelial Caco2-cell mRNA by degenerate RT-PCR based on the conserved amino acid sequence of RBD3. Multiple cDNA clones were identified as encoding Noc2. Northern blot analysis revealed that Noc2 mRNA was expressed not only in secretory tissues but also in epithelial tissues and cell lines. A pull-down assay demonstrated that Noc2 bound to Rab3B in a GTP-dependent manner. When Noc2 was co-expressed with the GTP-bound form of Rab3B, it was recruited from the cytosol to perinuclear membranes. Furthermore, overexpression of Noc2 inhibited the cell-surface transport of basolateral vesicular stomatitis virus glycoprotein. These results suggest that Noc2 functions as a potential Rab3B effector protein in epithelial cells.


Involvement of Cdc42 and Rac small G proteins in invadopodia formation of RPMI7951 cells.

Invadopodia are membrane protrusions into the extracellular matrix by aggressive tumour cells. These structures are associated with sites of matrix degradation and invasiveness of malignant tumour cells in an in vitro fibronectin degradation/invasion assay. The Rho family small G proteins, consisting of the Rho, Rac and Cdc42 subfamilies, are implicated in various cell functions, such as cell shape change, adhesion, and motility, through reorganization of the actin cytoskeleton. We studied the roles of the Rho family small G proteins in invadopodia formation. We first demonstrated that invadopodia of RPMI7951 human melanoma cells extended into the matrix substratum on a vertical view using a laser scanning confocal microscope system. We confirmed that invadopodia were rich in actin filaments (F-actin) and visualized clearly with F-actin staining on a vertical view as well as on a horizontal view. We then studied the roles of Rho, Rac, and Cdc42 in invasiveness of the same cell line. In the in vitro fibronectin degradation/invasion assay, a dominant active mutant of Cdc42 enhanced dot-like degradation, whereas a dominant active mutant of Rac enhanced diffuse-type degradation. Furthermore, frabin, a GDP/GTP exchange protein for Cdc42 with F-actin-binding activity, enhanced both dot-like and diffuse-type degradation. However, a dominant active mutant of Rho did not affect the fibronectin degradation. Moreover, inhibition of phosphatidylinositol-3 kinase (PI3K) disrupted the Rac and Cdc42-dependent actin structures and blocked the fibronectin degradation. These results suggest that Cdc42 and Rac play important roles in fibronectin degradation and invasiveness in a coordinate manner through the frabin-Cdc42/Rac-PI3K signalling pathway.


Rabring7, a novel Rab7 target protein with a RING finger motif

Rab7, a member of the Rab family small G proteins, has been shown to regulate intracellular vesicle traffic to late endosome/lysosome and lysosome biogenesis, but the exact roles of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins that function in the exocytic/endocytic pathway. We have isolated a new Rab7 target protein, Rabring7 (Rab7-interacting RING finger protein), using a CytoTrap system. It contains an H2 type RING finger motif at the C termini. Rabring7 shows no homology with RILP, which has been reported as another Rab7 target protein. GST pull-down and coimmunoprecipitation assays demonstrate that Rabring7 specifically binds the GTP-bound form of Rab7 at the N-terminal portion. Rabring7 is found mainly in the cytosol and is recruited efficiently to late endosomes/lysosomes by the GTP-bound form of Rab7 in BHK cells. Overexpression of Rabring7 not only affects epidermal growth factor degradation but also causes the perinuclear aggregation of lysosomes, in which the accumulation of the acidotropic probe LysoTracker is remarkably enhanced. These results suggest that Rabring7 plays crucial roles as a Rab7 target protein in vesicle traffic to late endosome/lysosome and lysosome biogenesis.


Distinct roles of Rab3B and Rab13 in the polarized transport of apical, basolateral, and tight junctional membrane proteins to the plasma membrane

Regulated transport of proteins to distinct plasma membrane domains is essential for the establishment and maintenance of cell polarity in all eukaryotic cells. The Rab family small G proteins play a crucial role in determining the specificity of vesicular transport pathways. Rab3B and Rab13 localize to tight junction in polarized epithelial cells and cytoplasmic vesicular structures in non-polarized fibroblasts, but their functions are poorly understood. Here we examined their roles in regulating the cell-surface transport of apical p75 neurotrophin receptor (p75NTR), basolateral low-density lipoprotein receptor (LDLR), and tight junctional Claudin-1 using transport assay in non-polarized fibroblasts. Overexpression of Rab3B mutants inhibited the cell-surface transport of LDLR, but not p75NTR and Claudin-1. In contrast, overexpression of Rab13 mutants impaired the transport of Claudin-1, but not LDLR and p75NTR. These results suggest that Rab3B and Rab13 direct the cell-surface transport of LDLR and Claudin-1, respectively, and may contribute to epithelial polarization.

Biochemical and Biophysical Research Communications


Rabconnection-3: A novel protein that binds GDP/GTP exchange protein and GTPase-activating protein for Rab3 small G protein family

Rab3A, a member of the Rab3 small G protein family, regulates Ca(2+)-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP), and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). It remains unknown how or in which step of the multiple exocytosis steps these regulators are activated and inactivated. We isolated here a novel protein that was co-immunoprecipitated with Rab3 GEP and GAP by their respective antibodies from the crude synaptic vesicle fraction of rat brain. The protein, named rabconnectin-3, bound both Rab3 GEP and GAP. The cDNA of rabconnectin-3 was cloned from a human cDNA library and its primary structure was determined. Human rabconnectin-3 consisted of 3,036 amino acids and showed a calculated M(r) of 339,753. It had 12 WD domains. Tissue and subcellular distribution analyses in rat indicated that rabconnectin-3 was abundantly expressed in the brain where it was enriched in the synaptic vesicle fraction. Immunofluorescence and immunoelectron microscopy revealed that rabconnectin-3 was concentrated on synaptic vesicles at synapses. These results indicate that rabconnectin-3 serves as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles.


Isolation of regulator proteins for the Rab3 subfamily GTPases.


Role of Rab3 GDP/GTP exchange protein in synaptic vesicle trafficking at the mouse neuromuscular junction

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca(2+)-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 approximately 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.


Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.


Purification and properties of Rab3 GTPase activating protein.


Rabphilin-3: a target molecule for Rab3 small G proteins.


Doc2a as a modulator of Ca2+-dependent exocytosis.


Role of Rab GDP dissociation inhibitor alpha in regulating plasticity of hippocampla neurotransmission

Rab GDP dissociation inhibitor alpha (Rab GDIalpha) is a regulator of the Rab small G proteins implicated in neurotransmission, and mutations of Rab GDIalpha cause human X-linked mental retardation associated with epileptic seizures. In Rab GDIalpha-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 GDIalpha-deficient mice showed hypersensitivity to bicuculline, an inducer of epileptic seizures. These results suggest that Rab GDIalpha plays a specialized role in Rab3A recycling to suppress hyperexcitability via modulation of presynaptic forms of plasticity.

Proceedings of the National Academy of Sciences of the United States of America


Rho Family GTPase Cdc42 Is Essential for the Actin-based Motility of Shigella in Mammalian Cells.

Shigella, the causative agent of bacillary dysentery, is capable of directing its movement within host cells by exploiting actin dynamics. The VirG protein expressed at one pole of the bacterium can recruit neural Wiskott-Aldrich syndrome protein (N-WASP), a downstream effector of Cdc42. Here, we show that Cdc42 is required for the actin-based motility of Shigella. Microinjection of a dominant active mutant Cdc42, but not Rac1 or RhoA, into Swiss 3T3 cells accelerated Shigella motility. In add-back experiments in Xenopus egg extracts, addition of a guanine nucleotide dissociation inhibitor for the Rho family, RhoGDI, greatly diminished the bacterial motility or actin assembly, which was restored by adding activated Cdc42. In N-WASP-depleted extracts, the bacterial movement almost arrested was restored by adding exogenous N-WASP but not H208D, an N-WASP mutant defective in binding to Cdc42. In pyrene actin assay, Cdc42 enhanced VirG-stimulating actin polymerization by N-WASP-actin-related protein (Arp)2/3 complex. Actually, Cdc42 stimulated actin cloud formation on the surface of bacteria expressing VirG in a solution containing N-WASP, Arp2/3 complex, and G-actin. Immunohistological study of Shigella-infected cells expressing green fluorescent protein-tagged Cdc42 revealed that Cdc42 accumulated by being colocalized with actin cloud at one pole of intracellular bacterium. Furthermore, overexpression of H208D mutant in cells interfered with the actin assembly of infected Shigella and diminished the intra- and intercellular spreading. These results suggest that Cdc42 activity is involved in initiating actin nucleation mediated by VirG-N-WASP-Arp2/3 complex formed on intracellular Shigella.


Physical and Functional Interaction of Rabphilin-11 with Mammalian Sec13 Protein.

Rab11a small G protein (Rab11p) is implicated in vesicle trafficking, especially vesicle recycling. We have previously isolated a downstream effector of Rab11p, named rabphilin-11. We found here that rabphilin-11 directly bound the mammalian counterpart of yeast Sec13 protein (mSec13p) in cell-free and intact cell systems. Yeast Sec13p is involved as a component of coat proteins II in the Sar1p-induced vesicle formation from the endoplasmic reticulum, but the precise role of mSec13p is unknown. The interaction of rabphilin-11 with mSec13p was enhanced by GTP-Rab11p. Rabphilin-11 localized on the vesicles in perinuclear regions and along microtubules oriented toward the plasma membrane, whereas mSec13p partly colocalized with rabphilin-11 in the perinuclear regions, most presumably the Golgi complex. Disruption of the rabphilin-11-mSec13p interaction by overexpression of the mSec13p-binding region of rabphilin-11 impaired vesicle trafficking. These results indicate that the rabphilin-11-mSec13p interaction is implicated in vesicle trafficking.


Stimulation of Rho GDI release by ERM proteins.


Doc2 is an activity-dependent modulator of excitatory synaptic transmission.


Doc2 is an activity-dependent presynaptic modulator of neurotransmitter release.


Similar and differential behavior between the nectin-afadin-ponsin and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells.

We have recently identified a novel cell-cell adhesion system, named NAP system, which is localized at cadherin-based cell-cell adherens junctions (AJs). The NAP system is composed of at least nectin, afadin and ponsin. Nectin is an immunoglobulin-like cell adhesion molecule. Afadin is an actin filament-binding protein which associates nectin with the actin cytoskeleton. Ponsin is an afadin-binding protein which furthermore binds to vinculin and provides a possible linkage of nectin-afadin to cadherin-catenin through vinculin. We compared here the behaviour of the NAP and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells. At the early stage of the formation of the polarized junctional alignment in MTD-1 A cells, primordial spot-like junctions were formed at the tips of thin cellular protrusions radiating from adjacent cells. Nectin, afadin, ponsin, cadherin and catenin were simultaneously recruited to these junctions. As the cell polarization proceeded, the spot-like junctions were gradually fused to form belt-like AJs where all these proteins were concentrated. The disruption of cell-cell AJs in MDCK cells by culturing at a low Ca2+ concentration caused rapid endocytosis of cadherin, but not that of nectin or afadin. Addition of 12-O-tetradecanoylphorbol-13-acetate to the cells formed a tight junction-like structure where nectin and afadin, but not cadherin, accumulated. These results indicate that the NAP and cadherin-catenin systems show similar and differential behaviour during the formation and disruption of the polarized junctional alignment in epithelial cells.


Rab11BP/Rabphilin-11 A dodwnstream target of rab11 small G protein implicated in vesicle recycling

Rab11 small G protein has been implicated in vesicle recycling, but its upstream regulators or downstream targets have not yet been identified. We isolated here a downstream target of Rab11, named rabphilin-11, from bovine brain. Moreover, we isolated from a rat brain cDNA library its cDNA, which encoded a protein with a M(r) of 100,946 and 908 amino acids (aa). Rabphilin-11 bound GTP-Rab11 more preferentially than GDP-Rab11 at the N-terminal region and was specific for Rab11 and inactive for other Rab and Rho small G proteins. Both GTP-Rab11 and rabphilin-11 were colocalized at perinuclear regions, presumably the Golgi complex and recycling endosomes, in Madin-Darby canine kidney cells. In HeLa cells cultured on fibronectin, both the proteins were localized not only at perinuclear regions but also along microtubules, which were oriented toward membrane lamellipodia. Treatment of HeLa cells with nocodazole caused disruption of microtubules and dispersion of GTP-Rab11 and rabphilin-11. Overexpression of the C-terminal fragment of rabphilin-11 (aa 607-730), lacking the GTP-Rab11 binding domain, in HeLa cells reduced accumulation of transferrin at perinuclear regions and cell migration. Rabphilin-11 turned out to be a rat counterpart of recently reported bovine Rab11BP. These results indicate that rabphilin-11 is a downstream target of Rab11 which is involved in vesicle recycling.


Localization of the Rab3 small G protein regulators in nerve terminals and their involvement in Ca2+-dependent exocytosis.

The Rab3 small G protein subfamily (Rab3) consists of four members, Rab3A, -B, -C, and -D. We have recently isolated and characterized the Rab3 regulators, GDP/GTP exchange protein (GEP) and GTPase activating protein (GAP), both of which are specific for the Rab3 subfamily. Rab3 GEP stimulates the conversion of the GDP-bound inactive form to the GTP-bound active form, whereas Rab3 GAP stimulates the reverse reaction. Of the four members of the Rab3 subfamily, evidence is accumulating that Rab3A is involved in Ca2+-dependent exocytosis, particularly in neurotransmitter release. We first analyzed the subcellular localization of Rab3 GEP and GAP in rat brain. Subcellular fractionation analysis showed that both Rab3 GEP and GAP were enriched in the synaptic soluble fraction. Immunocytochemical analysis in primary cultured rat hippocampal neurons showed that both Rab3 GEP and GAP were concentrated at the presynaptic nerve terminals. We then examined whether Rab3 GEP and GAP were involved in Ca2+-dependent exocytosis by use of human growth hormone (GH) co-expression assay system of cultured PC12 cells. Overexpression of the deletion mutant of Rab3 GEP possessing the catalytic activity reduced the high K+-induced GH release without affecting the basal GH release, whereas that of the deletion mutant lacking the catalytic activity showed no effect on the high K+-induced GH release. In contrast, overexpression of Rab3 GAP or its deletion mutant possessing the catalytic activity did not affect the high K+-induced GH release or the basal GH release. These results indicate that Rab3 GEP and GAP are colocalized with Rab3A at the synaptic release sites and suggest that they regulate the activity of Rab3A and are involved in Ca2+-dependent exocytosis.


Interaction of Doc2 with tctex-1, a light chain of cytoplasmic dynein. Implication in dynein-dependent vesicle transport.

Doc2 has one Munc13-interacting domain at the N-terminal region and two C2-like domains interacting with Ca2+ and phospholipid at the C-terminal region. Doc2 consists of two isoforms, Doc2alpha and -beta. Doc2alpha is specifically expressed in neuronal cells and implicated in Ca2+-dependent neurotransmitter release, whereas Doc2beta is ubiquitously expressed and its function is unknown. We show here that both Doc2alpha and -beta interact with rat tctex-1, a light chain of cytoplasmic dynein, in both cell-free and intact cell systems. Overexpression of the N-terminal fragment of Doc2 containing the tctex-1-interacting domain induces changes in the intracellular localization of cation-independent mannose 6-phosphate receptor and its ligand, cathepsin D, which are transported from trans-Golgi network to late endosomes. Overexpression of the C-terminal fragment containing two C2-like domains shows the similar effect, but to a lesser extent, whereas overexpression of full-length Doc2 or the C-terminal fragment of rabphilin3 containing two C2-like domains does not show this effect. Because dynein is a minus-end-directed microtubule-based motor protein, these results suggest that Doc2, especially Doc2beta, plays a role in dynein-dependent intracellular vesicle transport.


Interaction of Doc2 with Tctex-1, a light chain of cytoplasmic dynein Implication in dynein-dependent vesicle transport.


Molecular cloning and characterization of the noncatalytic subunit of the Rab3 subfamily-specific GTPase activating protein.


Role of the Doc2 alpha-Munc13-1 interaction in the neurotransmitter release process.

Doc2alpha and Munc13-1 proteins are highly concentrated on synaptic vesicles and the presynaptic plasma membrane, respectively, and have been implicated in Ca2+-dependent neurotransmitter release. Doc2alpha interacts with Munc13-1 through the N-terminal region of Doc2alpha (the Mid domain; amino acid residues 13-37). Here we examine whether the interaction between Doc2alpha and Munc13-1 is required for Ca2+-dependent neurotransmitter release from intact neuron. A synthetic Mid peptide (the Mid peptide), but not a control mutated Mid peptide or a scrambled Mid peptide, inhibited the interaction between Doc2alpha and Munc13-1 in vitro. Introduction of the Mid peptide into presynaptic neurons of cholinergic synapses, formed between rat superior cervical ganglion neurons, reversibly inhibited synaptic transmission evoked by action potentials. In contrast, the control peptides did not inhibit synaptic transmission. This inhibitory effect depended on the presynaptic activity and was affected by extracellular Ca2+ concentrations. The onset of the Mid peptide effect was shortened when the neuron was stimulated at a higher frequency, and the inhibition was more potent at 1 mM Ca2+ than at 5.1 mM Ca2+. These results suggest that the Doc2alpha-Munc13-1 interaction plays a role in a step before the final fusion step of synaptic vesicles with the presynaptic plasma membrane in the evoked neurotransmitter release process.

Proceedings of the National Academy of Sciences of the United States of America


Role of the Doc2a-Munc13-1 interaction in the neurotransmitter release process.


Rho and Rab small G proteins coordinately reorganize stress fibers and focal adhesions in MDCK cells.

The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility.


A novel brain-specific isoform of spectrin : isolation and its interaction with Munc13.

Munc13 is a component of the neurotransmitter release machinery which is specifically expressed in brain. Munc13 interacts with Doc2 and syntaxin which are also implicated in the neurotransmitter release process. Here we isolated another Munc13-interacting molecule from a rat brain cDNA library by use of the yeast two-hybrid system, identified it to be a novel type of beta spectrin, and named it beta SpIII sigma 1. beta SpIII sigma 1 was specifically expressed in brain, where it was enriched in the synaptic vesicle and plasma membrane fractions. Because spectrin has been shown to interact with the actin cytoskeleton which is involved in the exocytotic process, the present results suggest that the Munc13-beta SpIII sigma 1 interactions play a role in neurotransmitter release.

Biochemical and Biophysical Research Communications


Interaction of radixin with Rho small G protein GDP/GTP exchange protein Dbl.

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various actin cytoskeleton-dependent cell functions. The Rho subfamily members regulate ERM (ezrin, radixin and moesin)-dependent association of the actin cytoskeleton with the plasma membrane. Moreover, the N-terminal regions of ERM interact with Rho GDI, an inhibitory regulator of all the Rho family members, and reduce its inhibitory action, finally initiating the activation of the Rho family members. We show here that the N-terminal region of radixin furthermore interacts with Dbl, a stimulatory GDP/GTP exchange protein of the Rho family members. This interaction does not affect the Dbl activity to stimulate the GDP/GTP exchange reaction of RhoA, a member of the Rho subfamily. Dbl does not interact with radixin which is precomplexed with Rho GDI, and Rho GDI displaces Dbl from radixin. Thus, radixin plays an important role in activation of the Rho family members by recruiting their positive and negative regulators.


The rho small G protein family-rho GDI system as a temporal and spatial determinant for cytoskeletal control.

Recent extensive studies have clarified the functions of the small G protein superfamily, which consists of the Ras, Rho, Rab, Arf, Sar1, and Ran families (for reviews, Refs, 1 and 2). The Ras family regulates gene expression at least through the MAP kinase cascade; the Rho family mainly regulates reorganization of the actin cytoskeleton; the Rab, Arf, and Sar1 families regulate intracellular vesicle trafficking; and the Ran family regulates nuclear transport. Of these cellular functions, reorganization of the actin cytoskeleton, seen in the formation of filopodia, lamellipodia, and ruffles during cell motility, dynamically occurs at specific sites of cells. To regulate this type of dynamic cellular functions, temporal and spatial determination mechanisms of signal transduction would be important. Like other G proteins, small G proteins cycle between the GDP-bound inactive and GTP-bound active forms (1,2). They receive upstream signals through their regulators and transduce signals to downstream targets while they stay in the GTP-bound form. Thus, G proteins serve as timers. There are at least three types of regulators for small G proteins: GDP/GTP exchange protein (GEP) which stimulates conversion from the GDP-bound form to the GTP-bound form; GDP dissociation inhibitor (GDI) which inhibits this reaction; and GTPase activating protein (GAP) which stimulates conversion from the GTP-bound form to the GDP-bound form. Of these regulators, GDI has thus far been found for the Rho and Rab families. We have recently found that the Rho family-Rho GDI system plays an important role in spatial determination in the actin cytoskeletal control (3-6). We briefly describe here this function of the Rho family-Rho GDI system.


Interaction of Rho1p target Bni1p and F-Actin-binding elongation factor 1alpha - Implication in Rho1p-regulated reorganization of the actin cytoskeleton in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of mammalian RhoA small G protein in the yeast Saccharomyces cerevisiae. We have shown that Bni1p is one of the downstream targets of Rho1p and regulates reorganization of the actin cytoskeleton through the interaction with profilin, an actin monomer-binding protein. A Bni1p-binding protein was affinity purified from the yeast cytosol fraction and was identified to be Tef1p/Tef2p, translation elongation factor 1alpha (EF1alpha). EF1alpha is an essential component of the protein synthetic machinery and also possesses the actin filament (F-actin)-binding and -bundling activities. EF1alpha bound to the 186 amino acids region of Bni1p, located between the FH1 domain, the proline-rich profilin-binding domain, and the FH2 domain, of which function is not known. The binding of Bni1p to EF1alpha inhibited its F-actin-binding and -bundling activities. The BNI1 gene deleted in the EF1alpha-binding region did not suppress the bni1 bnr1 mutation in which the actin organization was impaired. These results suggest that the Rho1p-Bni1p system regulates reorganization of the actin cytoskeleton through the interaction with both EF1alpha and profilin.


Interactions of drebrin and gephyrin with profilin.

Profilin is an actin monomer-binding protein which stimulates actin polymerization. Recent studies have revealed that profilin interacts with VASP, Mena, Bnilp, Bnrlp, and mDia, all of which have the proline-rich domain. Here, we isolated three profilin-binding proteins from rat brain cytosol by glutathione S-transferase-profilin affinity column chromatography and identified them as Mena, drebrin, and gephyrin. These proteins had a proline-rich domain and directly interacted with profilin.


Rabphilin-3A : a multifunctional regulator of synaptic vesicle traffic.

We have investigated the function of the synaptic vesicle protein Rabphilin-3A in neurotransmitter release at the squid giant synapse. Presynaptic microinjection of recombinant Rabphilin-3A reversibly inhibited the exocytotic release of neurotransmitter. Injection of fragments of Rabphilin-3A indicate that at least two distinct regions of the protein inhibit neurotransmitter release: the NH2-terminal region that binds Rab3A and is phosphorylated by protein kinases and the two C2 domains that interact with calcium, phospholipid, and beta-adducin. Each of the inhibitory fragments and the full-length protein had separate effects on presynaptic morphology, suggesting that individual domains were inhibiting a subset of the reactions in which the full-length protein participates. In addition to inhibiting exocytosis, constructs containing the NH2 terminus of Rabphilin-3A also perturbed the endocytotic pathway, as indicated by changes in the membrane areas of endosomes, coated vesicles, and the plasma membrane. These results indicate that Rabphilin-3A regulates synaptic vesicle traffic and appears to do so at distinct stages of both the exocytotic and endocytotic pathways.


Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae.

We purified a novel actin filament (F-actin)-binding protein from the soluble fraction of Saccharomyces cerevisiae by successive column chromatographies by use of the 125I-labeled F-actin blot overlay method. The purified protein showed a minimum Mr of about 140 kDa on SDS-polyacrylamide gel electrophoresis and we named it ABP140. A search with the partial amino acid sequences of ABP140 against the Saccharomyces Genome Database revealed that the open reading frame of the ABP140 gene (ABP140) corresponded to YOR239W fused with YOR240W by the +1 translational frame shift. The encoded protein consisted of 628 amino acids with a calculated Mr of 71,484. The recombinant protein interacted with F-actin and showed the activity to crosslink F-actin into a bundle. Indirect immunofluorescence study demonstrated that ABP140 was colocalized with both cortical actin patches and cytoplasmic actin cables in intact cells. However, elimination of ABP140 by gene disruption did not show a deleterious effect on cell growth or affect the organization of F-actin. These results indicate that ABP140 is not required for cell growth but may be involved in the reorganization of F-actin in the budding yeast.


Involvement of Rabphilin3 in endocytosis through interaction with Rabaptin5.


Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP.

Cdc42 is a small GTPase of the Rho family which regulates the formation of actin filaments to generate filopodia. Although there are several proteins such as PAK, ACK and WASP (Wiskott-Aldrich syndrome protein) that bind Cdc42 directly, none of these can account for the filopodium formation induced by Cdc42. Here we demonstrate that before it can induce filopodium formation, Cdc42 must bind a WASP-related protein, N-WASP, that is richest in neural tissues but is expressed ubiquitously. N-WASP induces extremely long actin microspikes only when co-expressed with active Cdc42, whereas WASP, which is expressed in haematopoietic cells, does not, despite the structural similarities between WASP and N-WASP. In a cell-free system, addition of active Cdc42 significantly stimulates the actin-depolymerizing activity of N-WASP, creating free barbed ends from which actin polymerization can then take place. This activation seems to be caused by exposure of N-WASP's actin-depolymerizing region induced by Cdc42 binding.


Regulation of cell-cell adhesion by Rac and Rho small G proteins in MDCK cells.

The Rho small G protein family, consisting of the Rho, Rac, and Cdc42 subfamilies, regulates various cell functions, such as cell shape change, cell motility, and cytokinesis, through reorganization of the actin cytoskeleton. We show here that the Rac and Rho subfamilies furthermore regulate cell-cell adhesion. We prepared MDCK cell lines stably expressing each of dominant active mutants of RhoA (sMDCK-RhoDA), Rac1 (sMDCK-RacDA), and Cdc42 (sMDCK-Cdc42DA) and dominant negative mutants of Rac1 (sMDCK-RacDN) and Cdc42 (sMDCK-Cdc42DN) and analyzed cell adhesion in these cell lines. The actin filaments at the cell-cell adhesion sites markedly increased in sMDCK-RacDA cells, whereas they apparently decreased in sMDCK-RacDN cells, compared with those in wild-type MDCK cells. Both E-cadherin and beta-catenin, adherens junctional proteins, at the cell-cell adhesion sites also increased in sMDCK-RacDA cells, whereas both of them decreased in sMDCK-RacDN cells. The detergent solubility assay indicated that the amount of detergent-insoluble E-cadherin increased in sMDCK-RacDA cells, whereas it slightly decreased in sMDCK-RacDN cells, compared with that in wild-type MDCK cells. In sMDCK-RhoDA, -Cdc42DA, and -Cdc42DN cells, neither of these proteins at the cell-cell adhesion sites was apparently affected. ZO-1, a tight junctional protein, was not apparently affected in any of the transformant cell lines. Electron microscopic analysis revealed that sMDCK-RacDA cells tightly made contact with each other throughout the lateral membranes, whereas wild-type MDCK and sMDCK-RacDN cells tightly and linearly made contact at the apical area of the lateral membranes. These results suggest that the Rac subfamily regulates the formation of the cadherin-based cell- cell adhesion. Microinjection of C3 into wild-type MDCK cells inhibited the formation of both the cadherin-based cell-cell adhesion and the tight junction, but microinjection of C3 into sMDCK-RacDA cells showed little effect on the localization of the actin filaments and E-cadherin at the cell-cell adhesion sites. These results suggest that the Rho subfamily is necessary for the formation of both the cadherin-based cell- cell adhesion and the tight junction, but not essential for the Rac subfamily-regulated, cadherin-based cell- cell adhesion.


Direct interaction of the Rho GDP dissociation inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small G protein.


Physical and functional interactions of Doc2 and Munc13 in Ca2+ -dependent exocytotic machinery.


Rho regulates association of both the ERM family and vinculin with the plasma membrane in MDCK cells.


Molecular cloning of mouse Doc2alpha and distribution of its mRNA in adult mouse brain.


Isolation and characterization of a GTPase activating protein specific for the Rab3 subfamily of small G proteins.


Moclecular cloning of mouse Doc2and distribution of its mRNA in adult mouse brain.


Physical and functional interaction of Rabphilin-3A with alpha-Actinin.


Interaction of both the C2A and C2B domains of rabphilin3 with Ca2+ and pospholipid.

Biochemical and Biophysical Research Communications


Involvement of Rabphilin-3A in cortical granule exocytosis in mouse eggs.

Rabphilin-3A is a putative target protein for Rab3A, a member of the small GTP-binding protein superfamily that has been suggested to play a role in regulated exocytosis in presynapses. In this study we determined the expression and the function of Rabphilin-3A in mouse eggs at fertilization. Rabphilin-3A mRNA and protein were detected by reverse transcriptase-PCR and immunoblot analysis, respectively, in metaphase II mouse eggs. Immunofluorescence analysis showed that Rabphilin-3A protein was distributed in the cortical region in eggs. Sperm induces cortical granule (CG) exocytosis via an increase in cytosolic Ca2+ at fertilization. We microinjected the NH2- or COOH-terminal fragment of recombinant Rabphilin-3A into metaphase II eggs. Neither treatments altered the sperm-induced cytosolic Ca2+ increase, but both inhibited CG exocytosis in a dose-dependent manner. The NH2-terminal fragment was more effective than the COOH-terminal fragment. Full-length Rabphilin-3A did not affect CG exocytosis, but it attenuated the inhibition of CG exocytosis by the NH2-terminal fragment. These results show that Rabphilin-3A is involved in Ca(2+)-dependent CG exocytosis at fertilization in mouse eggs.


Regulation mechanism of ERM (Ezrin/Radixin/Moesin) protein/plasma membrane association Possible involvement of phosphatidylinositol turnover and Rho-dependent signaling pathway.

The ERM proteins, ezrin, radixin, and moesin, are involved in the actin filament/plasma membrane interaction as cross-linkers. CD44 has been identified as one of the major membrane binding partners for ERM proteins. To examine the CD44/ERM protein interaction in vitro, we produced mouse ezrin, radixin, moesin, and the glutathione-S-transferase (GST)/CD44 cytoplasmic domain fusion protein (GST-CD44cyt) by means of recombinant baculovirus infection, and constructed an in vitro assay for the binding between ERM proteins and the cytoplasmic domain of CD44. In this system, ERM proteins bound to GST-CD44cyt with high affinity (Kd of moesin was 9.3 +/- 1.6nM) at a low ionic strength, but with low affinity at a physiological ionic strength. However, in the presence of phosphoinositides (phosphatidylinositol [PI], phosphatidylinositol 4-monophosphate [4-PIP], and phosphatidylinositol 4.5-bisphosphate [4,5-PIP2]), ERM proteins bound with a relatively high affinity to GST-CD44cyt even at a physiological ionic strength: 4,5-PIP2 showed a marked effect (Kd of moesin in the presence of 4,5-PIP2 was 9.3 +/- 4.8 nM). Next, to examine the regulation mechanism of CD44/ERM interaction in vivo, we reexamined the immunoprecipitated CD44/ERM complex from BHK cells and found that it contains Rho-GDP dissociation inhibitor (GDI), a regulator of Rho GTPase. We then evaluated the involvement of Rho in the regulation of the CD44/ERM complex formation. When recombinant ERM proteins were added and incubated with lysates of cultured BHK cells followed by centrifugation, a portion of the recombinant ERM proteins was recovered in the insoluble fraction. This binding was enhanced by GTP gamma S and markedly suppressed by C3 toxin, a specific inhibitor of Rho, indicating that the GTP form of Rho in the lysate is required for this binding. A mAb specific for the cytoplasmic domain of CD44 also markedly suppressed this binding, identifying most of the binding partners for exogenous ERM proteins in the insoluble fraction as CD44. Consistent with this binding analysis, in living BHK cells treated with C3 toxin, most insoluble ERM proteins moved to soluble compartments in the cytoplasm, leaving CD44 free from ERM. These findings indicate that Rho regulates the CD44/ERM complex formation in vivo and that the phosphatidylinositol turnover may be involved in this regulation mechanism.


Involvement of Rho and Rac small G proteins and Rho GDI in Ca2+-dependent exocytosis from PC12 cells.

The Rho small G protein family, which includes the Rho, Rac and Cdc42 subfamilies, is implicated in various cell functions such as cell shape change, cell motility and cytokinesis, through the reorganization of actin filaments. Rho GDI is an inhibitory regulator of the Rho small G protein family and inhibits the Rho family dependent cell functions. Reorganization of actin filaments is also known to regulate Ca2+-dependent exocytosis. We have examined here whether the Rho family members are also involved in Ca2+-dependent exocytosis. We have found, by the use of the human growth hormone (GH) co-expression assay system on PC12 cells, that overexpression of Rho GDI inhibits high K+-induced, Ca2+-dependent GH release. This inhibitory action of Rho GDI is restored by co-expression of a dominant active mutant of RhoA or Rac1, but not of a dominant active mutant of Cdc42. C3 transferase, known to ADP-ribosylate Rho and to inhibit its function, also inhibits this GH release. Overexpression of a dominant active mutant of RhoA or Rac1 alone shows only a small effect on GH release. Moreover, immunocytochemical studies show that the overexpression of Rho GDI prevents a partial disruption of the cortical actin network which accompanies exocytosis. These results suggest that RhoA, Rac1 and Rho GDI are involved in Ca2+-dependent exocytosis at least partly through the reorganization of actin filaments, and that the activation of RhoA or Rac1 alone is not sufficient for this reaction.


Rab3A small GTP-binding protein in Ca2+ -dependent exocytosis.

There exists a small GTP-binding protein (G protein) superfamily, consisting of more than 50 members, from yeast to mammal. The Rab family belongs to this superfamily and is implicated in intracellular vesicle trafficking. Rab3A small G protein is a member of the Rab3 subfamily which belongs to this Rab family. The regulators and downstream targets of Rab3A have been isolated, and evidence is accumulating that Rab3A and these Rab3A-interacting proteins are involved in Ca(2+)-dependent exocytosis, particularly in neurotransmitter release from nerve terminals.


Doc2 enhances Ca2+-dependent exocytosis from PC12 cells.

We previously isolated a new protein having two C2-like domains which interacted with Ca2+ and phospholipid and named Doc2 (Double C2). Because Doc2 was abundantly expressed in brain where it was highly concentrated on the synaptic vesicle fraction, we have examined here whether Doc2 is involved in Ca2+-dependent exocytosis from cultured PC12 cells. For this purpose, we took advantage of the growth hormone (GH) co-expression assay system of PC12 cells in which GH is stored in dense core vesicles and released in response to high K+ in an extracellular Ca2+-dependent manner. Northern and Western blot analyses indicated that Doc2 is present in PC12 cells. Overexpression of hemagglutinin-tagged Doc2 stimulated the Ca2+-dependent, high K+-induced release of co-expressed GH without affecting the basal release. In the PC12 cells transfected with a plasmid with the coding sequence of Doc2 in the antisense orientation, the high K+-induced release of co-expressed GH was inversely inhibited. The Doc2 mutant expressing an N-terminal fragment or a C-terminal fragment containing two C2-like domains inhibited the high K+-induced release of co-expressed GH. These results indicate that Doc2 enhances Ca2+-dependent exocytosis of dense core vesicles from PC12 cells.


Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase C Its implication in regulating the interaction of Munc-18/n-Sec1/rbSec1 with Syntaxin.


Cholecystokinin-B/gastrin receptors mediate rapid formation of actin stress fibers.


Involvement of rabphilin-3A in Ca2+-dependent exocytosis from PC12 cells.

Rabphilin-3A, a putative target molecule of Rab3A small GTP-binding protein implicated in Ca2+-dependent exocytosis, consists of two functionally different domains: the N-terminal Rab3A-binding domain and the C-terminal two C2-like domains (C2A and C2B domains) interacting with Ca2+ and phospholipid. Here, we used the growth hormone (GH) co-expression assay system of PC12 cells in which expressed GH is released in response to high K+. Reduction of endogenous rabphilin-3A inhibited the Ca2+-dependent, high K+-induced GH release. Various rabphilin-3A mutants expressing an N-terminal, C-terminal, or C2B fragment, but not the rabphilin-3A mutant expressing a C2A fragment, inhibited the high K+-induced GH release. These results indicate that rabphilin-3A is involved at least in Ca2+-dependent exocytosis from PC12 cells and that the C2A and C2B domains have different functions.


A membrane-associated GDP/GTP exchange protein specific for Rho small GTP-binding protein Partial purification and characterization from rat brain.


Heterotetramer formation of prenylated Rab3A with two Rabphilin-3A molecules.

Biochemical and Biophysical Research Communications


Synergistic activation of rat brain phospholipase D by ADP-ribosylation factor and rhoA p21, and its inhibition by clostridium botulinum C3 exoenzyme.


Prenylcysteine analogs mimicking the C-terminus of GTP-binding proteins stimulate exocytosis from permeabilized HIT-T15 cells comparison with the effect of Rab3AL peptide.


Translocation of activated Rho from the cytoplasm to membrane ruffling area, cell-cell adhesion sites and cleavage furrows.


Purification and characterization of Rab GDI beta from rat brain.


Doc2: a novel brain protein having two repeated C2-like domains.

Two repeated C2-like domains interacting with Ca2+ and phospholipid are found in synaptotagmin and Rabphilin-3A which are implicated in neurotransmitter release. Here we have isolated a cDNA encoding a novel protein having two repeated C2-like domains from a human brain cDNA library. The isolated cDNA encodes a protein with 400 amino acids and a M(r) of 44,071. The purified recombinant protein indeed interacts with Ca2+ and phospholipid. We have named this protein Doc2 (Double C2). Doc2 is exclusively expressed in brain and is highly concentrated in the synaptic vesicle fraction. These results suggest that Doc2 is a novel brain protein and serves as a Ca2+ sensor in neurotransmitter release.


Effects of prenyl modifications on interactions of small G proteins with regulators.


Purification and properties of recombinant Rho-GDP dissociation inhibitor.


Cell motility assay and inhibition by Rho-GDP dissociation inhibitor.


Purification and Properties of bovine Rab-GDP dissociation inhibitor.


Phosphorylation of Rabphilin-3A by calmodulin-dependent protein kinase II.


Identification of a Rabphilin-3A-interacting protein as GTP cyclohydorase I in PC12 cells.


GDP/GTP exchange reaction-stimulating activity of Rabphilin-3A for Rab3A small GTP-binding protein.


Comparison of kinetic properties between MSS4 and Rab3A GRF GDP/GTP exchange proteins.


Localization of Rabphilin-3A on the synaptic vesicle.


rac p21 is involved in insulin-induced membrane ruffling and rho p21 is involved in hepatocyte growth factor- and 12-O-tetradecanoylphorbol-13-acetate(TPA)-induced membrane ruffling in KB cells.


The Dbl oncogene product as a GDP/GTP exchange protein for the Rho family its properties in comparison with those of Smg GDS.

Biochemical and Biophysical Research Communications


Involvement of Rho p21 small GTP-binding protein and its regulator in the HGF-induced cell motility.


Consequences of weak interaction of rho GDI with the GTP-bound forms of rho p21 and rac p21.


Rab3A GTPase-activating protein-inhibiting activity of Rabphilin-3A, a putative Rab3A target protein.


Interaction of Sec4 with GDI proteins from bovine brain, drosophila melanogaster and saccharomyces cerevisiae: conservation of GDI membrane dissociation activity.


Combination of arachidonic acid and guanosine 5-O-(3-thiotriphosphate) induce translocation of rac p21s to membrane and activation of NADPH oxidase in a cell-free system.


Rab GDP dissociation inhibitor as a general regulator for the membrane association of rab proteins.


The nucleotide exchange rates of Rho and Rac small GTP-binding proteins are enhanced to different extents by their regulatory protein Smg GDS.


Regulation of cytoplasmic division of Xenopus embryo by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI).

Evidence is accumulating that the rho family, a member of the ras p21-related small GTP-binding protein superfamily, regulates cell morphology, cell motility, and smooth muscle contraction through the actomyosin system. The actomyosin system is also known to be essential for cytoplasmic division of cells (cytokinesis). In this study, we examined the action of rho p21, its inhibitory GDP/GTP exchange protein, named rho GDI, its stimulatory GDP/GTP exchange protein, named smg GDS, and botulinum ADP-ribosyltransferase C3, known to selectively ADP-ribosylate rho p21 and to impair its function, in the cytoplasmic division using Xenopus embryos. The sperm-induced cytoplasmic division of Xenopus embryos was not affected by microinjection into the embryos of either smg GDS or the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of rhoA p21, one member of the rho family, but completely inhibited by microinjection of rho GDI or C3. Under these conditions, nuclear division occurred normally but the furrow formation, which was induced by the contractile ring consisting of actomyosin just beneath the plasma membrane, was impaired. Comicroinjection of rho GDI with the GTP gamma S-bound form of rhoA p21 prevented the rho GDI action. Moreover, the sperm-induced cytoplasmic division of Xenopus embryos was inhibited by microinjection into the embryos of the rhoA p21 pre-ADP-ribosylated by C3 which might serve as a dominant negative inhibitor of endogenous rho p21. These results indicate that rho p21 together with its regulatory proteins regulates the cytoplasmic division through the actomyosin system.


Regulation of morphology by rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in Swiss 3T3 cells.


Involvement of rho p21 and its inhibitory GDP/GTP exchange protein (rho GDI) in cell motility.


Post-translational processing of rac p21s is important both for their interaction with the GDP/GTP exchange proteins and for their activation of NADPH oxidase.


Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes.


Functional interactions of stimulatory and inhibitory GDP/GTP exchange proteins and their common substrate small GTP-binding protein.


Cooperative function of rho GDS and rho GDI to regulate rho p21 activation in smooth muscle.


Involvement of rho p21 in the GTP-enhanced calcium ion sensitivity of smooth muscle contraction.


Inhibition of the action of the stimulatory GDP/GTP exchange protein for smg p21 by the geranylgeranylated synthetic peptides designed from its C-terminal region.


The smg GDS-induced activation of smg p21 is initiated by cyclic AMP-dependent protein kinase-catalyzed phosphorylation of smg p21.

Biochemical and Biophysical Research Communications


A novel type of regulatory protein for the GDP/GTP exchange reaction of smg p25A, a ras p21-like small GTP-binding protein, in bovine brain cytosol.


Inhibition of the ras p21 GTPase-activating protein-stimulated GTPase activity of c-Ha-ras p21 by smg p21 having the same putative effector domain as ras p21s.


A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein.


Role of the C-terminal region of smg p25A in its interaction with membranes and the GDP/GTP exchange protein.


Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein.


Purification and characterization from bovine brain cytosol of two GTPase- activating proteins specific for smg p21, a GTP-binding protein having the same effector domain as c-ras p21s.