Biomedical subjects

Hiroaki Miki

Publications and source records attributed to Hiroaki Miki.

At least 19 recordsLinked to original sources

PubMed2005

WASP-related proteins, Abi1 and Ena/VASP are required for Listeria invasion induced by the Met receptor.

Internalisation of the pathogenic bacterium Listeria monocytogenes involves interactions between the invasion protein InlB and the hepatocyte growth factor receptor, Met. Using colocalisation studies, dominant-negative constructs and small interfering RNA (siRNA), we demonstrate a cell-type-dependent requirement for various WASP-related proteins in Listeria entry and InlB-induced membrane ruffling. The WAVE2 isoform is essential for InlB-induced cytoskeletal rearrangements in Vero cells. In HeLa cells, WAVE1, WAVE2 and N-WASP cooperate to promote these processes. Abi1, a key component of WAVE complexes, is recruited at the entry site in both cell types and its inactivation by RNA interference impairs InlB-mediated processes. Ena/VASP proteins also play a role in Listeria internalization, and their deregulation by sequestration or overexpression, modifies actin cups beneath entering particles. Taken together, these results identify the WAVE complex, N-WASP and Ena/VASP as key effectors of the Met signalling pathway and of Listeria entry and highlight the existence of redundant and/or cooperative functions among WASP-family members.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin.

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. We have studied the molecular mechanisms of invadopodium formation in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induced invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibited the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP-Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia.

Actin Depolymerizing Factors↗

PubMed2004

N-WASP and WAVE2 acting downstream of phosphatidylinositol 3-kinase are required for myogenic cell migration induced by hepatocyte growth factor.

During skeletal muscle regeneration caused by injury, muscle satellite cells proliferate and migrate toward the site of muscle injury. This migration is mainly induced by hepatocyte growth factor (HGF) secreted by intact myofibers and also released from injured muscle. However, the intracellular machinery for the satellite cell migration has not been elucidated. To examine the mechanisms of satellite cell migration, we utilized satellite cell-derived mouse C2C12 skeletal muscle cells. HGF induced reorganization of actin cytoskeleton to form lamellipodia in C2C12 myoblasts. HGF treatment facilitated both nondirectional migration of the myoblasts in phagokinetic track assay and directional chemotactic migration toward HGF in a three-dimensional migration chamber assay. Endogenous N-WASP and WAVE2 were concentrated in the lamellipodia at the leading edge of the migrating cells. Moreover, exogenous expression of wild-type N-WASP or WAVE2 promoted lamellipodial formation and migration. By contrast, expression of the dominant-negative mutant of N-WASP or WAVE2 and knockdown of N-WASP or WAVE2 expression by the RNA interference prevented the HGF-induced lamellipodial formation and migration. When the cells were treated with LY294002, an inhibitor of phosphatidylinositol 3-kinase, the HGF-induced lamellipodial formation and migration were abrogated. These results imply that both N-WASP and WAVE2, which are activated downstream of phosphati-dylinositol 3-kinase, are required for the migration through the lamellipodial formation of C2C12 cells induced by HGF.

Actins↗

PubMed2004

The pre-mRNA-splicing factor SF3a66 functions as a microtubule-binding and -bundling protein.

SF3a (splicing factor 3a) complex is an essential component of U2 snRNPs (small nuclear ribonucleoprotein particles), which are involved in pre-mRNA splicing. This complex consists of three subunits: SF3a60, SF3a66 and SF3a120. Here, we report a possible non-canonical function of a well-characterized RNA-splicing factor, SF3a66. Ectopic expression experiments using each SF3a subunit in N1E 115 neuroblastoma cells reveals that SF3a66 alone can induce neurite extension, suggesting that SF3a66 functions in the regulation of cell morphology. A screen for proteins that bind to SF3a66 clarifies that SF3a66 binds to beta-tubulin, and also to microtubules, with high affinity, indicating that SF3a66 is a novel MAP (microtubule-associated protein). Electron microscopy experiments show that SF3a66 can bundle microtubules, and that bundling of microtubules is due to cross-bridging of microtubules by high-molecular-mass complexes of oligomerized SF3a66. These results indicate that SF3a66 is likely to be a novel MAP, and can function as a microtubule-bundling protein independently of RNA splicing.

Animals↗

PubMed2004

IRSp53/Eps8 complex is important for positive regulation of Rac and cancer cell motility/invasiveness.

IRSp53 has been characterized as an adaptor protein that links Rho-family small GTPases, such as Rac, to reorganization of the actin cytoskeleton. Here, we search for other binding partners for the IRSp53 SH3 domain and identify Eps8 as the major binding protein in fibroblasts and various cancer cell lines. Eps8 has been shown to form a Rac-specific guanine nucleotide exchange factor complex with Abi-1 and Sos-1, which seems essential for ruffling formation induced by oncogenic Ras. We confirm the IRSp53/Eps8 complex formation in vivo and the direct association between Eps8 NH(2)-terminal proline-rich sequence and IRSp53 SH3 domain. This complex synergistically activates Rac by reinforcing the formation of the Eps8/Abi-1/Sos-1 Rac-guanine nucleotide exchange factor complex, which mediates positive regulation of Rac activity. In addition, IRSp53/Eps8 complex formation as determined by fluorescent resonance energy transfer analysis, occurs at the leading edge of motile cells, and the motility and invasiveness of HT1080 fibrosarcoma cells are suppressed by inhibiting complex formation. These findings implicate the importance of the IRSp53/Eps8 complex in Rac activation and metastatic behavior of the malignant tumor cells.

Actins↗

PubMed2004

WAVE/Scars in platelets.

Using specific antibodies against isoforms of WAVE (WASP [Wiskott-Aldrich syndrome protein] family Verprolin-homologous protein, also called Scar), we demonstrated that human platelets express all 3 isoforms. With the use of an in vitro pull-down technique, the src homology 3 (SH3) domain of insulin receptor substrate p53 (IRSp53) precipitated WAVE2 from platelet lysates more efficiently than did profilin I. The opposite was true for WAVE1, and neither precipitated WAVE3, suggesting that WAVE isoforms have different affinities to these ligands, while the SH3 domain of abl binds to all 3 isoforms. The 3 WAVE isoforms were distributed in the actin-rich Triton X-100-insoluble pellets following platelet aggregation induced by thrombin receptor-activating peptide. We also found that all 3 WAVE isoforms are substrates for calpain in vivo and in vitro. Although portions of these 3 isoforms were commonly distributed in the actin- and actin-related protein 2 and 3 (Arp2/3)-rich edge of the lamellipodia in spreading platelets, only WAVE2 remained in the cell fringe following detergent extraction or fixation of the cells. Finally, by mass spectrometry, we found that the proteins, which reportedly interact with WAVE/Scars, are present in platelets. These data suggest that the 3 WAVE isoforms exhibit common and distinct features and may potentially be involved in the regulation of actin cytoskeleton in platelets.

Actin Cytoskeleton↗

PubMed2004

Arp2/3 activity is necessary for efficient formation of E-cadherin adhesive contacts.

Classical cadherin adhesion molecules are fundamental determinants of cell-cell recognition that function in cooperation with the actin cytoskeleton. Productive cadherin-based cell recognition is characterized by a distinct morphological process of contact zone extension, where limited initial points of adhesion are progressively expanded into broad zones of contact. We recently demonstrated that E-cadherin ligation recruits the Arp2/3 actin nucleator complex to the plasma membrane in regions where cell contacts are undergoing protrusion and extension. This suggested that Arp2/3 might generate the protrusive forces necessary for cell surfaces to extend upon one another during contact assembly. We tested this hypothesis in mammalian cells by exogenously expressing the CA region of N-WASP. This fragment, which potently inhibits Arp2/3-mediated actin assembly in vitro, also effectively reduced actin assembly at cadherin adhesive contacts. Blocking Arp2/3 activity by this strategy profoundly reduced the ability of cells to extend cadherin adhesive contacts but did not affect cell adhesiveness. These findings demonstrate that Arp2/3 activity is necessary for cells to efficiently extend and assemble cadherin-based adhesive contacts.

Actin-Related Protein 2↗

PubMed2003

Possible involvement of Wiskott-Aldrich syndrome protein family in aberrant neuronal sprouting in Alzheimer's disease.

One of the pathological characteristics of Alzheimer's disease (AD) is the formation of dystrophic neurites accompanied by aberrant neuronal sprouting. Although a number of studies have focussed on the formation of amyloid plaques and neurofibrillary tangles, the mechanism of neuronal sprouting in AD is not fully understood. The protein levels of neural Wiskott-Aldrich syndrome protein (N-WASP), WASP interacting SH3 protein (WISH) and WASP family verprolin-homologous protein (WAVE) were significantly increased in AD brains. In addition, N-WASP, WISH and WAVE were co-localized with filamentous actin in abnormal dendrite-like processes sprouting from staurosporine-treated human SH-SY5Y cells. These results suggest that N-WASP, WISH and WAVE may participate in the neurodegenerative aberrant sprouting in AD neurons.

Adaptor Proteins, Signal Transducing↗

PubMed2003

WAVE2 is required for directed cell migration and cardiovascular development.

WAVE2, a protein related to Wiskott-Aldrich syndrome protein, is crucial for Rac-induced membrane ruffling, which is important in cell motility. Cell movement is essential for morphogenesis, but it is unclear how cell movement is regulated or related to morphogenesis. Here we show the physiological functions of WAVE2 by disruption of the WAVE2 gene in mice. WAVE2 was expressed predominantly in vascular endothelial cells during embryogenesis. WAVE2-/- embryos showed haemorrhages and died at about embryonic day 10. Deficiency in WAVE2 had no significant effect on vasculogenesis, but it decreased sprouting and branching of endothelial cells from existing vessels during angiogenesis. In WAVE2-/- endothelial cells, cell polarity formed in response to vascular endothelial growth factor, but the formation of lamellipodia at leading edges and capillaries was severely impaired. These findings indicate that WAVE2-regulated actin reorganization might be required for proper cell movement and that a lack of functional WAVE2 impairs angiogenesis in vivo.

Animals↗

PubMed2003

IRSp53 is colocalised with WAVE2 at the tips of protruding lamellipodia and filopodia independently of Mena.

The insulin receptor tyrosine kinase substrate p53 (IRSp53) links Rac and WAVE2 and has been implicated in lamellipodia protrusion. Recently, however, IRSp53 has been reported to bind to both Cdc42 and Mena to induce filopodia. To shed independent light on IRSp53 function we determined the localisations and dynamics of IRSp53 and WAVE2 in B16 melanoma cells. In cells spread well on a laminin substrate, IRSp53 was localised by antibody labelling at the tips of both lamellipodia and filopodia. The same localisation was observed in living cells with IRSp53 tagged with enhanced green florescence protein (EGFP-IRSp53), but only during protrusion. From the transfection of deletion mutants the N-terminal region of IRSp53, which binds active Rac, was shown to be responsible for its localisation. Although IRSp53 has been reported to regulate filopodia formation with Mena, EGFP-IRSp53 showed the same localisation in MVD7 Ena/VASP (vasodilator stimulated phosphoprotein) family deficient cells. WAVE2 tagged with DsRed1 colocalised with EGFP-IRSp53 at the tips of protruding lamellipodia and filopodia and, in double-transfected cells, the IRSp53 signal in filopodia decreased before that of WAVE2 during retraction. These results suggest an alternative modulatory role for IRSp53 in the extension of both filopodia and lamellipodia, through WAVE2.

Animals↗

PubMed2003

Essential role of the C. elegans Arp2/3 complex in cell migration during ventral enclosure.

Migration of cells through the reorganization of the actin cytoskeleton is essential for morphogenesis of multicellular animals. In a cell culture system, the actin-related protein (Arp) 2/3 complex functions as a nucleation core for actin polymerization when activated by the members of the WASP (Wiskott-Aldrich syndrome protein) family. However, the regulation of cell motility in vivo remains poorly understood. Here we report that homologues of the mammalian Arp2/3 complex and N-WASP in Caenorhabditis elegans play an important role in hypodermal cell migration during morphogenesis, a process known as ventral enclosure. In the absence of one of any of the C. elegans Arp2/3 complex subunits (ARX-1, ARX-2, ARX-4, ARX-5, ARX-6 or ARX-7) or of N-WASP (WSP-1), hypodermal cell migration led by actin-rich filopodia formation is inhibited during ventral enclosure owing to the reduction of filamentous actin formation. However, there is no effect on differentiation of hypodermal cells and dorsal intercalation. Disruption of the function of ARX-1 and WSP-1 in hypodermal cells also resulted in hypodermal cell arrest during ventral enclosure, suggesting that their function is cell autonomous. WSP-1 protein activated Arp2/3-mediated actin polymerization in vitro. Consistent with these results, the Arp2/3 complex and WSP-1 colocalized at the leading edge of migrating hypodermal cells. The stable localization of WSP-1 was dependent on the presence of Arp2/3 complex, suggesting an interaction between the Arp2/3 complex and WSP-1 in vivo.

Actin-Related Protein 2↗

PubMed2003

Differential localization of WAVE isoforms in filopodia and lamellipodia of the neuronal growth cone.

The formation and extension of filopodia in response to an extracellular stimulus by guidance cues determine the path of growth cone advance. Actin-filament bundling and actin polymerization at the tips supply the driving force behind the formation and elongation. We tried to clarify how signals in response to extracellular cues are transformed to induce filopodial generation and extension. Observations on the formation process of filopodia at growth cones in the neuroblastoma cell line NG108 showed that WAVE (WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein) isoforms played crucial and distinct roles in this process. WAVE1 was continuously distributed along the leading edge only and was not found in the filopodia. WAVE2 and WAVE3 discretely localized at the initiation sites of microspikes on the leading edge and also concentrated at the tips of protruding filopodia. We further found that WAVE isoforms localized at the filopodial tips through SHD (SCAR homology domain), next to its leucine zipper-like motif. Furthermore, time-lapse observations of filopodial formation in living cells showed that WAVE2 and WAVE3 were continuously expressed at the tips of filopodia during elongation. These results indicate that WAVE2 or WAVE3 may guide the actin bundles into the filopodia and promote actin assembly at the tips.

Actin Cytoskeleton↗

PubMed2003

Small GTPase Tc10 and its homologue RhoT induce N-WASP-mediated long process formation and neurite outgrowth.

Rho family small GTPases regulate multiple cellular functions through reorganization of the actin cytoskeleton. Among them, Cdc42 and Tc10 induce filopodia or peripheral processes in cultured cells. We have identified a member of the family, designated as RhoT, which is closely related to Tc10. Tc10 was highly expressed in muscular tissues and brain and remarkably induced during differentiation of C2 skeletal muscle cells and neuronal differentiation of PC12 and N1E-115 cells. On the other hand, RhoT was predominantly expressed in heart and uterus and induced during neuronal differentiation of N1E-115 cells. Tc10 exogenously expressed in fibroblasts generated actin-filament-containing peripheral processes longer than the Cdc42-formed filopodia, whereas RhoT produced much longer and thicker processes containing actin filaments. Furthermore, both Tc10 and RhoT induced neurite outgrowth in PC12 and N1E-115 cells, but Cdc42 did not do this by itself. Tc10 and RhoT as well as Cdc42 bound to the N-terminal CRIB-motif-containing portion of N-WASP and activated N-WASP to induce Arp2/3-complex-mediated actin polymerization. The formation of peripheral processes and neurites by Tc10 and RhoT was prevented by the coexpression of dominant-negative mutants of N-WASP. Thus, N-WASP is essential for the process formation and neurite outgrowth induced by Tc10 and RhoT. Neuronal differentiation of PC12 and N1E-115 cells induced by dibutyryl cyclic AMP and by serum starvation, respectively, was prevented by dominant-negative Cdc42, Tc10 and RhoT. Taken together, all these Rho family proteins are required for neuronal differentiation, but they exert their functions differentially in process formation and neurite extension. Consequently, N-WASP activated by these small GTPases mediates neuronal differentiation in addition to its recently identified role in glucose uptake.

3T3 Cells↗

PubMed2003

Regulation of actin dynamics by WASP family proteins.

Rapid reorganization of the actin cytoskeleton underlies morphological changes and motility of cells. WASP family proteins have received a great deal of attention as the signal-regulated molecular switches that initiate actin polymerization. The first member, WASP, was identified as the product of a gene of which dysfunction causes the human hereditary disease Wiskott-Aldrich syndrome. There are now five members in this protein family, namely WASP, N-WASP, WAVE/Scar1, 2, and 3. WASP and N-WASP have functional and physical associations with Cdc42, a Rho family small GTPase involved in filopodium formation. In contrast, there is evidence that links the WAVE/Scar proteins with another Rho family protein, Rac, which is a regulator of membrane ruffling. All WASP family members have a VCA domain at the C-terminus through which Arp2/3 complex is activated to nucleate actin polymerization. Analyses of model organisms have just begun to reveal unexpected functions of WASP family proteins in multicellular organisms.

Actins↗

PubMed2003

Involvement of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) and Rac1 in the phagocytosis of amyloid-beta(1-42) in rat microglia.

Alzheimer's disease (AD) is characterized by the accumulation of extracellular amyloid-beta (A beta) fibrils with microglia. Recently, there has been great interest in the microglial phagocytosis of A beta, because the microglial pathway is considered to be one of the A beta clearance pathways in the brain parenchyma. However, the mechanism of microglial phagocytosis of A beta is not fully understood and, thus, was investigated in this study. At one minute after exposure to A beta(1-42) (A beta 42), A beta immunoreactivity was detected at the cell surface of microglia. After 1 h, marked immunoreactivity was observed in the cytosolic vesicles. At 12 h, delayed phagocytosis of fibrillar A beta 42 was also observed with the formation of a large phagocytic cup. The microglial cell shape rapidly changed to an ameboid form during the process of phagocytosis. Although neither neural Wiskott-Aldrich syndrome protein (N-WASP) nor WASP interacting SH3 protein (WISH) immunoreactivity was co-localized with filamentous actin (F-actin) distribution, both WASP family verprolin-homologous protein (WAVE) and Rac1 immunoreactivity was co-localized with F-actin in the lamellipodia of phogocytic microglia. These results suggest that WAVE and Rac1 participate in the phagocytosis of A beta 42 by microglia.

Amyloid beta-Peptides↗

PubMed2002

Small GTPase Rah/Rab34 is associated with membrane ruffles and macropinosomes and promotes macropinosome formation.

Macropinocytosis is an efficient process for the uptake of nutrients and solute macromolecules into cells from the external environment. Macropinosomes, which are surrounded by actin, are formed from the cell surface membrane ruffles and migrate toward the cell center. We have cloned the entire coding sequence of a member of the Rab family small GTPases, Rah/Rab34. It lacked a consensus sequence for GTP-binding/GTPase domain. Although wild-type Rah exhibited extremely low GTPase activity in vitro, it exerted appreciable GTPase activity in vivo. In fibroblasts, Rah was colocalized with actin to the membrane ruffles and membranes of relatively large vesicles adjacent to the ruffles. These vesicles were identified as macropinosomes on the basis of several criteria. Rah and Rab5 coexisted in some, but not all, macropinosomes. Rah was predominantly associated with nascent macropinosomes, whereas Rab5 was present in endosomes at later stages. The number of macropinosomes in the cells overexpressing Rah increased about 2-fold. The formation of macropinosomes by the treatment of platelet-derived growth factor or phorbol ester was also facilitated by Rah but suppressed by a dominant-negative Rah. Rah-promoted macropinosome formation was retarded by dominant-negative mutants of Rac1 and WAVE2, which are essential for membrane ruffling. These results imply that Rah is required for efficient macropinosome formation from the membrane ruffles.

Amino Acid Sequence↗

PubMed2002

Gene therapy for Wiskott-Aldrich syndrome: rescue of T-cell signaling and amelioration of colitis upon transplantation of retrovirally transduced hematopoietic stem cells in mice.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency that is caused by mutations in the recently identified WASP gene. WASP plays an important role in T-cell receptor-mediated signaling to the actin cytoskeleton. In these studies we assessed the feasibility of using retroviral gene transfer into WASP-deficient hematopoietic stem cells (HSCs) to rescue the T-cell signaling defect that is characteristic of WAS. Upon transplantation of WASP-deficient (WKO) HSCs that have been transduced with WASP-expressing retroviruses, mature B and T cells developed in normal numbers. Most importantly, the defect in antigen receptor-induced proliferation was significantly improved in T cells. Moreover, the susceptibility of colitis by WKO HSCs was prevented or ameliorated in recipient bone marrow chimeras by retrovirus-mediated expression of WASP. A partial reversal of the T-cell signaling defect could also be achieved following transplantation of WASP-deficient HSCs expressing the WASP-homologous protein N-WASP. Furthermore, we have documented a selective advantage of WT over WKO cells in lymphoid tissue using competitive repopulation experiments and Southern blot analysis. Our results provide proof of principle that the WAS-associated T-cell signaling defects can be improved upon transplantation of retrovirally transduced HSCs without overt toxicity and may encourage clinical gene therapy trials.

Animals↗

PubMed2002

Two verprolin homology domains increase the Arp2/3 complex-mediated actin polymerization activities of N-WASP and WAVE1 C-terminal regions.

WASP family proteins induce actin polymerization through a C-terminal verprolin homology, cofilin homology, and acidic (VCA) region by activating the Arp2/3 complex. The N-WASP VCA region is the most potent activator of the Arp2/3 complex. In addition, full-length WAVE1 and a WAVE1 VCA fragment show differential activity. The mechanisms underlying these differences are poorly understood. We examined the activities of various N-WASP and WAVE1 VCA mutant proteins with several types of fusion moieties. When fused to GST, maltose-binding protein, or the WAVE1 proline-rich domain, N-WASP VCA and WAVE1 VCA mutant proteins with two V motifs showed stronger activities than wild-type WAVE1 VCA with one V motif, demonstrating the importance of two V motifs for strong VCA activity. A WAVE1 VCA fragment tagged with six histidines (His) showed markedly reduced activity compared to GST-fused VCA, whereas His-tagged N-WASP VCA showed similar activity to GST-fused VCA. An additional V motif failed to enhance WAVE1 VCA activity in the His-tagged form. Thus, the WAVE1 VCA fragment may exist in an unfavorable conformation to activate the Arp2/3 complex, implying the existence of a structural difference between WAVE1 and N-WASP VCAs in addition to the number of V motifs.

Actin-Related Protein 2↗