Key points are not available for this paper at this time.
Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-γ (PLC-γ) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-γ completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-γ activates Rac in a manner dependent on Iba1. Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-γ (PLC-γ) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-γ completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-γ activates Rac in a manner dependent on Iba1. Cell motility is a dynamic process driven by structurally and functionally coordinated reorganization of the actin cytoskeleton (1Stossel T.P. Science. 1993; 260: 1086-1094Crossref PubMed Scopus (905) Google Scholar,2Mitchison T.J. Cramer L.P. Cell. 1996; 84: 371-379Abstract Full Text Full Text PDF PubMed Scopus (1303) Google Scholar). Among various types of cells, macrophages are extremely motile to migrate rapidly to sites of infection or inflammation, suggesting that highly integrated systems should exist to regulate the actin cytoskeleton in macrophages (3Hartwig J.H. Yin H.L. Cell Motil. Cytoskeleton. 1988; 10: 117-125Crossref PubMed Scopus (35) Google Scholar, 4Downey G.P. Curr. Opin. Immunol. 1994; 6: 113-124Crossref PubMed Scopus (127) Google Scholar). In addition to circulating monocytes/macrophages, there are many types of tissue-resident macrophages, including Langerhans cells, Kupffer cells, dendritic cells, splenocytes, and microglia. In response to various pathological phenomena, microglia are activated to exhibit drastic changes in shape and the abilities to become locomotive and to phagocytose (5Nakajima K. Kohsaka S. Neurosci. Res. 1993; 7: 187-203Crossref Scopus (192) Google Scholar, 6Kreutzberg G.W. Trends Neurosci. 1996; 19: 312-318Abstract Full Text Full Text PDF PubMed Scopus (3723) Google Scholar). These cellular reactions are also profoundly underlaid by dynamic remodeling of the actin cytoskeleton. The Rho family GTPases, Cdc42, Rac, and Rho, are known to be molecular switches that organize remodeling of the actin cytoskeleton (7Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5200) Google Scholar). Among them, in fibroblasts, Rac is activated by receptor tyrosine kinases such as platelet-derived growth factor receptor (PDGFR), 1The abbreviations used are: PDGFRplatelet-derived growth factor receptorM-CSFmacrophage colony-stimulating factorPAEporcine aorta endothelialCHOChinese hamster ovaryPI3Kphosphatidylinositol-3 kinasePLCphospholipase CFCSfetal bovine serumWTwild typeGSTglutathione S-transferaseDMEMDulbecco's modified Eagle's mediumHRPhorseradish peroxidaseLPAlysophosphatidic acidPBSphosphate-buffered salineBSAbovine serum albuminFITCfluorescein isothiocyanatePKCprotein kinase CCa2+iintracellular calciumPAKp21-activated kinase1The abbreviations used are: PDGFRplatelet-derived growth factor receptorM-CSFmacrophage colony-stimulating factorPAEporcine aorta endothelialCHOChinese hamster ovaryPI3Kphosphatidylinositol-3 kinasePLCphospholipase CFCSfetal bovine serumWTwild typeGSTglutathione S-transferaseDMEMDulbecco's modified Eagle's mediumHRPhorseradish peroxidaseLPAlysophosphatidic acidPBSphosphate-buffered salineBSAbovine serum albuminFITCfluorescein isothiocyanatePKCprotein kinase CCa2+iintracellular calciumPAKp21-activated kinase leading to the formation of lamellipodia and membrane ruffles (8Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3066) Google Scholar). Dominant active RacV12 induces remarkable membrane ruffling, and dominant negative RacN17 completely inhibits peptide growth factor-induced membrane ruffling; therefore, Rac is recognized to be an essential component in this type of membrane ruffling (8Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3066) Google Scholar). Some studies describe signaling molecules capable of interacting with Rac; however, the processes by which receptor tyrosine kinases activate Rac are not fully understood. platelet-derived growth factor receptor macrophage colony-stimulating factor porcine aorta endothelial Chinese hamster ovary phosphatidylinositol-3 kinase phospholipase C fetal bovine serum wild type glutathione S-transferase Dulbecco's modified Eagle's medium horseradish peroxidase lysophosphatidic acid phosphate-buffered saline bovine serum albumin fluorescein isothiocyanate protein kinase C intracellular calcium p21-activated kinase platelet-derived growth factor receptor macrophage colony-stimulating factor porcine aorta endothelial Chinese hamster ovary phosphatidylinositol-3 kinase phospholipase C fetal bovine serum wild type glutathione S-transferase Dulbecco's modified Eagle's medium horseradish peroxidase lysophosphatidic acid phosphate-buffered saline bovine serum albumin fluorescein isothiocyanate protein kinase C intracellular calcium p21-activated kinase Previously, we identified a calcium-binding protein, Iba1, which is restrictedly expressed in macrophages/microglia (9Imai Y. Ibata I. Ito D. Ohsawa K. Kohsaka S. Biochem. Biophys. Res. Commun. 1996; 224: 855-862Crossref PubMed Scopus (670) Google Scholar), and showed that the expression of Iba1 is up-regulated in activated microglia following facial nerve axotomy (10Ito D. Imai Y. Ohsawa K. Nakajima K. Fukuuchi Y. Kohsaka S. Brain Res. Mol. Brain Res. 1998; 57: 1-9Crossref PubMed Scopus (1044) Google Scholar). In our recent study, Iba1 was further characterized by using a microglial cell line MG5 (11Ohsawa K. Imai Y. Nakajima K. Kohsaka S. Glia. 1997; 21: 285-298Crossref PubMed Scopus (71) Google Scholar) and loss of function Iba1 mutants, and it was demonstrated that mutant Iba1 effectively suppresses the membrane ruffling produced by stimulation with macrophage colony-stimulating factor (M-CSF) or by expression of dominant active RacV12 (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). These observations suggested that Iba1 was involved in the molecular basis of membrane ruffling of macrophages/microglia and interacted with the signaling of Rac, which is a key molecule in controlling membrane ruffling also in macrophages (13Allen W.E. Jones G.E. Pollard J.W. Ridley A.J. J. Cell Sci. 1997; 110: 707-720Crossref PubMed Google Scholar). Iba1 is therefore considered to be one of the candidate molecules underlying the extremely motile property of macrophages/microglia. In this study, to address this hypothesis, we introduced Iba1 in Swiss 3T3 fibroblasts, porcine aorta endothelial (PAE) cells, and Chinese hamster ovary (CHO) cells, none of which expresses endogenous Iba1, and examined the formation of membrane ruffles, chemotaxis, and profiles of intracellular signaling molecules, including PDGFR, phosphatidylinositol-3 kinase (PI3K), phospholipase C-γ (PLC-γ), and Rac. Swiss 3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS). Mouse iba1 cDNA (9Imai Y. Ibata I. Ito D. Ohsawa K. Kohsaka S. Biochem. Biophys. Res. Commun. 1996; 224: 855-862Crossref PubMed Scopus (670) Google Scholar) was inserted into the tetracycline-regulated expression vector pTet-Splice (Invitrogen) to construct pTet-iba1. The cells were transfected with pTet-iba1, transactivator pTet-tTAK, and pSV2-neo by calcium phosphate coprecipitation, and stably transfected clones were isolated by selection with 400 μg/ml G418 (Invitrogen). PAE cells (14Westermark B. Siegbahn A. Heldin C.-H. Claesson-Welsh L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 128-132Crossref PubMed Scopus (132) Google Scholar), kindly provided by Dr. C.-H. Heldin (Ludwig Institute for Cancer Research, Sweden) and Dr. Koutaro Yokote (Chiba University, Japan) were cultured in Ham's F12 medium (Invitrogen) supplemented with 10% FCS. pLXSN plasmids carrying wild type (WT) and a series of mutant human β-PDGFRs (15Valius M. Kazlauskas A. Cell. 1993; 73: 321-334Abstract Full Text PDF PubMed Scopus (570) Google Scholar) were kindly provided by Dr. A. Kazlauskas (Schepens Eye Research Institute, Harvard Medical School, Boston, MA). F5 mutant PDGFR, which was constructed by the substitution of phenylalanines for five tyrosine residues that are required for the of and is to with of mutants of were by tyrosine residues sites for of the (15Valius M. Kazlauskas A. Cell. 1993; 73: 321-334Abstract Full Text PDF PubMed Scopus (570) Google Scholar). PAE cells were transfected with the tetracycline-regulated Iba1-expressing and as Iba1-expressing cells were transfected with or the add-back series of mutants by the and by μg/ml of PAE were in Ham's F12 for of carrying and which were kindly provided by Dr. J.W. A. B. J. Cell. Full Text PDF PubMed Scopus Google Scholar) of and PAE cells were maintained for to protein plasmids for glutathione S-transferase and Y. J. 1992; Full Text PDF PubMed Google Scholar) were kindly provided by Dr. and Dr. K. of Medical of The were into the and for The cells were with PDGF for cells were maintained in medium supplemented with 10% FCS. cells were transfected using (Invitrogen) with pLXSN carrying PDGFR, carrying or mutant and carrying or mutant (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). microglial cell was maintained as (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). were in 10% μg/ml and The was by for protein and to were to an which was with and The membrane was with (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar), with a horseradish peroxidase and using an Swiss 3T3 or PAE were on a cultured for for in or in Ham's F12 with (PAE) in the presence or absence of μg/ml The cells were with human or lysophosphatidic acid for with in phosphate-buffered saline The cells were for with for with and for with serum and bovine serum albumin The cells were for in μg/ml with and for in μg/ml fluorescein isothiocyanate and The cells were with Japan) or Cell was by a modified K. S. Siegbahn A. L. Heldin C.-H. Claesson-Welsh L. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) using a The that were with of and PDGF were with an with μg/ml type the the were and with of with cells The was for The cells on the of the were and the cells that to the of the were and in 10% and Cell were in are of The was as (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar, L. I. L. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). were in of saline and The were by with protein (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar, L. I. L. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) and for and in activated Rac was by with an and using the PAE and MG5 cells were with PDGF and The cells were in for was by and the cell were for protein The were with μg/ml or and with protein The were to with an and using the In this study, to analyze the of Iba1, we transfected a construct into Swiss a cell line expressing endogenous Iba1. a we five clones of Iba1 with the demonstrated that the expression of Iba1 was the presence of whereas or the of expression of Iba1 was induced In Swiss 3T3 cells, and PDGF are known to activate Cdc42, Rho, and Rac, and the cells to and membrane ruffles (7Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5200) Google Scholar). the of Iba1 on the cells were with and and with to the actin cytoskeleton. Iba1 expression was suppressed in the presence of the cells formed and membrane ruffles in response to and as for Swiss 3T3 cells (8Ridley A.J. Paterson H.F. Johnston C.L. Diekmann D. Hall A. Cell. 1992; 70: 401-410Abstract Full Text PDF PubMed Scopus (3066) Google Scholar, A.J. Hall A. Cell. 1992; 70: Full Text PDF PubMed Scopus Google Scholar, S. A. L. Mol. Cell. PubMed Scopus Google Scholar, Hall A. Cell. Full Text PDF PubMed Scopus Google Scholar). Iba1 expression was induced by the cells also formed and shown in the absence of Iba1 expression stimulation with and contrast, in response to the Iba1-expressing cells formed enhanced membrane ruffles in with the Iba1-nonexpressing cells the cells were with and the PDGF Iba1 was shown to be the sites of membrane ruffles, with but Iba1 did not with in or induced by or stimulation not clones of the enhanced membrane ruffling not that Iba1 membrane ruffling in Swiss 3T3 membrane ruffling is considered to be to cell motility J. PubMed Scopus Google Scholar), we the of Iba1-expressing cells by the K. S. Siegbahn A. L. Heldin C.-H. Claesson-Welsh L. J. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) using PDGF as a shown in Swiss 3T3 cells and the Iba1-nonexpressing showed motile PDGF in a whereas the Iba1-expressing cells a in results were in clones of Iba1 on PDGF-induced in Swiss 3T3 cells not These results indicate that Iba1 is also to the of Swiss 3T3 The signaling pathway is to be for PDGF-induced membrane ruffling of Swiss 3T3 cells L. Hall A. J. Cell Sci. PubMed Google Scholar). this pathway is also required for the Iba1-dependent enhancement of membrane ruffling, the of and on PDGF-induced membrane ruffling was examined in Iba1-nonexpressing and treatment with the formed membrane ruffles as a of PDGF but the of formation was in Iba1-expressing cells in Iba1-nonexpressing cells the membrane ruffling of Iba1-nonexpressing was completely that the formation of membrane ruffles of Iba1-nonexpressing Swiss 3T3 cells on the signaling pathway. contrast, the Iba1-expressing cells formed membrane ruffles that membrane ruffling of Iba1-expressing cells on a signaling pathway in addition to PI3K. The results were using These observations to that the enhanced membrane ruffling with Iba1 is by a pathway. the that Iba1 is involved in the signaling we the add-back mutants of PDGFR, which were transfected into PAE cells endogenous (14Westermark B. Siegbahn A. Heldin C.-H. Claesson-Welsh L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 128-132Crossref PubMed Scopus (132) Google Scholar). with various signaling molecules tyrosine acid and whereas and PLC-γ and These signaling molecules are to to the F5 mutant, in which of the five were by phenylalanines (15Valius M. Kazlauskas A. Cell. 1993; 73: 321-334Abstract Full Text PDF PubMed Scopus (570) Google Scholar). PAE cells, which did not Iba1, were with the construct and expression were for the in the as by cell not stimulation with the of the cells was in the presence and absence of Iba1 expression not the Iba1-nonexpressing cells were with membrane ruffles were formed in the cells with receptor or mutant, which is capable of in with a S. Siegbahn A. Yokote K. Heldin C.-H. S. Claesson-Welsh L. 1994; Google Scholar). In contrast, cells expressing F5 or Tyr-1021 receptor did not to PDGF These observations indicate the of signaling for PDGF-induced membrane ruffling in the absence of Iba1. the the of Iba1, membrane ruffles were formed in the cells transfected with Tyr-1021 mutant, capable of of ruffling was also in the Iba1-expressing cells with the or mutant, as in the Iba1-nonexpressing cells expressing or kinase receptor did not PDGF-induced membrane ruffling of Iba1 expression not These observations that PLC-γ is the key signaling molecule in Iba1-dependent and membrane ruffling. Iba1 was demonstrated to function with Rac in the membrane ruffling of and Rac was shown to be activated during membrane ruffling (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). Rac is also activated in and membrane ruffling, the of Rac was by with the of L. I. L. Mol. Cell. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) using PAE In the absence of Iba1, PDGF stimulation Rac into the in the cells expressing and the mutant but not in the cells expressing the Tyr-1021 or not mutant. of Rac. These results indicate that Rac was activated through the pathway in the absence of Iba1. in the presence of Iba1, in addition to or cells, cells also showed Rac in response to PDGF These observations indicate the existence of an and pathway that the formation of membrane the of PLC-γ in Iba1-dependent and Rac we the of PLC-γ mutants that as dominant negative forms endogenous phenylalanines for and and the to the of endogenous J.W. A. B. J. Cell. Full Text PDF PubMed Scopus Google Scholar). or mutant was expressed in the PAE cells expressing Tyr-1021 and Iba1. the cells were with PDGF and with membrane ruffles were formed in cells that were by the of contrast, PDGF-induced membrane ruffling was in cells the cells were with an the cells that were recognized to the cells not is a protein of and a but and is to whereas the but not PLC-γ signaling Y. J. 1992; Full Text PDF PubMed Google Scholar). the PAE cells expressing Tyr-1021 and Iba1, we or protein with to the cells that were The cells with protein were with PDGF and with PDGF-induced membrane ruffling was in the cells, whereas the cells with were not we mutant Iba1-dependent Rac activation. induced the expression of PDGFR, and or mutant Iba1 and in cells, which expression J. 1994; Full Text PDF PubMed Google Scholar), and examined Rac by the we in cells expressing Rac was not activated by PDGF stimulation and and membrane ruffles were not formed not In the cells expressing Iba1 and PDGFR, Rac was activated and and membrane ruffles were formed not in response to that cells also the pathway Iba1-dependent Rac activation. In cells expressing Iba1 and PDGFR, expression of the mutant completely suppressed Rac in response to PDGF and These results indicate the of PLC-γ in Iba1-dependent Rac activation. the cells expressing and were with Rac was induced and whereas this Rac was by expression of mutant which effectively suppresses the membrane ruffling of MG5 cells (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar) and PDGF did not Rac in the cells expressing and mutant or mutant and These results a PLC-γ and Iba1 in membrane ruffling and Rac activation. the PLC-γ is known to be by receptor tyrosine kinases A. Science. 1990; PubMed Scopus Google we the tyrosine of PLC-γ in response to growth membrane ruffling. In types of PLC-γ are and shown in PAE cells whereas the microglial cell line MG5 expresses The PAE cells expressing Iba1 and were with with an and with an of was PDGF and the was MG5 cells were with of by contrast, the of was in MG5 cells stimulation These observations indicate in response to peptide growth and are in Iba1-expressing cells, including and during membrane ruffling. In our (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar), Iba1 was to be a macrophage/microglia-specific protein responsible for and membrane ruffling. In this study, to analyze the of Iba1 in we introduced an Iba1 expression construct into Swiss and cells, the cells with and examined signaling profiles leading to Rac and membrane ruffling. a we a novel signaling pathway in the presence of Iba1, Rac is activated in a manner dependent on In response to Iba1-expressing Swiss 3T3 cells enhanced membrane ruffling and in with Iba1-nonexpressing Swiss 3T3 cells and suggested that a in receptor tyrosine membrane ruffling and in many types of cells L. Hall A. J. Cell Sci. PubMed Google Scholar, S. Siegbahn A. Yokote K. Heldin C.-H. S. Claesson-Welsh L. 1994; Google Scholar, K. K. K. H. Y. H. A. A. B. M. M. J. 1994; PubMed Scopus Google Scholar, D. A. J. Immunol. 1998; PubMed Scopus Google Scholar, Claesson-Welsh L. S. Yokote K. Siegbahn A. Heldin C.-H. Cell Res. 1997; PubMed Scopus Google Scholar). Among them, in Swiss 3T3 and PAE cells, is to be responsible for Rac L. Hall A. J. Cell Sci. PubMed Google Scholar, S. Siegbahn A. Yokote K. Heldin C.-H. S. Claesson-Welsh L. 1994; Google Scholar, L. Siegbahn A. M. K. Heldin C.-H. J. Full Text Full Text PDF PubMed Scopus Google A. D. S. Claesson-Welsh L. M. L. Curr. Full Text Full Text PDF PubMed Scopus Google Scholar). treatment with or effectively suppressed the PDGF-induced membrane ruffling of Iba1-nonexpressing Swiss 3T3 cells In addition, in the absence of Iba1, PDGF not Rac and membrane ruffling in PAE cells expressing mutants of activating of contrast, membrane ruffling of Iba1-expressing Swiss 3T3 cells was not by or Furthermore, in the presence of Iba1, PDGF stimulation induced Rac and membrane ruffling in PAE cells expressing the mutant that is capable of activating PLC-γ but not and These indicate the existence of an pathway leading to Rac and membrane ruffling, and the of PLC-γ in this pathway. In dominant negative forms of PLC-γ Iba1-dependent membrane ruffling and and Rac PLC-γ was further shown to be during membrane ruffling These observations indicate that PLC-γ is involved in Iba1-dependent Rac and membrane ruffling and that Iba1 is the molecule responsible for the signaling of Rac and studies provided for Rac activation. macrophages induced ruffling the stimulation J. Cell 1996; PubMed Scopus Google induced and Rac in J. Full Text Full Text PDF PubMed Scopus Google Scholar, H. H. J. Full Text Full Text PDF PubMed Scopus Google Scholar, S. L. PubMed Google Scholar). In our with did not membrane ruffling of microglial cell line MG5 and cultured microglia not These observations the existence of a pathway leading to Rac and membrane ruffling. Kazlauskas A. 1994; PubMed Scopus Google Scholar) showed that the mutant PDGFR, which the for not expressed in of mutant that induces PLC-γ by PDGF showed in PAE cells L. Siegbahn A. M. K. Heldin C.-H. J. Full Text Full Text PDF PubMed Scopus Google Scholar). is that PLC-γ are required for by A. H. A. M. S. J. 1998; PubMed Scopus Google Scholar) that PLC-γ and Rac are involved in cell of suggesting that PLC-γ and Rac are to in a signaling to the molecules that PLC-γ and Rac completely Iba1 or molecules in that Rac activation. cells expressing did not to PDGF in Rac however, of PLC-γ induced Rac in response to PDGF Furthermore, Rac was suppressed by expression of a mutant of Iba1. In with the results shown with mutant PLC-γ and Iba1, the of Iba1 and PLC-γ in Rac are considered to be is that Iba1 the signaling pathway. membrane ruffling, are constructed by Iba1, and signaling molecules, including Rac, which in we to Iba1, and Rac. are that PLC-γ to the of the cell membrane and the of phosphatidylinositol to and which are capable of activating protein kinase C and intracellular calcium In our membrane ruffling of Iba1-expressing Swiss 3T3 cells was not suppressed by with to not the membrane ruffling of MG5 cells was by and was completely by of calcium with (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). Iba1 calcium-binding an Iba1 mutant calcium-binding suppressed membrane ruffling (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). of indicate the of calcium signaling in actin remodeling (3Hartwig J.H. Yin H.L. Cell Motil. Cytoskeleton. 1988; 10: 117-125Crossref PubMed Scopus (35) Google Scholar, 1994; PubMed Scopus Google Scholar). These observations to the of in membrane ruffling and that is a candidate for PLC-γ and Iba1 Iba1 also to the cell membrane during membrane ruffling (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). In our Iba1 was demonstrated to to in the presence of calcium not suggesting that Iba1 to the of the cell membrane in a Iba1 was further shown to to phosphatidylinositol the of Iba1 or of PLC-γ to the cell membrane and and Rac by the of of PLC-γ in cells enhanced of Rac it with mutant Iba1 suppressed membrane ruffling induced by activated RacV12 in our (12Ohsawa K. Imai Y. Kanazawa H. Sasaki Y. Kohsaka S. J. Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar). mutant Iba1 loss of to to the cell and actin not RacV12 showed a to be the where mutant Iba1 not In our hypothesis, Iba1 also function in of Rac, and mutant Iba1 membrane ruffling by of are extremely this macrophages to highly integrated that regulate dynamic reorganization of the actin cytoskeleton. In addition to the Rac and are to Rac in we shown that PLC-γ activates Rac and membrane ruffling in the presence of Iba1. that macrophages/microglia to regulate Rac and motile by In studies and PLC-γ L. Siegbahn A. M. K. Heldin C.-H. J. Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. J. 1998; PubMed Scopus Google Scholar). Iba1 is therefore considered to be a molecule for the function of activated macrophages/microglia. studies are required to the molecular underlying and of Rac and membrane ruffling. Dr. Heldin (Ludwig Institute for Cancer and Dr. Koutaro Yokote (Chiba for with PAE cells, Dr. Kazlauskas Medical for pLXSN plasmids carrying the human or mutant Dr. of and for plasmids carrying the or mutant and Dr. and Dr. of Medical of for expression plasmids of and also Dr. Koutaro Yokote and Dr. for
Kanazawa et al. (Wed,) studied this question.