1997;324:283C287. the LBPs used in our study; interestingly, in the latter publication, 20 ABPs were found to be enriched in these Triton X-100Cresistant fractions. Whether ezrin or moesin is in this portion remains to be decided, but it is usually interesting to note that the amount of Triton X-100Cnonextractable ezrin is usually Pyrindamycin B higher on cell (ezrin) activation (Berryman em et al. /em , 1995 ; Lamb em et al. /em , 1997 ). Finally, type III PI 4-kinase and a PI-phosphatase have been found in raft fractions (observe Payrastre em et al. /em , 2001 ). The Arp2/3 complex, N-WASP, Cdc42, and partners that unquestionably nucleate actin under some conditions (Machesky and Gould, 1999 ; Cooper and Schafer, 2000 ; Pollard em et al. /em , 2000 ) are present on phagosomes (Garin em et al. /em , 2001 ; our unpublished data), but this whole complex is usually unlikely to be involved in the LBP actin assay, because no GTP is present. Further, GTPS, toxin B, and C3 toxin (which cleave or inactivate Rho proteins), as well as the N-WASP WA Pyrindamycin B domain name (a potent regulator of the Arp2/3 system, which stimulated cytosolic actin assembly in our hands) experienced no effect (our unpublished data). We suggest that in the cell, the ezrin/moesinCfacilitated process is responsible for the primary membrane nucleation of actin on phagosomes, whereas the Arp2/3 system might drive secondary nucleation (branching) from your sides of these main actin filaments (observe Amann and Pollard, 2001 , and recommendations therein). Actin assembly by the phagosomal membrane is likely to be a highly complex process, even as it occurs in a technically simple, GTP-free in vitro system. The results shown here nevertheless spotlight an important role for two phosphoinositides in the regulation of this process. Our more recent data lengthen this complexity by showing that a large cascade of signaling lipids and enzymes communicate with the PIPs and sphingolipids in the LBP Pyrindamycin B membrane. An eventual understanding of this process will require a more total deciphering of all the relevant proteinCprotein, proteinClipid, and lipidClipid interactions, as well as a detailed structural analysis of these components on their specific membrane subdomains. It will also require signaling network analysis, which is now in progress. ACKNOWLEDGMENTS This study was greatly supported by a network grant of the Human Frontier Science Program Business to G.G. H.D. was supported by a Marie Curie fellowship; E.B. by a Talent Stipendium of the Dutch Business of Scientific Research; P.M. by a grant from l’Association pour la Recherche sur le Malignancy; and C.B. by La Ligue Nationale Fran?aise contre le Malignancy. We are grateful to Dr. Heinz Faulstich and Dr. Sergei Kuznetsov for their continued support and encouragement. We also thank Dr. Tom Martin for his helpful discussions and suggestions and Dr. Andreas Mayer for providing the phospholipase C protocol. We thank Dr. N. Schmidt of Assay Designs Inc., Dr. K. Fukami, and Dr. T. Yoshioka for their help in deciphering the lineage of the anti-PI(4,5)P2 antibody KT10 (further information can be provided by Dr. N. Schmidt, Assay Designs Inc.), as well as Ann Atzberger, who helped with the FACS analysis. Dr Yoshioka kindly provided anti-PIP2. We also appreciate the nice gift of chemically synthesized thymosin 4 from Dr. W. Voelter Rabbit polyclonal to APEH and Dr. H. Eichner. Abbreviations used: ABPactin-binding proteinERMezrin/radixin/moesinHPLChigh-pressure liquid chromatographyLBPlatex bead phagosomeN-WASPneural Wiskott-Aldrich syndrome proteinPIphosphatidylinositolPIPphosphoinositidePI(4)Pphosphatidylinositol-4-phosphatePI(4,5)P2phosphatidylinositol-4,5-bisphosphatePI(3,4,5)P3phosphatidylinositol-3,4,5-trisphosphate Footnotes Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.01C06C0314. Article and publication date are at www.molbiolcell.org/cgi/doi/10.1091/mbc.01C06C0314. Recommendations Al-Haddad A, et al. Myosin Va bound to phagosomes binds to F-actin and delays microtubule-dependent motility. Mol Biol Cell. 2001;12:2742C2755. [PMC free article] [PubMed] [Google Scholar]Amann KJ, Pollard TD. The Arp2/3 complex.