Coronin-1 function is required for phagosome formation.
Coronin-1 is an actin-associated protein whose function in actin dynamics has remained obscure. All coronin proteins have a variable N-terminal domain, followed by WD repeats and a C-terminal coiled-coil dimerization domain. Transfection of coronin-1-GFP into RAW 264.7 cells revealed that coronin rapidly and transiently associates with the phagosome. To determine if coronin is involved in mammalian phagocytosis we used a dominant-negative approach by expressing only the central WD domains. However, this caused cell rounding and dissociation from the substratum, hampering analysis of their phenotype. We therefore developed TAT-fusion constructs of coronin-1 WD domains to acutely introduce the recombinant protein fragment into live cells. We show that although TAT-WD has no effect on binding of opsonized RBCs to RAW 264.7 cells, receptor clustering or several downstream signaling events, lamellipodial extensions, and actin accumulation at the base of the bound particle were diminished. Furthermore, Arp3 accumulation at the phagosome was impaired after TAT-WD treatment. Interestingly, whereas coronin-1 also accumulates at the sites of actin remodeling associated with Salmonella invasion, TAT-WD had no effect on this process. Together, our data demonstrates that coronin-1 is required for an early step in phagosome formation, consistent with a role in actin polymerization.
Yan
Ming
M
Programme in Cell Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Collins
Richard F
RF
Grinstein
Sergio
S
Trimble
William S
WS
D000199:Actins / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002448:Cell Adhesion; D002460:Cell Line; D003176:Complement C3 / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D019281:Dimerization; D004912:Erythrocytes / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000502:physiology*; D008855:Microscopy, Electron, Scanning; D017830:Octoxynol / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism*; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D011951:Receptors, Complement / Q000378:metabolism; D012480:Salmonella Infections / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D001616:beta-Galactosidase / Q000378:metabolism
D000199:Actins; D003176:Complement C3; D003902:Detergents; D015696:Gene Products, tat; D008840:Microfilament Proteins; D010455:Peptides; D034741:RNA, Small Interfering; D011951:Receptors, Complement; C072061:coronin proteins; D049452:Green Fluorescent Proteins; D017830:Octoxynol; D004247:DNA; D001616:beta-Galactosidase