RESUMEN
Professional phagocytic cells ingest microbial intruders by engulfing them into phagosomes, which subsequently mature into microbicidal phagolysosomes. Phagosome maturation requires sequential fusion of the phagosome with early endosomes, late endosomes, and lysosomes. Although various phosphoinositides (PIPs) have been detected on phagosomes, it remained unclear which PIPs actually govern phagosome maturation. Here, we analyzed the involvement of PIPs in fusion of phagosomes with various endocytic compartments and identified phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 3-phosphate [PI(3)P], and the lipid kinases that generate these PIPs, as mediators of phagosome-lysosome fusion. Phagosome-early endosome fusion required PI(3)P, yet did not depend on PI(4)P. Thus, PI(3)P regulates phagosome maturation at early and late stages, whereas PI(4)P is selectively required late in the pathway.
Asunto(s)
Lisosomas/metabolismo , Fagosomas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Animales , Línea Celular , Sistema Libre de Células/metabolismo , Cromatografía Líquida de Alta Presión , Endosomas/metabolismo , Immunoblotting , Membranas Intracelulares/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Espectrometría de Masas , Fusión de Membrana , Ratones , Microscopía Fluorescente , Microesferas , Fagocitosis , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
Lamellipodia are sheet-like protrusions formed during migration or phagocytosis and comprise a network of actin filaments. Filament formation in this network is initiated by nucleation/branching through the actin-related protein 2/3 (Arp2/3) complex downstream of its activator, suppressor of cAMP receptor/WASP-family verprolin homologous (Scar/WAVE), but the relative relevance of Arp2/3-mediated branching versus actin filament elongation is unknown. Here we use instantaneous interference with Arp2/3 complex function in live fibroblasts with established lamellipodia. This allows direct examination of both the fate of elongating filaments upon instantaneous suppression of Arp2/3 complex activity and the consequences of this treatment on the dynamics of other lamellipodial regulators. We show that Arp2/3 complex is an essential organizer of treadmilling actin filament arrays but has little effect on the net rate of actin filament turnover at the cell periphery. In addition, Arp2/3 complex serves as key upstream factor for the recruitment of modulators of lamellipodia formation such as capping protein or cofilin. Arp2/3 complex is thus decisive for filament organization and geometry within the network not only by generating branches and novel filament ends, but also by directing capping or severing activities to the lamellipodium. Arp2/3 complex is also crucial to lamellipodia-based migration of keratocytes.
