Cell polarization is essential for targeting signaling elements and organelles to active plasma membrane regions. In a few specialized cell types, cell polarity is enhanced by reorientation of the MTOC and associated organelles toward dynamic membrane sites. Phagocytosis is a highly polarized process whereby particles >0.5 m are internalized at stimulated regions on the cell surface of macrophages. Here we provide detailed evidence that the MTOC reorients toward the site of particle internalization during phagocytosis. We visualized MTOC proximity to IgG-sRBCs in fixed RAW264.7 cells, during live cell imaging using fluorescent chimeras to label the MTOC and using frustrated phagocytosis assays. MTOC reorientation in macrophages is initiated by Fc␥R ligation and is complete within 1 h. Polarization of the MTOC toward the phagosome requires the MT cytoskeleton and dynein motor activity. cdc42, PI3K, and mPAR-6 are all important signaling molecules for MTOC reorientation during phagocytosis. MTOC reorientation was not essential for particle internalization or phagolysosome formation. However Golgi reorientation in concert with MTOC reorientation during phagocytosis implicates MTOC reorientation in antigen processing events in macrophages. INTRODUCTIONPhagocytosis is a specialized mechanism for cells of the innate immune system to clear pathogens and dying cells from the body. Phagocytosis is initiated at the site of particle/pathogen attachment, creating a polarized region of activity within the macrophage. This process is a rapid, highly orchestrated event that recruits a multitude of signaling proteins, mobilizes organelles, and causes dramatic cytoskeletal rearrangements. Phagocytosis is initiated by ligation of Fc␥ receptors to IgG-opsonins on the target cell. Engaged Fc␥Rs cluster at the site of particle contact, which in turn recruits Src, Syk, and PI3K (Greenberg and Grinstein, 2002). Local activation of cdc42 and rac1 initiates actin remodeling that coincides with the growth of membrane pseudopods (Greenberg and Grinstein, 2002). Within minutes, the particles are engulfed by the pseudopods into the cytoplasm as a membrane-bound phagosome. Particle contents within the phagosome are then degraded by fusion with the macrophage endocytic machinery. Phagolysosome formation is concomitant with retrograde translocation of the phagosome along the microtubule (MT) cytoskeleton (Blocker et al., 1997;Harrison and Grinstein, 2002;Harrison et al., 2003). Over the next several hours, particulate antigens from the phagosome will bind to specialized antigen-presentation proteins that will become displayed on the cell surface to initiate the adaptive immune response. Exogenous antigens bind to Major Histocompatibility Complex II (MHCII) proteins, which are largely delivered to phagosomes from Golgi-derived endocytic organelles (Ramachandra and Harding, 2000). Macrophages are also capable of cross-presenting antigens that become complexed with MHC I molecules in the endoplasmic reticulum (ER), before surface presentation (G...
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