Mobility of late endosomal and lysosomal markers on phagosomes analyzed by fluorescence recovery after photobleaching

During phagosome maturation, the late endosomal marker Rab7 and the lysosomal marker LAMP1 localize to the phagosomes. We investigated the mobility of Rab7 and LAMP1 on the phagosomes in macrophages by fluorescence recovery after photobleaching (FRAP) analysis. Rab7 was mobile between the phagosomal membrane and the cytosol in macrophages that ingested latex beads during phagosome maturation. The addition of interferon-gamma (IFN-gamma) restricted this mobility, suggesting that Rab7 is forced to bind to the phagosomal membrane by IFN-gamma-mediated activation. Immobilization of LAMP1 on the phagosomes was observed irrespective of IFN-gamma-activation. We further examined the mobility of Rab7 on the phagosomes containing mycobacterium bovis BCG by FRAP analysis. The rate of fluorescence recovery for Rab7 on mycobacterial phagosomes was lower than that on the phagosomes containing latex beads, suggesting that mycobacteria impaired the mobility of Rab7 and arrested phagosome maturation. (C) 2011 Elsevier Inc. All rights reserved.