Supplementary MaterialsSupplementary Movie 1 7601407s1. effector of Rab7, thus preventing fusion
May 15, 2019
Supplementary MaterialsSupplementary Movie 1 7601407s1. effector of Rab7, thus preventing fusion with lysosomes (Harrison (Scianimanico remaining for a long period of time within a phagosome, its vacuole should be able to remodel and receive a constant supply of membrane and nutrients. This process is usually presently not comprehended, although Rab5 has been indirectly implicated, most likely through its effects on Rab5-dependent aspects of endocytosis (Kelley and Schorey, 2003). Although there are over 60 Rabs identified in mammalian PGE1 manufacturer cells, only a few of them have been studied in terms of phagosomal biogenesis. We have initiated a systematic functional analysis of Rab proteins in the regulation of trafficking events affecting phagosomal organelles. A recent report showed the participation of Rab14 in trafficking between the Golgi complex and the early endosomes (Junutula phagosome in its immature early endosomal-like stage. Results Differential recruitment of Rab14 to phagosomes formed by live or lifeless mycobacteria Rab14 expression in macrophages was examined in RAW 264.7 cells. Rab14 was detected at both the RNA and protein levels (Supplementary Physique S1A and B). Identical intracellular distribution of endogenous Rab14 and the EGFP-Rab14 probe used in subsequent experiments was observed in transfected cells (Supplementary Physique S1CCE). Macrophages expressing EGFP-Rab14 were infected with PGE1 manufacturer live or heat killed BCG (BCG) labeled with Texas Red in a procedure preserving viability and properties of the bacilli (Chua and Deretic, 2004). We also verified that Texas Red labeling did not affect uptake by CR3 or other receptors (Supplementary Physique 1F). Rab14 dynamics in relationship to phagosomes was analyzed by fluorescence microscopy. PGE1 manufacturer Upon entry, the majority (75%) of live mycobacterial phagosomes recruited and maintained Rab14 at 30 min postinfection (Physique 1A). Application of four-dimensional (4D) live confocal microscopy, by examining volume over time using published procedures (Chua and Deretic, 2004; Vergne indicate bacteria under observation. Physique 1B displays quantification of phagosomal membrane associated EGFP-Rab14 fluorescence relative to that of the cytosol (excludes the vacuolar proton pump from its phagosome (Sturgill-Koszycki H37Rv and phagosome maturation PGE1 manufacturer measured by accumulation of Lysotracker Red. Bars, meanss.e.m. (3 impartial experiments); H37Rv (Physique 4D). Rab14 was knocked down by siRNA in human U937 macrophage-like cell line (Physique 4A). The reduction in Rab14 levels resulted in increased acidification of H37Rv phagosomes Rabbit Polyclonal to CCDC102A (from 37.41.2 to 72.93.3), detected by the acidotropic dye LysoTracker Red (Via phagosome maturation arrest. Active Rab14 rescues phagosomes harboring lifeless mycobacteria from maturation into phagolysosomes The phagosomes made up of lifeless mycobacteria normally mature into phagolysosomes (Armstrong and Hart, 1971; Vergne phagosomeCendosome fusion assay (Mayorga fusion assay, as previously described (Vergne and promotes phagosome maturation. Conversely, overexpression of constitutively active mutant of Rab14 was able to block maturation of lifeless mycobacterial phagosomes. Rab14 was demonstrated to be important for phagosomeCearly endosome fusion while inhibiting phagosomeClate endosome fusion. Thus, Rab14 is critical for the maintenance of the early endocytic nature of the phagosome. The role of Rab14 is most likely impartial and additive with the previously reported action of on blocking PI3P production (Vergne phagosomes and prevent them from maturing into the phagolysosome. Several lines of evidence indicate that this mycobacterial phagosome maintains an early endosomal niche in infected macrophages. Firstly, the phagosome associates with early endosomal Rabs or transferrin receptor but not the late endosomal GTPase Rab7 or the late endosomal tetraspanin CD63 (Clemens and Horwitz, 1995; Via access to iron (Kelley and Schorey, 2003). Since Rab14 had no effect on trasferrin recycling kinetics and seems to shuttle between the Golgi apparatus and early endosome (Junutula (Hashim and (Van Ooij H37Rv were produced in 7H9 broth. Mycobacterium was heat killed for 10 min at 90C. For fluorescent labeling, live or.