Flotillin-1-enriched Lipid Raft Domains Accumulate on Maturing Phagosomes

Abstract

Flotillin-1 was recently shown to be enriched on detergent-resistant domains of the plasma membrane called lipid rafts. These rafts, enriched in sphingolipids and cholesterol, sequester certain proteins while excluding others. Lipid rafts have been implicated in numerous cellular processes including signal transduction, membrane trafficking, and molecular sorting. In this study, we demonstrate both morphologically and biochemically that lipid rafts are present on phagosomes. These structures are enriched in flotillin-1 and devoid of the main phagosomes membrane protein lysosomal-associated membrane protein (LAMP1). The flotillin-1 present on phagosomes does not originate from the plasma membrane during phagocytosis but accumulates gradually on maturing phagosomes. Treatment with bafilomycin A1, a compound that inhibits the proton pump ATPase and prevents the fusion of phagosomes with late endocytic organelles, prevents the acquisition of flotillin-1 by phagosomes, indicating that this protein might be recruited on phagosomes from endosomal organelles. A proteomic characterization of the lipid rafts of phagosomes indicates that actin, the ␣-and ␤-subunits of heterotrimeric G proteins, as well as subunits of the proton pump V-ATPase are among the constituents of these domains. Remarkably, the intracellular parasite Leishmania donovani can actively inhibit the acquisition of flotillin-1-enriched lipid rafts by phagosomes and the maturation of these organelles. These results indicate that specialized functions required for phagolysosome biogenesis may occur at focal points on the phagosome membrane, and therefore represent a potential target of intracellular pathogens.Lateral assemblies of lipids, termed lipid rafts, have been postulated to represent a general feature of the plasma membrane of eukaryotic cells (1, 2). Rafts apparently form because of the biophysical properties of sphingolipids and cholesterol, which pack tightly into liquid-ordered (l o ) 1 domains that partition away from the more disorganized glycerophospholipids in the bulk of the membrane (3). Lipid-modified proteins and some transmembrane proteins are concentrated in the rafts while other proteins are excluded. Lipid rafts have been implicated in many important cellular processes, such as polarized sorting of apical membrane proteins in epithelial cells and signal transduction (4). Recent evidence further indicates that a raft-based mechanism might be involved in the sorting of SNAREs to the plasma membrane and in their function in apical membrane docking and fusion events (5). As this is in no way an exhaustive list of the potential function of lipid rafts, it appears that membrane subdomains represent important sites conferring specialized properties to foci within biological membranes.In the present study, we provide evidence showing that lipid rafts are present on phagosomes. These specialized regions, devoid of the major phagosomal protein LAMP1, are enriched in flotillin-1. The phagosomal lipid rafts are unlikely to be simply transferred ...

The Protein Tyrosine Phosphatase SHP-1 Regulates Phagolysosome Biogenesis

“…Consistent with the results obtained by confocal immunofluorescence microscopy, we found reduced levels of LAMP-1 in phagosome extracts from SHP-1 deficient at all time points tested. Flotillin is recruited to phagosomes from compartments distinct from those bearing LAMP-1 (35). As shown in Fig.…”

“…Little is known concerning the mechanism(s) regulating the phagosomal recruitment of the v-ATPase as well as the regulation of its activity. Previous studies revealed that phagosomal acidification starts early during phagosome formation, before acquisition of lysosomal features such as LAMP-1 and cathepsins (8,53,54), which are recruited to phagosomes through distinct kinetics and mechanisms (7,35,55). Recently, it was shown that early recruitment of the a3 subunit of the v-ATPase to young phagosomes occurs via tubular structures extending from the perinuclear lysosomes along microtubules (56).…”

Raft association and lipid droplet targeting of flotillins are independent of caveolin

“…Despite a striking sequence similarity between flotillin-1 and flotillin-2, specific recruitment of flotillin-1 to lipid bodies upon fatty-acid loading could also underline a specific function in lipid regulation or trafficking towards these organelles. Flotillin-1, but not flotillin-2, has been shown to be enriched in maturing phagosomes (Dermine et al, 2001) and exosomes (Rajendran et al, 2006), and only flotillin-1 is specifically upregulated at least 10-fold during adipocyte differentiation (Bickel et al, 1997), suggesting that flotillin-1 could be involved in storage pathways. Finally, another possibility is that flotillins could function as a raft organizer by means of homo/hetero-oligomerization (Neumann-Giesen et al, 2004), and thus would organize or shape scaffold domains at the level of lipid droplet membranes.…”

HIV envelope induces a cascade of cell signals in non-proliferating target cells that favor virus replication

“…Finally we note the induction of flotillin-1 (Fig. 2), a protein that is enriched in lipid rafts (44), which are membrane structures that facilitate fusion of HIV with plasma membranes of target cells (45). Discussion HIV-1 pathogenesis is regulated by a complex interaction between viral and cellular factors.…”