Quantitative Analysis of Membrane Remodeling at the Phagocytic Cup

Abstract

Nascent phagosomes, which are derived from the plasma membrane, acquire microbicidal properties through multiple fusion and fission events collectively known as maturation. Here we show that remodeling of the phagosomal membrane is apparent even before sealing, particularly when large particles are ingested. Fluorescent probes targeted to the plasma membrane are cleared from the region lining the particle before engulfment is completed. Extensive clearance was noted for components of the inner as well as outer monolayer of the plasmalemma. Segregation of lipid microdomains was ruled out as the mechanism underlying membrane remodeling, because markers residing in rafts and those that are excluded were similarly depleted. Selective endocytosis was also ruled out. Instead, several lines of evidence indicate that endomembranes inserted by exocytosis at sites of ingestion displace the original membrane constituents from the base of the phagosomal cup. The Fc␥ receptors that trigger phagocytosis remain associated with their ligands. By contrast, Src-family kinases that are the immediate effectors of receptor activation are flushed away from the cup by the incoming membranes. Together with the depletion of phosphoinositides required for signal transduction, the disengagement of receptors from their effectors by bulk membrane remodeling provides a novel means to terminate receptor signaling.

WASH drives early recycling from macropinosomes and phagosomes to maintain surface phagocytic receptors

“…6F). phagosomal and macropinosome membranes, the specificity of protein internalization is unclear (49)(50)(51). To assess this lack of specificity, we labeled surface proteins by biotinylation and looked for their presence in purified early phagosomes.…”

Cholesterol-Binding Peptides and Phagocytosis

“…It is recognized that successful phagosome formation requires local actin polymerization/depolymerization (Aderem & Underhill, 1999;Tjelle et al, 2000;May & Machesky, 2001;Greenberg, 2001;Lee at al., 2007) that dependes on the phosphatidylinsiotol metabolism. A detailed quantitative assessment of membrane remodeling during FcγR-mediated phagocytosis revealed marked changes in membrane composition that concerned the localization and metabolism of phosphoinositides (Botelho et al, 2000;Lee at al., 2007;Fairn et al, 2010).…”

“…A detailed quantitative assessment of membrane remodeling during FcγR-mediated phagocytosis revealed marked changes in membrane composition that concerned the localization and metabolism of phosphoinositides (Botelho et al, 2000;Lee at al., 2007;Fairn et al, 2010). It was found in particular that at the onset of phagocytosis phosphatidylinositol (PI) 4,5-bisphosphate (PI4,5P 2 ) accumulates at sites where pseudopods are formed.…”

“…Lee at al., 2007 reported that there was an obvious clearance of the raft marker YFP-GPI from the base of forming phagosomes within minutes of particle contact and that this clearance resulted from the focal insertion of unlabeled endomembranes that are delivered focally by directed exocytosis. The authors interpreted these results as evidence against the raft involvement in the phagocytic process.…”

“…Therefore, understanding the molecular mechanisms of phagocytosis is very important and should help to solve a number of medicinal problems and elaborate new approaches for regulation and control/correction of the phagocytic process. B y n o w , i t i s a c c e p t e d t h a t m e c h a n i s m o f phagocytosis implicates such processes as exocytosis, endocytosis, and adhesion (Aderem & Underhill, 1999;Botelho et al, 2000;Booth et al, 2001;Dunina-Barkovskaya, 2004;Lee at al., 2007;Fairn et al, 2010). A detailed list of the molecular participants that accomplish the initial membrane reorganization after the contact with the particle, subsequent formation and pinching-off of the phagosomal vesicle, and the components involved in the phagolysosome maturation and recycling has been created (Araki et al, 1996;Hackam et al, 1998;Morrissette et al, 1999;Garin et al, 2001;May & Machesky, 2001;Booth et al, 2001;Grinstein, 2010).…”