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Recruitment of Syt V to GM1-containing microdomains of phagosome membranes is prevented by LPG. A, BMM were allowed to internalize Zym for 30 min, fixed and stained for endogenous Syt V (green) and GM1 (red). White arrowheads indicate examples of colocalization between Syt V and GM1-positive microdomains, indicating a Syt V enrichment on these microdomains. B and C, BMM were either left untreated or treated with 10 mmol/L MßCD for 1 h before the internalization of Zym for 30 and 120 min. Cells were then fixed and stained for Syt V and LAMP-1. Representative confocal images of Syt V recruitment on cells with or without MßCD treatment is presented (B), white arrowheads indicate phagosomes. Syt V acquisition is expressed as a percentage of phagosome recruitment for Syt V. At least 100 phagosomes for each condition were assessed. Three independent experiments were performed and the bars show the standard deviations of one representative triplicate (C) (**, p=0.005). D, BMM were allowed to internalize Zym-LPG for 30 min, fixed and stained for LPG (green) and GM1 (red). White arrowheads indicate a colocalization between LPG and GM1-positive rafts. BMM were allowed to internalize Zym (E, upper panel) or LPG-Zym (E, lower panel) for 30 min, fixed and stained for Syt V (blue), LPG (green) and GM1 (red). Blue arrowheads indicate a local Syt V acquisition on phagosome membrane and yellow arrowheads indicate a local colocalization between GM1 microdomains and LPG. A rim around each phagosome was manually traced with a one pixel width and fluorescence intensity profile of Syt V in blue, LPG in green and GM1 in red were represented in a graph (F). Bars, 3 µm (A, B and D) or 1 µm (E).
From: Vinet AF, Fukuda M, Turco SJ, Descoteaux A (2009) The Leishmania donovani Lipophosphoglycan Excludes the Vesicular Proton-ATPase from Phagosomes by Impairing the Recruitment of Synaptotagmin V. PLoS Pathog 5(10): e1000628.