Fig. 4. Calflagin Tb24 distribution reflects membrane raft stability. (A) The flagellum of bloodstream form T. brucei is enriched in cholesterol and calflagin Tb24. Filipin staining of untreated cells shows enrichment in the flagellum. The flagellar localization of calflagin Tb24 is resistant to 1% Triton X-100 at 4°C. The region of the flagellum connector (arrows, center panel) is particularly rich in staining for this DRM marker. If cholesterol is depleted by preincubation in 20 mM MBCD for 30 minutes, flagellar concentration of calflagins Tb24 is lost. (B) MBCD treatment leads to loss of buoyancy of calflagin Tb24. Bloodstream form T. brucei treated with MBCD and untreated control cells were analyzed by Optiprep gradient centrifugation and western blotting with calflagin Tb24-specific antiserum as described in Fig. 1. W, whole-cell lysate. (C) Chemical modulation of membrane fluidity. Washed procyclic form trypanosomes were treated with 10% DMSO for 1 minute to stabilize the liquid ordered phase or 1% diethyl ether for 30 seconds to fluidize the liquid ordered phase. Cells were then fixed and processed for immunofluorescence microscopy using anti-calflagin Tb24. Bar, 5 µm.