Hisae Tsuboi

Membrane molecules mobile even after chemical fixation

Lyn, H-ras, Cbp and TfR (we observed ~70% immobilization of GPI-anchored proteins, on average, depending strongly on the protein; Fig. 1b and Supplementary Figs. 3–5). Increasing glutaraldehyde concentration to 0.2% or methanol (100%) fixation resulted in immobilization of >80% GPI-anchored proteins but not of phospholipids or cholesterol. We then investigated antibody-induced clustering under different fixation conditions by measuring the fluorescence intensities of individual spots (Supplementary Fig. 6). Whereas treatment with 4% paraformaldehyde with or without 0.1% glutaraldehyde did not itself induce clustering, paraformaldehyde treatment did not block antibody-induced clustering of Halo-GPI and Halo–H-ras. Including 0.1% glutaraldehyde in addition to 4% paraformaldehyde suppressed clustering but did not entirely inhibit it. These results indicate that, in interpreting immunolocalization data, antibody-induced clustering Membrane molecules mobile even after chemical fixation

Raft-based interactions of gangliosides with a GPI-anchored receptor

Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59s transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.