Manami Miyahara

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

Ultrafast Diffusion of a Fluorescent Cholesterol Analog in Compartmentalized Plasma Membranes

Cholesterol distribution and dynamics in the plasma membrane (PM) are poorly understood. The recent development of Bodipy488‐conjugated cholesterol molecule (Bdp‐Chol) allowed us to study cholesterol behavior in the PM, using single fluorescent‐molecule imaging. Surprisingly, in the intact PM, Bdp‐Chol diffused at the fastest rate ever found for any molecules in the PM, with a median diffusion coefficient (D) of 3.4 µm2/second, which was ∼10 times greater than that of non‐raft phospholipid molecules (0.33 µm2/second), despite Bdp‐Chols probable association with raft domains. Furthermore, Bdp‐Chol exhibited no sign of entrapment in time scales longer than 0.5 milliseconds. In the blebbed PM, where actin filaments were largely depleted, Bdp‐Chol and Cy3‐conjugated dioleoylphosphatidylethanolamine (Cy3‐DOPE) diffused at comparable Ds (medians = 5.8 and 6.2 µm2/second, respectively), indicating that the actin‐based membrane skeleton reduces the D of Bdp‐Chol only by a factor of ∼2 from that in the blebbed PM, whereas it reduces the D of Cy3‐DOPE by a factor of ∼20. These results are consistent with the previously proposed model, in which the PM is compartmentalized by the actin‐based membrane‐skeleton fence and its associated transmembrane picket proteins for the macroscopic diffusion of all of the membrane molecules, and suggest that the probability of Bdp‐Chol passing through the compartment boundaries, once it enters the boundary, is ∼10× greater than that of Cy3‐DOPE. Since the compartment sizes are greater than those of the putative raft domains, we conclude that raft domains coexist with membrane‐skeleton‐induced compartments and are contained within them.