Ultrafast diffusion of a fluorescent cholesterol analog in compartmentalized plasma membranes.
Bottom Line: 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.
Affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS) and Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.Show MeSH
Mentions: The effects of actin-modifying drugs on macroscopic diffusion, even that of phospholipids and transmembrane proteins, have been found to be generally small 19,45, consistent with the data shown here (Figures 4 and 10). This is probably the reason why many scientists have previously been misled to conclude that the actin cytoskeleton is not involved in regulating the diffusion of membrane molecules, and that phospholipids undergo simple-Brownian diffusion, e.g. in PtK2-cell, HASM-cell and COS-7-cell PMs 12,23,36. However, using ultra high-speed single-molecule tracking with time resolutions better than 25 microseconds, hop diffusion of phospholipids has been clearly detected by statistical analyses (see Figures 7 and 11B for HASM- and COS-7-cell PMs, and Murase et al. 19 for the PtK2-cell PM), and the effects of actin-modifying drugs on the compartment size and the residency time within a compartment have also been clearly detected here, even for the HASM-cell PM [see Figure 8 and Table5; see Note superscript ‘a’ in Table5 for the equation to calculate residency time from the compartment size determined by ultrafast single Gold-PE tracking and the macroscopic diffusion coefficient of fluorescently labeled molecules. Table5 addresses our aims (iv-b) and (iv-c)].
Affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS) and Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.