Both MHC class II and its GPI-anchored form undergo hop diffusion as observed by single-molecule tracking.
Previously, investigations using single-fluorescent-molecule tracking at frame rates of up to 65 Hz, showed that the transmembrane MHC class II protein and its GPI-anchored modified form expressed in CHO cells undergo simple Brownian diffusion, without any influence of actin depolymerization with cytochalasin D. These results are at apparent variance with the view that GPI-anchored proteins stay with cholesterol-enriched raft domains, as well as with the observation that both lipids and transmembrane proteins undergo short-term confined diffusion within a compartment and long-term hop diffusion between compartments. Here, this apparent discrepancy has been resolved by reexamining the same paradigm, by using both high-speed single-particle tracking (50 kHz) and single fluorescent-molecule tracking (30 Hz). Both molecules exhibited rapid hop diffusion between 40-nm compartments, with an average dwell time of 1-3 ms in each compartment. Cytochalasin D hardly affected the hop diffusion, consistent with previous observations, whereas latrunculin A increased the compartment sizes with concomitant decreases of the hop rates, which led to an approximately 50% increase in the median macroscopic diffusion coefficient. These results indicate that the actin-based membrane skeleton influences the diffusion of both transmembrane and GPI-anchored proteins.
Umemura
Yasuhiro M
YM
Membrane Mechanisms Project, International Cooperative Research Project, Japan Science and Technology Agency, Institute for Integrated Cell-Material Sciences, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Vrljic
Marija
M
Nishimura
Stefanie Y
SY
Fujiwara
Takahiro K
TK
Suzuki
Kenichi G N
KG
Kusumi
Akihiro
A
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004058:Diffusion; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques
D017261:Glycosylphosphatidylinositols; D000949:Histocompatibility Antigens Class II