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Ultrafast diffusion of a fluorescent cholesterol analog in compartmentalized plasma membranes.

Hiramoto-Yamaki N, Tanaka KA, Suzuki KG, Hirosawa KM, Miyahara MS, Kalay Z, Tanaka K, Kasai RS, Kusumi A, Fujiwara TK - Traffic (2014)

Bottom Line: 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-Chol's probable association with raft domains.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS) and Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.

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In the COS-7-cell PM, Bdp-Chol diffused as rapidly as in the PtK2- and HASM-cell PMs, and the COS-7-cell PM, like the PtK2- and HASM-cell PMs, is compartmentalized for phospholipid diffusion. A) The distribution of DeffMACRO values of Bdp-Chol in COS-7 intact PM (n = 164). The arrowhead indicates the median value. B) The distribution of the compartment sizes in the COS-7-cell PM, obtained by hop-diffusion fitting. The arrowhead indicates the median value.
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fig11: In the COS-7-cell PM, Bdp-Chol diffused as rapidly as in the PtK2- and HASM-cell PMs, and the COS-7-cell PM, like the PtK2- and HASM-cell PMs, is compartmentalized for phospholipid diffusion. A) The distribution of DeffMACRO values of Bdp-Chol in COS-7 intact PM (n = 164). The arrowhead indicates the median value. B) The distribution of the compartment sizes in the COS-7-cell PM, obtained by hop-diffusion fitting. The arrowhead indicates the median value.

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)].


Ultrafast diffusion of a fluorescent cholesterol analog in compartmentalized plasma membranes.

Hiramoto-Yamaki N, Tanaka KA, Suzuki KG, Hirosawa KM, Miyahara MS, Kalay Z, Tanaka K, Kasai RS, Kusumi A, Fujiwara TK - Traffic (2014)

In the COS-7-cell PM, Bdp-Chol diffused as rapidly as in the PtK2- and HASM-cell PMs, and the COS-7-cell PM, like the PtK2- and HASM-cell PMs, is compartmentalized for phospholipid diffusion. A) The distribution of DeffMACRO values of Bdp-Chol in COS-7 intact PM (n = 164). The arrowhead indicates the median value. B) The distribution of the compartment sizes in the COS-7-cell PM, obtained by hop-diffusion fitting. The arrowhead indicates the median value.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265843&req=5

fig11: In the COS-7-cell PM, Bdp-Chol diffused as rapidly as in the PtK2- and HASM-cell PMs, and the COS-7-cell PM, like the PtK2- and HASM-cell PMs, is compartmentalized for phospholipid diffusion. A) The distribution of DeffMACRO values of Bdp-Chol in COS-7 intact PM (n = 164). The arrowhead indicates the median value. B) The distribution of the compartment sizes in the COS-7-cell PM, obtained by hop-diffusion fitting. The arrowhead indicates the median value.
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)].

Bottom Line: 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-Chol's probable association with raft domains.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Integrated Cell-Material Sciences (WPI-iCeMS) and Institute for Frontier Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.

Show MeSH