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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: 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.

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 HASM-cell PM, Bdp-Chol diffused ∼9× faster than Cy3-DOPE in the intact PM (comparison by median values), probably due to the higher sensitivity of Cy3-DOPE diffusion to the actin-based membrane skeleton, as compared to that of Bdp-Chol diffusion. The distribution of the effective diffusion coefficient, DeffMACRO, for each trajectory of Bdp-Chol (top) or Cy3-DOPE (bottom) in the intact PM (both left and right), after partial actin-depolymerization (right), after actin stabilization (right) and in the blebbed PM (left) of HASM cells. Arrowheads indicate median values. p values indicate the Mann–Whitney U-test results against the results in the intact PM. See the summary in Tables2 and 3 (including the numbers of trajectories inspected).
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fig10: In the HASM-cell PM, Bdp-Chol diffused ∼9× faster than Cy3-DOPE in the intact PM (comparison by median values), probably due to the higher sensitivity of Cy3-DOPE diffusion to the actin-based membrane skeleton, as compared to that of Bdp-Chol diffusion. The distribution of the effective diffusion coefficient, DeffMACRO, for each trajectory of Bdp-Chol (top) or Cy3-DOPE (bottom) in the intact PM (both left and right), after partial actin-depolymerization (right), after actin stabilization (right) and in the blebbed PM (left) of HASM cells. Arrowheads indicate median values. p values indicate the Mann–Whitney U-test results against the results in the intact PM. See the summary in Tables2 and 3 (including the numbers of trajectories inspected).

Mentions: After partial actin depolymerization by the latrunculin-B treatment, virtually all of the Cy3-PEs still exhibited simple-Brownian diffusion in the time scale of ≥1 second, with a ∼20% increase in DeffMACRO (Table3, the distribution for Cy3-DOPE is shown in Figure 10, bottom right), whereas actin stabilization by the jasplakinolide treatment led to a ∼35% decrease in DeffMACRO (the distribution for Cy3-DOPE is shown in Figure 10, bottom right) for all Cy3-PEs (means for Cy3-PEs, see Table3, which also includes the data obtained at 24°C). These results clearly indicate that the actin-based membrane skeleton, rather than the half-micron-sized domains that confine and concentrate saturated phospholipids proposed previously 36,37, is involved in slowing the phospholipid diffusion in the HASM-cell PM, strongly supporting that the actin-induced fence-picket model is applicable to the PM of the HASM cell.


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 HASM-cell PM, Bdp-Chol diffused ∼9× faster than Cy3-DOPE in the intact PM (comparison by median values), probably due to the higher sensitivity of Cy3-DOPE diffusion to the actin-based membrane skeleton, as compared to that of Bdp-Chol diffusion. The distribution of the effective diffusion coefficient, DeffMACRO, for each trajectory of Bdp-Chol (top) or Cy3-DOPE (bottom) in the intact PM (both left and right), after partial actin-depolymerization (right), after actin stabilization (right) and in the blebbed PM (left) of HASM cells. Arrowheads indicate median values. p values indicate the Mann–Whitney U-test results against the results in the intact PM. See the summary in Tables2 and 3 (including the numbers of trajectories inspected).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig10: In the HASM-cell PM, Bdp-Chol diffused ∼9× faster than Cy3-DOPE in the intact PM (comparison by median values), probably due to the higher sensitivity of Cy3-DOPE diffusion to the actin-based membrane skeleton, as compared to that of Bdp-Chol diffusion. The distribution of the effective diffusion coefficient, DeffMACRO, for each trajectory of Bdp-Chol (top) or Cy3-DOPE (bottom) in the intact PM (both left and right), after partial actin-depolymerization (right), after actin stabilization (right) and in the blebbed PM (left) of HASM cells. Arrowheads indicate median values. p values indicate the Mann–Whitney U-test results against the results in the intact PM. See the summary in Tables2 and 3 (including the numbers of trajectories inspected).
Mentions: After partial actin depolymerization by the latrunculin-B treatment, virtually all of the Cy3-PEs still exhibited simple-Brownian diffusion in the time scale of ≥1 second, with a ∼20% increase in DeffMACRO (Table3, the distribution for Cy3-DOPE is shown in Figure 10, bottom right), whereas actin stabilization by the jasplakinolide treatment led to a ∼35% decrease in DeffMACRO (the distribution for Cy3-DOPE is shown in Figure 10, bottom right) for all Cy3-PEs (means for Cy3-PEs, see Table3, which also includes the data obtained at 24°C). These results clearly indicate that the actin-based membrane skeleton, rather than the half-micron-sized domains that confine and concentrate saturated phospholipids proposed previously 36,37, is involved in slowing the phospholipid diffusion in the HASM-cell PM, strongly supporting that the actin-induced fence-picket model is applicable to the PM of the HASM cell.

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.

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