Figure 6.
Lyn-FG, FGH-Ras, and other lipid-anchored raftophilic molecules were recruited at CD59 clusters but not at non–cross-linked CD59. The distributions (histograms) of the colocalization durations for the “correct” and “shifted” overlays, shown in semilog plots. The histograms for shifted overlays were fitted by a single exponential function (dashed line), and those for the correct overlays were fitted by the sum of two exponential functions (solid line), with the shorter time constant set to τ1 obtained from the histogram of the shifted overlay. The boxes highlighted in orange contain histograms that could be better fitted with the sum of two exponential decay functions rather than a single exponential function. The values of τ1 and τ2 are indicated in each box. See Table S1 for statistical parameters. (A) Lyn-FG was recruited at CD59 clusters but not at non–cross-linked CD59 (a, b). (B) Recruitment of Lyn-related molecules and other lipid-anchored cytoplasmic model proteins at CD59 clusters: myrpal-N20LynGFP (a) and palpal-N16GAP43-GFP (c) were recruited, but TM-Lyn-GFP (b) and GFP-C5Rho-gerger (d) were not. (C) FGH-Ras was recruited at CD59 clusters but not at non–cross-linked CD59 (a, b), and FGH-Ras recruitment at CD59 clusters depended on the PM cholesterol (c). Meanwhile, GFP-tH was recruited at CD59 clusters. (D) Summary of the bound lifetimes (τ2) of Lyn-FG, FGH-Ras, and other cytoplasmic lipid-anchored signaling molecules at CD59 clusters. The differences in τ2 values are nonsignificant. ND, not detected. The MβCD treatments (4 mM at 37°C for 30 min; see part C, c) have been controversial. However, the involvement of raft domains was examined in a variety of methods in the present research, including the use of various lipid-anchoring chains and the TM domain of a prototypical nonraft molecule, LDLR, and a prototypical nonraft phospholipid DOPE. In the past, we employed the MβCD treatments together with other control experiments (using TM artificial mutants of GPI-ARs, saponin treatment, cholesterol repletion after the MβCD treatment) and found that the MβCD treatment with 4 mM MβCD at 37°C for 30 min reproducibly gave the results consistent with the results obtained by using other methods of testing the raft involvement.

Lyn-FG, FGH-Ras, and other lipid-anchored raftophilic molecules were recruited at CD59 clusters but not at non–cross-linked CD59. The distributions (histograms) of the colocalization durations for the “correct” and “shifted” overlays, shown in semilog plots. The histograms for shifted overlays were fitted by a single exponential function (dashed line), and those for the correct overlays were fitted by the sum of two exponential functions (solid line), with the shorter time constant set to τ1 obtained from the histogram of the shifted overlay. The boxes highlighted in orange contain histograms that could be better fitted with the sum of two exponential decay functions rather than a single exponential function. The values of τ1 and τ2 are indicated in each box. See Table S1 for statistical parameters. (A) Lyn-FG was recruited at CD59 clusters but not at non–cross-linked CD59 (a, b). (B) Recruitment of Lyn-related molecules and other lipid-anchored cytoplasmic model proteins at CD59 clusters: myrpal-N20LynGFP (a) and palpal-N16GAP43-GFP (c) were recruited, but TM-Lyn-GFP (b) and GFP-C5Rho-gerger (d) were not. (C) FGH-Ras was recruited at CD59 clusters but not at non–cross-linked CD59 (a, b), and FGH-Ras recruitment at CD59 clusters depended on the PM cholesterol (c). Meanwhile, GFP-tH was recruited at CD59 clusters. (D) Summary of the bound lifetimes (τ2) of Lyn-FG, FGH-Ras, and other cytoplasmic lipid-anchored signaling molecules at CD59 clusters. The differences in τ2 values are nonsignificant. ND, not detected. The MβCD treatments (4 mM at 37°C for 30 min; see part C, c) have been controversial. However, the involvement of raft domains was examined in a variety of methods in the present research, including the use of various lipid-anchoring chains and the TM domain of a prototypical nonraft molecule, LDLR, and a prototypical nonraft phospholipid DOPE. In the past, we employed the MβCD treatments together with other control experiments (using TM artificial mutants of GPI-ARs, saponin treatment, cholesterol repletion after the MβCD treatment) and found that the MβCD treatment with 4 mM MβCD at 37°C for 30 min reproducibly gave the results consistent with the results obtained by using other methods of testing the raft involvement.

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