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. 2016 Jan 21;113(6):E772–E781. doi: 10.1073/pnas.1514530113

Fig. 4.

Fig. 4.

The actin cytoskeleton actively arrests diffusing α-GalCer–loaded hCD1d nanoclusters on the cell membrane. (A) Snapshot of a dual-color TIRFM movie displaying Lifeact-GFP–labeled actin (green) and α-GalCer–loaded hCD1d nanoclusters (red) labeled with the iNKT-TCR-QD conjugate. (Scale bar: 2 µm.) (B and C) Representative magnified TIRFM images at two different time sequences with an example of a 2D trajectory of α-GalCer–loaded hCD1d diffusing inside (black line) and outside (white line) actin-rich regions, with the diffusing particle outlined with a red dashed circle. In B, hCD1d is inside actin, whereas C shows the time sequence when hCD1d is outside actin. (Scale bar: 500 nm.) (D) Distributions of the D2–4 values for α-GalCer–loaded WT-hCD1d and α-GalCer–loaded TD-hCD1d nanoclusters inside and outside high-actin regions. The D2–4 values reported here are somewhat different from the median values reported in Fig. 1, because a preselected number of trajectories that diffused outside and inside actin-rich regions was chosen for this analysis. ns, Not significant (Student’s t test). *P < 0.0001 (Student’s t test). (E and F) Cumulated cartography maps containing 8,000 localizations over a recording time of 20 s from (E) α-GalCer–loaded WT-hCD1d and (F) TD-hCD1d overlaid onto a Lifeact-GFP TIRFM image. The white dots correspond to the different spatial positions explored by hCD1d during the observation time. The pseudocolored image corresponds to the actin intensity, going from blue (actin-poor) to red (actin-rich). The black arrows point to hCD1d diffusion on actin-poor regions, whereas black-outlined arrows point to hCD1d diffusing close to actin-rich regions. (Scale bar: 1 µm.) (G) Fractional occurrence of hCD1d localization positions vs. changes in actin intensity: ∆actin for α-GalCer–loaded WT-hCD1d (black) and α-GalCer–loaded TD-hCD1d (red). Data were fitted with a straight line, and the slope was obtained. SPT data are from at least 87 trajectories on 30 different cells over five different experiments. Cartography maps data contain at least 8,000 localizations. Analysis has been performed on six different cells and three different experiments per condition.