Figure 7.

Actin-bound proteins LFA-1 treadmill backward at the cytoplasmic membrane, whereas nonactin-bound protein MHC-1 diffuses without net transport. (A and B) Backward transports of actin and actin-bound LFA-1 transmembrane protein are shown. (i) Maximal intensity projection of TIRF sequences on adhesion-free cells transduced with RFP-actin (A) and stained with antibody M24 that binds the actin-bound integrin LFA-1 in its high-affinity state (B). Scale bars, 5 μm. (ii) Representative kymographs along the yellow line in (i), with the front advance highlighted in magenta and clusters retrograde flow highlighted in blue. See also Videos S13 and S14. (iii) Schematics representing a side view of a nonadherent cell observed with a TIRF objective and highlighting the retrograde flow of internal actin (green units) and external LFA-1 integrins (orange bars). (C and D) Diffusive dynamics of nonactin-bound transmembrane protein MHC-1 and lipidic DiO marker. TIRF-FRAP experiments on adhesion-free cells (C), stained with anti-HLA-ABC that binds the nonactin-bound MHC-1 type I proteins and (D) with membrane lipidic marker DiO. Yellow circles indicate frapped regions used to calculate fluorescence recovery. Scale bars, 5 μm. (E) Quantification of transmembrane proteins advection and diffusion. Left: speed values for retrograding actin were measured by cluster tracking as in (D) (n = 7 cells, 51 clusters tracked) and by FRAP as in Supporting Materials and Methods (n = 8 cells) and for LFA-1 clusters (n = 16 cells, 106 clusters tracked), all normalized to the front of the cell. Error bars stand for standard deviation. Right: shown are the averaged FRAP curves for DiO (blue, n = 10 cells) and MHC-1 (green, n = 14 cells). All values were normalized and corrected by a nonbleached cell. Diffusion coefficient is of 3.1 ± 1.8 μm²/s for DiO and 0.26 ± 0.22 μm²/s for MHC-1. (F) Single cell evidence of average backward advection for actin-bound LFA-1 and average absence of motion for nonactin-bound MHC-1. (i) Schematics representing a side view of a nonadherent cell double stained for LFA-1 (orange) and MHC (blue). (ii) TIRF images after FRAP bleaching of a line pattern reveal actin-bound activated LFA-1 in its high-affinity state (left) and nonactin-bound MHC-1 (right). (iii) Kymographs in the yellow rectangle in (ii) with the motion of clusters and the center of mass of the FRAP region highlighted in blue for, respectively, LFA-1 and MHC-1. See also Video S15. To see this figure in color, go online.