Figure 1
Figure 1. Requirement for Rap1, RAPL, and talin in LFA-1–mediated arrest under shear flow. (A) Knockdown of talin, RAPL, and Rap1 by shRNA. Western blots of total cell lysates from BAF/LFA-1/L-selectin cells with lentiviruses encoding control shRNA or shRNA targeting Rap1a/b-specific (Rap1KD), RAPL (RAPLKD), talin (TalinKD) are shown. Tubulin was used as a loading control. Western blots of total lysates from the double knockdown cells with lentiviruses encoding RAPL (RAPLKD) and talin (TalinKD) are also shown in the right panel. (B) Effects of anti–L-selectin, anti–LFA-1, Rap1KD, RAPLKD, TalinKD, and double KD on the interactions of BAF/LFA-1/L-selectin cells with LS12 endothelial cells. Control cells were pretreated with or without anti–L-selectin or anti–LFA-1 antibody. Then the cells perfused at 2 dyne/cm2 on LS12 monolayers, which were immobilized with CXCL12. The digital images of interactions of BAF/LFA-1/L-selectin cells with LS12 endothelial cells were taken at 30 frames/second. The adhesive events of more than 100 cells were measured and categorized as described in the supplemental Methods. Data represent the mean ± SD of 3 independent experiments. *P < .001, compared with control cells. **P < .01, compared with RAPL or Talin KD cells. (C) Time-displacement profiles of individual cell movement over LS12 endothelial monolayers under shear flow. BAF/LFA-1/L-selectin cells were perfused at 2 dyne/cm2 on LS12 monolayers immobilized with CXCL21. Representative profiles of the displacements over time are shown for “stable arrest” of the cells on the CXCL12 immobilized endothelium, “rolling” in the presence of anti–LFA-1 antibody (CXCL12/anti–LFA-1), “rolling” of those depleting Rap1 (CXCL12/Rap1KD), and “transient arrest” of those depleting talin (CXCL12/TalinKD) and depleting RAPL (CXCL12/RAPL). Each line represents individual cell tracking. (D) The noninteracting and rolling velocities of control BAF/LFA-1/L-selectin cell movements on LS12 in the presence of anti–L-selectin (anti–L-selectin) and anti–LFA-1 (anti–LFA-1) antibodies as well as the Rap1 knockdown cells (Rap1KD). Data represent the mean ± SD of 3 independent experiments. (E) Stopping time of control (ct), RAPLKD or talinKD, or RAPL/talin double KD BAF/LFA-1/L-selectin cells arrested on LS12 endothelial cells are shown. More than 100 cells were measured in 3 independent experiments, and representative distribution of stopping time is shown. *P < .02, compared with RAPL or talin KD cells. (F) Epitope expression of mAb KIM127 on control (ct) BAF/LFA-1 cells and the Rap1 (Rap1KD), RAPL (RAPLKD), or talin (Talin KD) knockdown cells in the absence (None) or presence of CXCL12. *P < .05, compared with control cells. Data are normalized for LFA-1 expression detected by TS1/18.

Requirement for Rap1, RAPL, and talin in LFA-1–mediated arrest under shear flow. (A) Knockdown of talin, RAPL, and Rap1 by shRNA. Western blots of total cell lysates from BAF/LFA-1/L-selectin cells with lentiviruses encoding control shRNA or shRNA targeting Rap1a/b-specific (Rap1KD), RAPL (RAPLKD), talin (TalinKD) are shown. Tubulin was used as a loading control. Western blots of total lysates from the double knockdown cells with lentiviruses encoding RAPL (RAPLKD) and talin (TalinKD) are also shown in the right panel. (B) Effects of anti–L-selectin, anti–LFA-1, Rap1KD, RAPLKD, TalinKD, and double KD on the interactions of BAF/LFA-1/L-selectin cells with LS12 endothelial cells. Control cells were pretreated with or without anti–L-selectin or anti–LFA-1 antibody. Then the cells perfused at 2 dyne/cm2 on LS12 monolayers, which were immobilized with CXCL12. The digital images of interactions of BAF/LFA-1/L-selectin cells with LS12 endothelial cells were taken at 30 frames/second. The adhesive events of more than 100 cells were measured and categorized as described in the supplemental Methods. Data represent the mean ± SD of 3 independent experiments. *P < .001, compared with control cells. **P < .01, compared with RAPL or Talin KD cells. (C) Time-displacement profiles of individual cell movement over LS12 endothelial monolayers under shear flow. BAF/LFA-1/L-selectin cells were perfused at 2 dyne/cm2 on LS12 monolayers immobilized with CXCL21. Representative profiles of the displacements over time are shown for “stable arrest” of the cells on the CXCL12 immobilized endothelium, “rolling” in the presence of anti–LFA-1 antibody (CXCL12/anti–LFA-1), “rolling” of those depleting Rap1 (CXCL12/Rap1KD), and “transient arrest” of those depleting talin (CXCL12/TalinKD) and depleting RAPL (CXCL12/RAPL). Each line represents individual cell tracking. (D) The noninteracting and rolling velocities of control BAF/LFA-1/L-selectin cell movements on LS12 in the presence of anti–L-selectin (anti–L-selectin) and anti–LFA-1 (anti–LFA-1) antibodies as well as the Rap1 knockdown cells (Rap1KD). Data represent the mean ± SD of 3 independent experiments. (E) Stopping time of control (ct), RAPLKD or talinKD, or RAPL/talin double KD BAF/LFA-1/L-selectin cells arrested on LS12 endothelial cells are shown. More than 100 cells were measured in 3 independent experiments, and representative distribution of stopping time is shown. *P < .02, compared with RAPL or talin KD cells. (F) Epitope expression of mAb KIM127 on control (ct) BAF/LFA-1 cells and the Rap1 (Rap1KD), RAPL (RAPLKD), or talin (Talin KD) knockdown cells in the absence (None) or presence of CXCL12. *P < .05, compared with control cells. Data are normalized for LFA-1 expression detected by TS1/18.

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