Figure 10
FRAP analysis of JAM-C and Cadm3 adhesion dynamics in migrating neurons. CGNs were transfected with expression vectors encoding JAM-C-pHluorin or Cadm3-pHluorin, and FRAP was used to track cell surface adhesion receptor diffusion and recruitment. Blebbistatin (50 μM) or jasplakinolide (5 μM) potently retarded the recruitment of JAM-C (A, B) and Cadm3 (C, D) to photobleached adhesion sites within the neuronal leading process. Blebbistatin and jasplakinolide increased the t1/2 of recovery of JAM-C-pHluorin fluorescence from 1.94 ± 0.75 sec to 17.34 ± 3.16 sec (n = 30 cells; P = 9.64 × 10-22, t-test) and to 10.63 ± 3.88 sec (n = 25 cells; P = 1.07 × 10-11, t-test), respectively. Blebbistatin and jasplakinolide increased the t1/2 of Cadm3-pHluorin fluorescence recovery from 4.26 ± 0.98 sec to 23.28 ± 4.77 sec (n = 30 cells; P = 1.83 × 10-19, t-test) and to 11.19 ± 4.52 sec (n = 30 cells, P = 1.69 × 10-8, t-test), respectively. Scale bar = 10 μm. A bracket highlights the photobleached region of interest in each representative image.