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Fig. 7. Rac1 activity is required for bulk plasma-membrane endocytosis in the growth cones. (A) Growth cones of 3-DIV neurons coexpressing YFP (green in the merged images) and either the dominant-negative mutant Rac1-N17 (top panels) or the constitutively active mutant Rac1-V12 (middle panels), or expressing only YFP (bottom panels), incubated in KRH containing FM4-64 (red in the merged images) for 1 minute and fixed. The Rac1 mutants are detected with an anti-FLAG antibody (blue in the merged images). Expression of Rac1-N17 inhibits bulk FM4-64 internalization whereas endocytosis via smaller vesicles is unaffected (arrowheads). (B) Quantification of FM4-64 uptake in Rac1-N17- or Rac1-V12-expressing growth cones treated as shown in A (mean ± s.e.m.; n=27 growth cones per condition; *P<0.001, Dunnett's test for neurons expressing either Rac1-N17 or Rac1-V12 vs control, i.e. growth cones expressing YFP only). The total growth-cone area was reduced by Rac1-N17 expression (mean ± s.e.m.: control, 44±2.5 µm²; Rac1-N17, 35±2.6 µm²; P<0.05, Dunnett's test vs control), but not by Rac1-V12 expression (mean ± s.e.m.: 42±2.7 µm²). Scale bar: 10 µm.

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