Fig. 1. Myosin Va is distributed in the cortex of PC12 cells and colocalises with rCgB. (A-G) Non-transfected PC12 cells were cultured for 2 days in the absence (A-D) or presence (E-G) of NGF. Then, the cells were fixed and immunostained against rCgB (B,D,F) and myosin Va with DIL1 (A) and DIL2 (C,E) antibody, respectively. Single corresponding confocal sections (AB, CD, EF) are shown. G shows a Nomarski image of the same cells as in E and F. Arrowheads in A-D, show cortical subdomains enriched in both rCgB and myosin Va; arrows in E-G indicate growth cones. Bars, 10 µm. (H-H") Cells were transfected with hCgB-GFP(S65T). After incubation of cells for 2 hours at 20°C and then for 180 minutes at 37°C, a PNS was prepared and SGs were sedimented on coverslips by differential centrifugation, fixed, and immunostained with DIL2 antibody against myosin Va. Subsequently, single confocal sections of GFP-fluorescent SGs (H) and the corresponding immunosignals (H") were recorded. Arrows indicate immuno-positive, and arrowheads immuno-negative, GFP-fluorescent SGs (compare H and H'). Bar, 1 µm. The colocalisation of fluorescent SGs with myosin Va was quantified (H"). Black bar, colocalisation of GFP-fluorescence and immuno-signals form corresponding frame pairs (correct); white bar, random colocalisation of GFP-and immuno-signals from non-corresponding frame pairs (random). Error bars, s.e.m. (n=20 images, each 60x60 µm) corresponding to >500 fluorescent SGs.