Figure 5.

Microspheres move rearward on growth cones and progressively accumulate L1–GFP molecules. Microspheres coated with L1–Fc, Ncad–Fc, or antibodies against L1 were placed for 10 s at the periphery of growth cones from L1–GFP-transfected neurons, by using an optical trap. (A and B) The movement of the bead and the fluorescence accumulation at the bead contact (arrowheads) were followed for 10 min. The black trace on the white field upper image indicates the bead trajectory, and the bead position is shown at the end of the experiment (for better contrast, we superimposed the white field image of the bead to a DIC image of the growth cone obtained at the end of the sequence). Cells were either left untreated (A) or pretreated with thrombin for 1 min, and then they were rinsed in the presence of PPACK before optical tweezers manipulation (B). The fluorescence level around the bead was normalized by that on adjacent regions and plotted over time for untreated cells (C) or cells pretreated with thrombin (D). Data are expressed as mean ± SEM, and they are fit with a first-order kinetics model (plain curves) (Thoumine et al., 2006). (E) The instantaneous bead velocity is plotted over time for the different bead coatings, pooling data from conditions with or without thrombin, which did not differ in terms of velocity. The number of experiments (between 10 and 20) is given in Table 1.