Supplemental Materials

This article contains the following supporting material:

• Movie 01 - Movement of L1-GFP vesicles in a growth cone initially treated with thrombin. Time is indicated in seconds. Bar = 5 µm.
• Movie 02 - Movement of L1-GFP vesicles in a growth cone initially treated with thrombin and in the presence of 1 µM cytochalasin D. Note that vesicle movement is faster when compared to movie 1. Time is indicated in seconds. Bar = 5 µm.
• Movie 03 - Movement of anti-GFP conjugated Quantum dots bound to L1-GFP molecules on a growth cone surface. The Quantum dots (in red) are super-imposed on the L1-GFP signal (green) taken at the beginning of the recording. Time is indicated in seconds. Bar = 10 µm.
• Movie 04 - Recruitment of L1-GFP by an L1-Fc coated microsphere, in the absence of thrombin. The 4 µm microsphere is placed on the growth cone at time zero with optical tweezers (first transmission image), then L1-GFP fluorescence accumulation is followed for 10 min. Time is given in minutes:seconds. Bar = 5 µm.
• Movie 05 - Neurons transfected with L1-GFP were incubated for 30 min with L1-Fc coated microspheres, leading to steady state accumulation of L1-GFP. Then cells were mounted on the microscope and observation was started. A growth cone with a bead having with recruited L1-GFP is shown in false colour. Thrombin was applied for 100 sec at time 2 min, leading to a dramatic shedding of L1-GFP signal at the microsphere. We observed very little recovery over a 30 min period, indicating a lack of exocytosis to these stable contacts. Time is given in minutes:seconds. Bar = 5 µm.
• Movie 06 - Same experiment as in Movie 5 except that thrombin was applied continuously starting at time 2 min. One can observe the appearance of vesicles at the bead contact, then their sudden extinction (e.g. at time 6 min). These events likely correspond to vesicle exocytosis, the exposure of L1-GFP at the cell surface resulting in immediate cleavage of the GFP. Time is given in minutes:seconds. Bar = 10 µm.
• Supplemental Figure 1 - Quantification of exogenous L1-GFP versus endogenous L1. Neurons transfected with L1-GFP were immuno-stained under non-permeabilizing conditions with anti-L1 antibodies followed by anti-rabbit conjugated to Alexa⁵⁶⁸. An L1-GFP expressing cells is shown on the left and non-transfected counterparts in the middle and right panels. (A) Phase contrast image. (B) GFP fluorescence signal. (C) Anti-L1 staining or non-specific background obtained with no primary antibody (right panel).
• Supplemental Figure 2 - Images of L1-GFP behaviour at anti-L1 and Ncad-Fc coated microspheres. Microspheres coated with Ncad-Fc (A, B) or antibodies against L1 (C, D) were placed at the periphery of growth cones from L1-GFP transfected neurons, using an optical trap. The movement of the bead and the L1-GFP fluorescence 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. Cells were either left untreated (A, C) or pre-treated with thrombin for 1 min before optical tweezers manipulation (B). Note the accumulation of L1-GFP at anti-L1 coated microspheres (arrowheads) compared to an absence of recruitment for Ncad-Fc coated beads.
• Supplemental Figure 3 - Turnover of L1 adhesions formed spontaneously between older neurons. (A) Neurons transfected with L1-GFP were imaged at 15 DIV, at which time we could observe L1-GFP enrichment at sites of crossing between neurites (arrowheads). We performed FRAP experiments at these contacts (arrow), or on control areas showing no enrichment, and quantified the enrichment factor as described for microspheres. (B) The recovery showed a similar trend as for L1-Fc coated microspheres on younger neurons (Fig. 7B), giving a comparable turnover rate (9 ± 5 hr^-1). Data are mean ± sem of 8 individual experiments fitted with the diffusion/reaction model (plain curves).