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First published online 6 February 2003
doi: 10.1242/jcs.00285

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Regulation of membrane expansion at the nerve growth cone

¹ Department of Cellular and Structural Biology, University of Colorado School of Medicine, and University of Colorado Cancer Center, Denver, CO 80262, USA
² Departmento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, and CIQUIBIC, CONICET, Córdoba, Argentina
³ Instituto Investigación Médica Mercedes y Martín Ferreyra (INIMEC-CONICET), Córdoba, Argentina

View larger version (25K): [in a new window]   Fig. 1. Confocal fluorescence micrographs of a growth cone doublelabeled for F-actin (red) and TrkB (green). The merged image shows almost complete overlap of the two labels (yellow), indicating predominant localization of the receptor in the growth cone periphery. The growth cone is from a culture of hippocampal pyramidal neurons grown for 24 hours in vitro.

View larger version (13K): [in a new window]   Fig. 2. Fluorescence micrographs of hippocampal neurons, labeled with BODIPY-ceramide for 30 minutes at room temperature and chased for 2.5-3 hours at 37°C. The images were recorded in the red channel, which reveals only highly concentrated label. Intense labeling of the Golgi complex and of vesicle clusters in neurites and growth cones is evident. The growth cones of these neurons are shown in the time series in Fig. 4.

View larger version (68K): [in a new window]   Fig. 3. Confocal fluorescence image of the unusually large, live axonal growth cone of a BODIPY/ceramide-labeled hippocampal neuron (24 hours in culture). The labeling protocol was as described for Fig. 2. (A) A merged image showing the BODIPY label recorded separately in the red (high concentration) and green (low concentration) channels. The many red puncta show Golgi-derived vesicles or vesicle clusters, whereas the green fluorescence outlines the growth cone's plasmalemma, labeled with small amounts of BODIPY. As soon as possible (30 seconds) after recording BODIPY fluorescence, a phase-contrast image was taken of the same growth cone and then superimposed on the red BODIPY image. The merged image shows the relationship of Golgi-derived vesicles to structures seen under phase contrast.

View larger version (47K): [in a new window]   Fig. 4. Fluorescence micrographs (recorded in the red channel) of the axonal growth cones of hippocampal neurons from membrane addition experiments. Neurons were labeled with BODIPY-ceramide for 30 minutes at room temperature and then chased for 2.5-3 hours at 37°C. Growth cones were challenged with control medium, IGF-1 or BDNF, after factor deprivation, for the time indicated in minutes. Whereas fluorescent vesicle clusters persist in controls (vehicle only; growth cone of the neuron shown in Fig. 2A) and in the presence of BDNF, red fluorescence rapidly dissipates upon challenge with 20 nM IGF-1 (growth cone of the neuron shown in Fig. 2B).

View larger version (15K): [in a new window]   Fig. 5. Relative numbers of fluorescent puncta (vesicles or clusters recorded in the red channel) in growth cones for controls and IGF-1 (normalized to 1 at the onset of challenge). The values for t1/2 are >14 minutes and 6 minutes, respectively. Data points are means ± s.e.m. from ten growth cones.

View larger version (16K): [in a new window]   Fig. 6. Effects of trophic factors on membrane expansion in GCPs. The ordinate of the plot shows the percentage of the internal membrane pool added to the surface (determined by 125I-WGA binding). The incubation was for 6 minutes at 36°C. Data points are means ± s.e.m. from five to ten determinations.

View larger version (78K): [in a new window]   Fig. 7. TrkB in isolated growth cones. (A) Western blot of fetal rat brain (F18) subfractions probed with antibody to the external domain of TrkB. Equal amounts of protein were loaded in each lane. Arrowheads point at intact TrkB (140 kDa). (B) TrkB immunoprecipitated from GCPs incubated in control medium or with BDNF (0.2 nM) for 1 minute or 5 minutes. Blots were probed with anti-TrkB (external domain) antibody to show the amount of receptor in each lane (top; Cy3 label) as well as with anti-P-tyr to demonstrate phosphorylation (bottom; Cy5). Although receptor levels are equal in all lanes, there is a substantial increase in P-tyr with BDNF relative to control at 5 minutes' incubation.

View larger version (26K): [in a new window]   Fig. 8. Double immunofluorescence micrographs showing the distribution of tyrosinated -tubulin (A,C,E,G,I) and ßgc (B,D,F,H,J) in hippocampal pyramidal neurons after 24 hours (A,B,E,F) or 36 hours (C,D,G-J) in culture, in the absence (A-D) or the presence (E-J) of 5 ng ml-1 BDNF. In the absence of BDNF, neurons exhibit faint ßgc immunofluorescence in the growth cones (B and D, arrows). In the presence of BDNF, ßgc immunofluorescence is strong in the growth cones and distal third of the axons (F and H, arrows). The addition of anti-sense KIF2 (asKIF2), even in the presence of BDNF (I and J, 36 hours in culture), greatly reduces axonal growth and abolishes distal ßgc. Bar, 10 µm.

View larger version (37K): [in a new window]   Fig. 9. ßgc and KIF2 protein levels as determined by western blot of homogenates of hippocampal neurons cultured in the absence (-) or the presence (+) of 50 ng ml-1 BDNF for 36 hours. Whereas ßgc levels are unchanged, there is a striking increase in KIF2 production in the cells treated with BDNF.