JCS03159 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. GSK-3 inhibition is most effective at the stage 2/ 3 transition. To identify at which developmental stage GSK-3 inhibition is most effective, we treated hippocampal neurons for different time periods with SB216763 and fixed cells 24, 48 or 96 hours later. Inhibition of GSK-3 during the early stages of neurite budding and initial neurite outgrowth (2-4 hour, followed by wash out) had no significant effect on polarity establishment observed at 48 hours. Inhibitor treatment during 4-24 hours, followed by wash out, produced an increase in the number of neurons with multiple, axon-like processes, but the most effective time period was between 24 and 48 hours. Also GSK-3 inhibition at the stage at which most neurons were already polarized was less effective and neurons with multiple axons mostly developed from stage 2 neurons still present at 48 hours (compare controls 48 hour and 96 hour with the treatment from 48-96 hours). The percentage of neurons bearing one single axon, multiple axons or no axons was calculated for each condition. For each treatment 200-300 neurons were counted from different hippocampal preparations (means ± s.e.m., **P<0.001; *P<0.01).

• Supplemental Figure 2 -

  Fig. S2. Specificity of anti-GSK-3β serine9 antibody. Antibody specificity was tested using neurons isolated from a genetically altered knockin mouse, where the Ser9 phosphorylation site of both alleles of GSK-3β had been altered to alanine (GSK-3α/β ^{21A/21A/9A/9A}). As shown in the lower panel, no signal could be detected in these neurons, while, as shown in the upper panels, in wild type neurons a signal similar to the one obtained using a total GSK-3β antibody was seen.

• Supplemental Figure 3 -

  Fig. S3. Sub-cellular localization of GSK-3. (A) Sub-cellular fractionation using a continuous sucrose gradient. Cortical neurons were scraped into cold MES buffer (25 mM MES, 5 mM dithiothreitol, 2 mM EDTA, protease inhibitors; pH 7.0) and left for 15 minutes on ice to allow osmotic swelling to occur. Extracts were obtained by homogenization and centrifuged for 5 minutes at 1000 g and 4°C. The supernatant was carefully layered on top of a continuous 55%-10% sucrose gradient in MES buffer in a SW40 ultracentrifuge tube (Beckman Instruments). After centrifugation for 12 hours at 200,000 g, 25 fractions were collected from the top to the bottom of the tube and proteins analysed by SDS-PAGE and immunoblotting using a GM-130 antibody as a marker for Golgi fractions. (B) Neurons were solubilized for 5 minutes on ice in 0.1% Triton X 100. The soluble fraction was saved and the remainder was scraped from the plates and loaded on a 12% SDS-gel as insoluble fraction. Rho-GDI was used as a soluble, cytoplasmic marker protein. (C) Quantification of neurite lengths after neurons were treated for 48 hours with SB216763 (n=30) in comparison with control neurons (n=30; means±s.e.m.). (D) The distribution of new membrane carriers exiting from the TGN, using BODIPY ceramide (Molecular probes), a lipophilic dye that integrates specifically into the Golgi. Control and SB216763 treated hippocampal neurons were loaded with BODIPY ceramide as described (Bradke and Dotti, 1997) and images taken after a 10 minute incubation. Fluorescence along the neurites was measured, normalized and sorted according to intensity. Labelled vesicles normally concentrated in the axon of control cells, were after treatment with a GSK-3 inhibitor homogeneously distributed in all axon-like processes.

• Supplemental Figure 4 - Fig. S4 and Movies. Traffic of membrane carriers along neurites becomes symmetrically distributed after GSK-3 inhibition. The traffic of membrane carriers in individual living neurons after 48 hours in culture was imaged by phase contrast microscopy. The upper left panel shows a stage 3 control neuron. The indicated proximal segments from a minor neurite and the axon are shown at a higher magnification in the galleries besides during 4 different consecutive time points. The different symbols indicate individually tracked carriers which are either moving anterograde (arrowhead), retrograde (arrow), or remain static (star). A similar experiment is shown in the panel below but here GSK-3 was inhibited for 48 hours before imaging. The quantification of the amount and speed of membrane traffic are shown in Fig. 4E and F respectively. In the supplementary movies the same insets as indicated here are shown. One image is taken every 1.3 seconds for a total observation period of 120 seconds. The names of the movies are according to the names used in this figure.

• Movie 1
• Axon Movie
• Minor Movie
• Neurite2 Movie