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First published online 26 July 2005
doi: 10.1242/jcs.02490

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PI3K activation by IGF-1 is essential for the regulation of membrane expansion at the nerve growth cone

¹ Departamento de Química Biológica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba y CIQUIBIC, CONICET, Córdoba 5000, Argentina
² Department of Cell and Developmental Biology, University of Colorado School of Medicine and University of Colorado Cancer Center, Aurora, CO 80045, USA
³ Instituto de Investigaciones Mercedes y Martin Ferreyra (INIMEC-CONICET), Córdoba, 5016, Argentina

View larger version (61K): [in a new window]   Fig. 1. IGF-1 stimulates the phosphorylation and SHP-2 binding of IRS-2 in growth cones. GCPs were stimulated with IGF-1 or incubated in control conditions for 1 or 5 minutes. IRS-2 was immunoprecipitated, and blots of the precipitates were probed with antibodies to pTyr, SHP-2 and IRS-2 as a loading control.

View larger version (58K): [in a new window]   Fig. 2. IGF-1 increases phosphorylation of the PI3K subunit p85 in growth cones. Phosphotyrosine (PY20) immunoprecipitates were prepared from GCPs incubated in control medium or with 1 nM IGF-1 for 5 minutes. Blots were probed with anti-p85 antibody. IP, immunoprecipitate; SN, supernatant polypeptides after immunoprecipitation. A substantial increment of p85 is evident in the immunoprecipitate of GCPs stimulated with IGF-1. Relative optical densities of p85 are shown at the bottom of the figure (arbitrary units; means±s.e.m. of three independent experiments).

View larger version (41K): [in a new window]   Fig. 3. IGF-1 enhances Akt phosphorylation in growth cones. GCPs were incubated in control conditions or stimulated with IGF-1 for 30 seconds, 1 minute or 5 minutes. Western blots of polypeptides were probed with anti-p85 and with a phosphorylation-specific antibody to Akt (AktS473). p85 served as a loading control. The relative O.D. (A.U.) of AktS473 normalized over p85 signal are shown at the bottom of the figure. Those results are the means±s.e.m. of three (30 seconds and 5 minutes) or two (1 minute) independent experiments.

View larger version (52K): [in a new window]   Fig. 4. (A) The phosphorylated regulatory subunit of PI3K, pTyr-p85, is associated primarily with the growth cone cytoskeleton. Western blots of GCP subfractions (S, cytosol; M, membranes; and C, cytoskeletal fraction). GCPs were preincubated in control medium, with IGF-1 (10 nM, 5 minutes) or with BDNF (0.2 nM, 5 minutes). Blots were probed with anti p85 (middle) or anti-pTyr-p85 (top and bottom). Equal amounts of protein were loaded in each lane. Note the significant activation of PI3K with IGF-1 (top), but not with BDNF (bottom). pTyr-p85 was highly enriched in the cytoskeletal fraction and, to a lesser extent, in the membrane fraction. (B) Western blot of GCP polypeptides probed with anti-p110 antibody. Note mono-specificity of the antibody.

View larger version (86K): [in a new window]   Fig. 5. (A) Double immunofluorescence micrographs showing the distributions of tyrosinated -tubulin (TUB-IA2) and of the p110 subunit of PI3K in hippocampal pyramidal neurons after 18 hours in culture (in the presence of 50 ng ml–1 BDNF). Cells were incubated for 5 minutes in control medium (bottom) or with 10 nM IGF-1 (top and middle), permeabilized in microtubule-stabilizing conditions, and then fixed for antibody labeling. Note that stimulation with IGF-1 triggers the association of p110 with microtubules primarily in the distal axon (top) and the axonal growth cone (middle). Bar, 20 µm (top and bottom) and 5 µm (middle). (B) Double immunofluorescence micrographs showing the distribution of the p110 subunit of PI3K in hippocampal pyramidal neurons after 36 hours in culture (in the presence of 50 ng ml–1 BDNF). Cells were incubated for 5 minutes in control medium (right) or with 10 nM IGF-1 (left), permeabilized in microtubule-stabilizing conditions, and then fixed for antibody labeling. Bar, 20 µm (top) and 5 µm (bottom). (C) Double immunofluorescence micrographs showing the distribution of p110 and F-actin at the growth cone of an hippocampal pyramidal cell culture for 18 hours in the presence of 50 ng ml–1 BDNF and incubated for 5 minutes with 10 nM IGF-1. Note that most of p110 immunostaining does not co-localize with F-actin. Bar, 5 µm.

View larger version (31K): [in a new window]   Fig. 6. (A) Double immunofluorescence micrographs showing the distributions of pTyr-p85 and tyrosinated -tubulin in hippocampal pyramidal neurons after 24 hours in culture. Cells were incubated in control medium or in the presence of 10 nM IGF-1 or insulin for 5 minutes. pTyr-p85, indicating activation of PI3K, is present only in the cells stimulated with IGF-1 and is evident especially in the distal axon and growth cone. Bar, 20 µm. (B) Inmunofluorescence micrographs showing the distribution of pTyr-p85 in hippocampal pyramidal neurons after 36 hours in culture and incubated for 5 minutes with 10 nM IGF-1 (top) or in control medium (bottom). Note the intense staining of the distal third and the growth cone of the axon. Bar, 20 µm (left), 5 µm (right).

View larger version (24K): [in a new window]   Fig. 7. Stimulation and inhibition of growth cone PI3K activity by IGF-1 and by LY/Wm, respectively. GCPs, in the presence or absence of 20 µM LY or 100 nM Wm, were pre-incubated for 5 minutes at 37°C with or without IGF-1 (4 nM). PI3K was immunoprecipitated and subjected to kinase assay, using PI as a substrate. PI3P was isolated by TLC and phosphorylation assessed by storage phosphor imaging. LY reduced control levels and inhibited IGF-1-stimulation of PI3K activity. Wm reduced IGF-1-stimulated kinase activity to below control levels.

View larger version (43K): [in a new window]   Fig. 8. LY and Wm inhibit IGF-1 stimulation of membrane expansion at the growth cone. Representative fluorescence micrographs (recorded in the red channel) of the axonal growth cones of hippocampal neurons in culture are shown. 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 10 nM IGF-1 in the absence (control) or the presence of the PI3K inhibitors Wm (1 µM) or LY (20 µM). Note the rapid dissipation of fluorescence upon challenge with IGF-1 versus the persistence of fluorescent puncta in the presence of the PI3K inhibitors. Bar, 3 µm.

View larger version (17K): [in a new window]   Fig. 9. Relative number of fluorescent puncta (vesicles or clusters recorded in the red channel) in growth cones challenged with 10 nM IGF-1 in control conditions or in the presence of 1 µM Wm or 20 µM LY. Values were normalized to 1 at the onset of challenge. Data points are means + s.e.m. from more than three independent growth cone assays.

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