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First published online December 21, 2005
doi: 10.1242/10.1242/jcs.02710

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Functional analysis of SIRP in the growth cone

Department of Cell and Developmental Biology, University of Colorado School of Medicine and University of Colorado Cancer Center, Aurora, CO 80010, USA

View larger version (128K): [in a new window]   Fig. 1. (A-F) Immunolocalization of SIRP in growth cones of cortical neurons cultured either on laminin (A-D) or poly-D-lysine (E,F). Bar, 10 µm. (G-I) Double-immunofluorescence of SIRP and CD81 in a growth cone on laminin. Bar, 5 µm. Growth cones were double-labeled with anti-SIRP antibody (Alexa Fluor® 594-conjugated secondary antibody; B,D,E,F,G,I) and FITC-phalloidin (C,D,F) or anti-CD81 (Alexa Fluor® 488-conjugated secondary antibody; H,I). D is the merged image of B and C (overlap appears yellow), and A is the corresponding phase-contrast image. F is the merged image of E with that of phalloidin labeling (not shown separately). I is the merged image of G and H and shows extensive co-localization (yellow) of SIRP and CD81. Images are 0.1 µm optical sections obtained by digital deconvolution. The arrows in D point to SIRP immunoreactivity in filopodial tips. The arrows in I indicate filopodial tips with prominent SIRP and CD81 overlap.

View larger version (47K): [in a new window]   Fig. 2. Distribution of SIRP in lipid microdomains. Growth cone particles (GCPs) were treated with different detergents (indicated on the left of each panel) and fractionated in sucrose density gradients (A-E); the sucrose concentrations of the fractions are given at the top of each lane. Fractions were collected from top to bottom of the gradients. Fractions were analyzed by western blot with the indicated antibodies (IB, right; =anti). (C) The experiment without detergent serves as a control for non-solubilizing conditions. Anti-transferrin receptor (TfR) blot is a control for Brij 98, showing that transmembrane proteins such as TfR are solubilized under these conditions. (D) Saponin was added before the Brij 98 extraction of GCPs to disrupt cholesterol interactions. The controls without saponin treatment produced the same results as in C and, therefore, are not shown again. (E) MßCD was used to extract cholesterol. In these experiments, samples of different density (range indicated on the top) were pooled to represent high-, middle- and low-density fractions. For detailed description, see Results.

View larger version (39K): [in a new window]   Fig. 3. The addition of laminin to GCPs in suspension activates Src kinase. (A) Western blot of laminin-treated or control GCPs, probed with the indicated antibodies (IB; =anti). p85 serves as a loading control. Numbers below the blot are the average ratios for phospho-Src over p85 (arbitrary units normalized to control ± s.e.m.; n=2). (B) Cytoskeleton fractions prepared from GCPs with or without prior laminin treatment (each lane was loaded with the total cytoskeletal preparation from equal GCP samples). Samples were subjected to western blot with the indicated antibodies (IB). The numbers below the blot represent quantitation of Src (arbitrary units normalized to control ± s.e.m.; n=2). Note that SIRP was not detected in the cytoskeleton fraction, but that SHP-2 was present.

View larger version (43K): [in a new window]   Fig. 4. Laminin, IGF-1 and BDNF stimulate transient phosphorylation of SIRP and its association with SHP-2 in GCPs. (A) GCPs with or without stimulation were immunoprecipitated with anti-SIRP antibody after 1 minute of incubation. Immunoprecipitates were analyzed by western blot with anti-pTyr and anti-SHP-2 to examine SIRP phosphorylation and SHP-2 co-immunoprecipitation, respectively (=anti). The bar graphs show quantitation of SIRP phosphorylation (left) and of SHP-2 co-immunoprecipitation (right), expressed as the percentage change over control level. Results are means ± s.e.m. of at least four independent experiments. *P0.01 compared to control. (B) The same experiment as in A, but 5 minutes incubation time. The bar graphs show decreases in phosphorylation and SHP-2 co-immunoprecipitation relative to control. (C,D) Effects of Src family kinase inhibitor on laminin-, IGF-1- and BDNF-stimulated tyrosyl phosphorylation (C) and SHP-2 binding of SIRP (D). The Src kinase inhibitor, PP2, was added to GCPs prior to incubation in control or stimulated conditions. PP3 added in control conditions served as a negative control for PP2. After 1 minute incubation, GCPs were solubilized and SIRP immunoprecipitated. Western blots of SIRP immunoprecipitates were probed with anti-pTyr to examine SIRP phosphorylation (C) or with anti-SHP-2 to reveal SHP-2 co-immunoprecipitation (D). In addition, they were probed with anti-SIRP to control for SIRP loading. Note that PP2, even with stimulation, reduced SIRP phosphorylation and co-immunoprecipitation of SHP-2 to below control levels without PP2 treatment (C and D) or with PP3 treatment (C). The numbers below the blots are mean ratios ± s.e.m. (n=2 in C; n=3 in D) of pTyr or SHP-2 label over SIRP label (arbitrary units, normalized to control without PP2 treatment).

View larger version (16K): [in a new window]   Fig. 5. Phosphorylation shifts SIRP from lipid microdomains (LMDs). The bar charts show the percentage of SIRP (upper) and, for comparison, of Src (lower) recovered in LMDs under different experimental conditions. Data were normalized to the value in PP2-treated GCPs. The values are expressed as means ± s.e.m. for three independent experiments.

View larger version (67K): [in a new window]   Fig. 6. cSIRP expressed in neurons is phosphorylated and binds SHP-2. Cortical neurons transfected with GFP-cSIRP were cultured for 48 hours. Cells and neurites were harvested, permeabilized with ß-escin and incubated for 10 minutes at 37°C in the presence or absence of ATP and vanadate. GFP-cSIRP was immunoprecipitated from a TX100 extract of the samples and analyzed by western blot (IB; =anti). Probing with anti-pTyr antibody (Cy5-conjugated secondary antibody) revealed greatly increased phosphorylation of a single band in the ATP plus vanadate-treated samples (top panel). The blot was stripped, checked for absence of remaining Cy5 signal, and re-probed with anti-GFP (middle panel). This confirmed (i) presence of approximately equal amounts of GFP-cSIRP in both samples, and (ii) the identity of the pTyr-positive band. The same blot was also probed with anti-SHP-2 (Cy3-conjugated secondary antibody), revealing SHP-2 co-precipitation in ATP plus vanadate samples only. Representative image of two independent experiments are shown.

View larger version (112K): [in a new window]   Fig. 7. (A-C) GFP-positive neurons express cSIRP in co-transfected cultures. (A) GFP fluorescence of neurons; (B) the same neurons labeled with anti-cSIRP, an anti-SIRP raised against C-terminal peptide (Alexa Fluor® 594-conjugated secondary antibody); (C) merger of the two images. Anti-cSIRP labels non-transfected neurons (abundant in the field) only very weakly so that they are not visible. Bar, 20 µm. (D-I) Overexpression of mutant SIRP in the growth cone. (D-F) Representative phase-contrast images of growth cones transfected with: GFP only (D), cSIRP plus GFP (E), or cSIRP-FYFF plus GFP (F). (G) Distribution of endogenous SIRP (labeled with an antibody to the external domain) in a growth cone overexpressing cSIRP. (H) Immunolocalization of cSIRP overexpressed in the growth cone (labeled with anti-cSIRP). (I) The phase-contrast image of the growth cone in H. A process of a non-transfected neuron, visible only in phase-contrast, runs across the image. It is not detectable by immunofluorescence. Secondary antibodies were Alexa Fluor® 594 conjugated. Bar, 10 µm.

View larger version (40K): [in a new window]   Fig. 8. Overexpression of cSIRP affects axonal growth in cortical neurons. (A) The axons of two neurons cultured on laminin were visualized by GFP fluorescence (top) and phase-contrast (middle and bottom). (Left) Neuron transfected with GFP only; (right) neuron transfected with cSIRP plus GFP. The GFP and t=0 phase-contrast images were recorded immediately after the addition of IGF-1. Phase-contrast images were also recorded 1 hour after the addition of IGF-1 (t=1h). The arrows in the images indicate the distance that the growth cons had advanced during 1 hour of observation. Note that the growth cone shown in the left panels advanced much faster than the ones in the right panels. Bar, 10 µm. (B) Quantitation of axon growth rates for neurons cultured on laminin or poly-D-lysine. Data are shown for neurons transfected with GFP only (control), with cSIRP plus GFP, or with mutant cSIRP-FYFF plus GFP. Results are means ± s.e.m. for the number of independent experiments indicated above each bar. Error bars indicate s.e.m. *P<0.05 compared to the growth rate measured for control neurons grown on laminin (the left-most bar).

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