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First published online 6 March 2007
doi: 10.1242/jcs.03413

Journal of Cell Science 120, 1168-1177 (2007)
Published by The Company of Biologists 2007
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Expression of a synapsin IIb site 1 phosphorylation mutant in 3T3-L1 adipocytes inhibits basal intracellular retention of Glut4

Joseph M. Muretta, Irina Romenskaia, Patrick A. Cassiday and Cynthia Corley Mastick*

Department of Biochemistry and Molecular Biology, Mailstop 330, University of Nevada, Reno, NV 89557, USA


Figure 1
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  		Fig. 1. Synapsin IIb mRNA and protein are expressed in adipocytes. (A) Northern blots of RNA samples (10 µg) from mouse brain (Br) or 3T3-L1 adipocytes (Ad) probed with a 185 bp pan-synapsin C domain probe, a 649 bp synapsin II C domain probe or a probe from the synapsin IIb 3'-untranslated region (UTR). (B,C) Anti-synapsin western blotting with two affinity-purified antibodies raised against the C domain of synapsin IIb (antibodies 69 and 83) from samples (10 µg) of (B) mouse brain post-nuclear supernatant or (C) 3T3-L1 adipocyte (L1), primary adipocyte (1°), or liver low density microsomes. Solid boxes indicate that the samples were run on the same gel; dashed lines indicate that the order of samples on the gel was altered in the image used in the figure.

 

Figure 2
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  		Fig. 2. Distribution of synapsin IIb protein in subcellular fractions. (A) Fractions from basal and insulin stimulated primary adipocytes; M/N, mitochondria/nuclei; PM, plasma membrane, HDM, high density microsomes; LDM, low density microsomes; cyt, cytosol. (B) Soluble (S) and particulate (P) fractions from basal L1 adipocytes (Ad) prepared by differential centrifugation (1 K=1000 g). (C) Subcellular fractions from L1 fibroblasts (Fib). Equal protein from each fraction was analyzed (10 µg for synapsin and 1 µg for Glut4) and proteins were detected using antibodies specific for Glut4 or synapsin IIb (Syn; two antibodies used, 83 and 69). In the synapsin blots using antibody 69, an additional non-specific band was detected that was also observed when the blots were probed with secondary antibody alone (2°).

 

Figure 3
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  		Fig. 3. Synapsin IIb and Glut4 are co-localized. L1 cells were differentiated on coverslips, then infected with virus expressing HA-Glut4-GFP alone (G4), or coexpressed with WT-Flag-synapsin IIb (G4/S), S10A Flag-synapsin IIb (G4/S10A), or S10D Flag-synapsin IIb (G4/S10D). Two days post-infection they were fixed, permeabilized, and labeled with anti-Flag antibody. Representative confocal images of Glut4 (GFP: green) and Flag-synapsin (Cy3: red) are shown. Images are maximum projections of 10-0.5 µm slices through the center of the cell (montages of the individual slices are shown in supplementary material Fig. S3).

 

Figure 4
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  		Fig. 4. S10A synapsin redistributes Glut4 in adipocytes. L1 adipocytes prepared as described in Fig. 3 were treated with or without insulin (100 nM, 30 minutes), then fixed and analyzed. zstack confocal images of GFP fluorescence were collected from ~60 infected adipocytes of each type and maximum projections were analyzed. (A) Representative images. (B) Percentage of cells in the maximum projections with Glut4 at or near the plasma membrane (rim + diffuse fluorescence) in basal (white) or insulin-stimulated (black) cells. (C) Average object sizes (contiguous area with green intensity greater than the threshold in maximum projections; mean ± s.e.m., standardized to Glut4 basal). (D) Histograms of intensity distributions (shown on z-axis) of GFP fluorescence in the representative images shown in A (G4, G4/S, G4/S10A, left to right).

 

Figure 5
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  		Fig. 5. S10A synapsin inhibits basal intracellular retention of Glut4 in adipocytes. L1 adipocytes differentiated on culture dishes were infected as described in Fig. 3. Cells were treated ± insulin (100 nM, 30 minutes), placed on ice, then labeled with anti-HA (detected with PC7). After washing, cells were lifted from the plate with collagenase at 4°C, and analyzed by flow cytometry. MFR, mean fluorescence ratio (PC7/GFP). ~5000 cells/sample. Mean values from uninfected cells were used to correct for autofluorescence and non-specific binding. MFRs were standardized to G4 basal samples. (A) Average MFR ± s.d. from three experiments. (B) Average foldstimulation (MFR Insulin/MFR basal) ± s.d. from three experiments. (C) Comparison of the effects of S10A synapsin and S10A dsRED, a fusion protein with the first 118-amino acids of S10A synapsin fused to dsRED. White, basal; black, insulin; grey, ratio. Statistical analysis (Bonferroni, Scheffe's and Tukey tests; Origin 7.5) was done after standardizing to either the basal or insulin stimulated controls (***P<0.01 significant difference; ns, no significant difference, P>0.05).

 

Figure 6
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  		Fig. 6. S10A synapsin does not inhibit general endocytic trafficking. (A) Surface transferrin receptor content in basal (white) or insulinstimulated (black) cells. L1 adipocytes differentiated, infected and treated with insulin as described in Fig. 6 were labeled with PE-antitransferrin receptor antibody at 4°C and mean fluorescence intensity (MFI) determined by flow cytometry. Average MFI ± s.d. of samples from four experiments, standardized to G4 basal. (B) Infected fibroblasts were labeled for 5 minutes (surface Glut4, white) or 120 minutes (total cycling pool, grey) with anti-HA antibody at 37°C, fixed, permeabilized and anti-HA detected with Cy3-labeled secondary antibody. Samples were analyzed by wide-field microscopy. MFRs were calculated for 18 fields of cells (six fields per experiment, three experiments, 20-50 cells/field). Average MFRs ± s.d., expressed as % total cycling HA-Glut4.

 

Figure 7
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  		Fig. 7. Synapsin is phosphorylated at site 1 in basal adipocytes. Western blotting with site-1-specific phospho-synapsin (pSyn) and pansynapsin (Syn) antibodies. (A) Samples from mouse brain (Br) or 3T3-L1 adipocytes expressing WT-Flag-synapsin IIb (L1). (B) Samples from basal or insulin-stimulated (100 nM, 30 minutes) 3T3-L1 adipocytes expressing WT or S10A Flag-synapsin IIb. (C) Anti-Flag immunoprecipitates of WTFlag-synapsin incubated ± {lambda}-phosphatase ({lambda}-PPase) ± phosphatase inhibitors.

 




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