Figure 1.
Expression, localization and stimulus-dependent regulation of β2-syntrophin.
A) Confocal microscopy for β2-syntrophin in INS-1 cells and for GFP-β2-syntrophin or GFP in transfected INS-1 cells. β2-syntrophin was detected with the pan-syntrophin antibody 1351. White arrows indicate the enrichment of β2-syntrophin at the cell periphery. Bars: 5 µm. B) Western blots (WB) with anti-syntrophin or anti-GFP antibodies on extracts of INS-1 cells (left panel) or GFP-β2-syntrophin INS-1 cells (right panel). Cells were previously kept at rest (0 mM glucose, 5 mM KCl) or stimulated with high (25 mM) glucose and low (5 mM) KCl (HGLK), low (0 mM) glucose and high (55 mM) KCl (LGHK) or high glucose and high KCl (HGHK).
Figure 2.
Insulin content and secretion of INS-1 cells in relation to β2-syntrophin expression levels.
A) Immunoblots with the anti-syntrophin antibody on immunoprecipitates obtained with the same antibody from extracts of INS-1 cells and three INS-1 cell clones (G1, G6 and G8) stably transfected with GFP-β2-syntrophin. B) Insulin content (IC) and basal insulin secretion (BIS) of INS-1 cells, GFP-β2-syntrophin INS-1 cell clones G1, G6, G8 and INS-1 cells stably transfected with GFP. C) Insulin secretion Stimulation Index (SI) of INS-1 cells, GFP-β2-syntrophin INS-1 cell clones G1, G6, G8 and GFP INS-1 cells. D) Immunoblots with anti-syntrophin and anti-γ-tubulin antibodies on extracts of INS-1 cells transfected with a scrambled shRNA (scr shRNA) or an shRNA for the knockdown of β2-syntrophin (β2-syn shRNA). E) IC and BIS of INS-1 cells transfected with β2-syn or scr shRNAs. F) SI of INS-1 cells transfected with β2-syn or scr shRNAs. n = number of independent experiments; *, p = 0.05; **, p = 0.01; ***, p = 0.005. p-values in B and C are relative to INS-1 cells, while in E and F they are relative to INS-1 cells transfected with scr shRNA.
Figure 3.
Granule density and mobility in GFP-β2-syntrophin INS-1 cells.
A–D) Frames from TIRFM movies tracking the motion of CgB-mRFP1+ granules (red) in resting INS-1 cells expressing GFP (GFP/CgB-mRFP1 INS-1) (A, C) or GFP-β2-syntrophin (GFP-β2-syn/CgB-mRFP1 INS-1) (B, D). In C and D each blue line shows the motion of an individual CgB-mRFP1+ granule tracked for 40 sec. Scale bars: 4 µm. E, F) Mean speed velocity (E) and square displacement (F) of CgB-mRFP1+ granules in resting INS-1 cells expressing GFP (black) or GFP-β2-syntrophin (red). In F the green and blue lines show the weighted least square fit of the initial (first 4 sec) mean square displacement points. The mean square displacement slope of linear dependency is K = 4D, where D is the diffusion coefficient in µm2 sec−1. G) Density of granules expressed as the number of granules/100 µm2 in resting (black bars) and stimulated (red bars) GFP/and GFP-β2-syntrophin/CgB-mRFP1 INS-1 cells. H, I) Mean speed velocities (H) and diffusion coefficients (I) of CgB-mRFP1+ granules in resting (black bars) or HGHK-stimulated (red bars) INS-1 cells expressing GFP or GFP-β2-syntrophin. m = number of movies examined for each condition from four independent experiments.
Figure 4.
Phosphorylation/dephosporylation of β2-syntrophin.
A) Immunoblots with the anti-syntrophin or anti-GFP antibodies following immunoprecipitations with the same antibodies from extracts of INS-1 and GFP-β2-syntrophin INS-1 cells, respectively. Alkaline phosphatase (AP) was added to the immunoprecipitates, while okadaic acid (OA) was added before cell extraction. β2-syntrophin species the levels of which are reduced upon AP treatment are marked with a gray arrow, while black arrows indicated species increased upon OA incubation. B) The two panels on the left show the autoradiographies of 32P-GFP-β2-syntrophin immunoprecipitated with the rabbit anti-GFP antibody from extracts of GFP-β2-syntrophin INS-1 cells labeled with 32P kept in culture media. A control immunoprecipitation from the same cells was performed using rabbit control IgG. The right panel shows the immunoblot with the anti-GFP antibody on the same immunoprecipitated material visualized by autoradiography. C) Domain structure of β2-syntrophin, including the phosphoserines identified by mass spectrometry. PH = Pleckstrin Homology domain, PDZ = PSD95/Dlg/ZO-1 domain, SU = Syntrophin Unique domain.
Figure 5.
Characterization of β2-syntrophin phosphomutants.
A) Confocal microscopy images of GFP-β2-syntrophin and mutants with single or dual replacements of serine (S) 75, 90 and 213 with either aspartate (D) or alanine (A). Bars: 5 µm. B) Expression pattern of GFP-β2-syntrophin variants in INS-1 cells as detected by western blotting with the anti-GFP antibody.
Figure 6.
Binding of β2-syntrophin phosphomutants to ICA512 and impact on granule density, mobility and insulin secretion.
A) Autoradiography of in vitro transcribed-translated 35S-labeled His-β2-syntrophin (His-β2-syn) S/D and S/A mutants following pull down assay with GST-ICA512601–979 or GST. B) Insulin content (IC), basal insulin secretion (BIS) and insulin secretion Stimulation Index (SI) of INS-1 cells transfected with the S75 and S90 GFP-β2-syntrophin mutants. Wild-type and GFP INS-1 cells were used as controls. n = number of independent experiments; *, p = 0.05; **, p = 0.01; ***, p = 0.005; p-values of IC, BIS, and SI were calculated relative to those in INS-1 cells. C–E) Density (C), mean speed velocity (D) and diffusion coefficient (E) of CgB-mRFP1+ granules in resting INS-1 cells expressing GFP, GFP-β2-syntrophin or the GFP-β2-syntrophin S75D and S90D phosphomutants. m = number of movies examined for each condition from six independent experiments.
Figure 7.
Phosphorylation of endogenous and GFP-β2-syntrophin and the S/D mutants by Cdk5.
A) Immunoblots with anti-syntrophin or anti-GFP antibodies on rat islet extracts (right panel) or immunoprecipitates obtained with the same antibodies from extracts of INS-1 cells (left panel) or GFP-β2-syntrophin INS-1 cells (middle panel) treated with the Cdk5 inhibitor roscovitine or the Erk1/2 inhibitor PD98059. Asterisks indicate the β2-syntrophin species sensitive to roscovitine. B) Immunoblots with the anti-GFP antibody on immunoprecipitates obtained with the same antibody from extracts of INS-1 cells expressing GFP-β2-syntrophin variants and treated with roscovitine. Gray and black arrows point to β2-syntrophin species the levels of which decreased or increased in response to roscovitine, respectively. C) Autoradiographies and immunoblots of β2-syntrophin (left panels) and GFP-β2-syntrophin (right panels) immunoprecipitated with anti-syntrophin or anti-GFP antibodies from INS-1 and GFP-β2-syntrophin INS-1 cells, respectively. Immunoprecipitates were incubated with or without the Cdk5/p25 complex in the presence of 32P-γ-ATP. D) Autoradiography of GFP-β2-syntrophin variants immunoprecipitated with the anti-GFP antibody from GFP-β2-syntrophin INS-1 cells and incubated with the Cdk5/p25 complex as in C. An asterisk indicates the β2-syntrophin species that is lacking in the GFP-β2-syntrophin S75D mutant.
Figure 8.
Expression of β2-syntrophin species upon Cdk5 depletion.
A) Immunoblots with anti-syntrophin, anti-Cdk5 and anti-γ-tubulin antibodies on extracts of INS-1 (left panels), GFP-β2-syntrophin INS-1 (middle panels) and GFP-β2-syntrophin S75D INS-1 (right panels) cells following the knockdown of Cdk5 (Cdk5 shRNA). As control, INS-1 cells were transfected with a scrambled shRNA. Gray and black arrows point to the β2-syntrophin species the levels of which decreased or increased upon Cdk5-depletion, respectively. The β2-syntrophin species were numbered from top to bottom B) Quantification of the immunoblots shown in A. p-values are relative to cells transfected with scr shRNA. n = number of experiments. *, p = 0.05; **, p = 0.01; ***, p = 0.005.