Journal: bioRxiv

Article Title: Novel signaling hub of insulin receptor, dystrophin glycoprotein complex and plakoglobin regulates muscle size

doi: 10.1101/517789

Figure Lengend Snippet: Affinity-based purification of plakoglobin-containing multiprotein assembly. A plasmid encoding 6His-tagged plakoglobin was electroporated into mouse TA mus cle, and 6His-plakoglobin-bound proteins were isolated with Nickel beads and identified by mass spectrometry. His-plakoglobin-bound proteins were eluted from Nickel column in (a) with a Histidine gradient (50-250 mM) and analyzed by immunoblotting. Elution fraction #2 was subjected to mass spectrometry (one experiment was performed and data compared to the experiment presented in ). Analysis of high MW protein peak fractions eluted from size exclusion chromatography (as in ) by SDS-PAGE and immunoblotting. n= two independent experiments. Plakoglobin was immunoprecipitated from the high MW protein peak, and bound proteins were detected by immunoblotting. Plakoglobin, DGC components (including glycosylated-β-dystroglycan), the insulin receptor, caveolin-1, desmin, and MuSK sediment to the same glycerol gradient fractions (marked bya red rectangle). Membrane-cytoskeleton fraction from mouse muscle was separated on 10-40% glycerol gradient, and alternate protein eluates were analyzed by Western blotting. n= two independent experiments. In normal muscle, plakoglobin, DGC components, the insulin receptor, and desmin interact. Left: proteins co-purified with anti-plakoglobin from glycerol gradient fraction #16 illustrated in (e) , and detected by immunoblotting. MuSK did not bind plakoglobin although it sedimented to the same fractions in (e). Right: a reciprocal immunoprecipitation with a-1-syntrophin antibody from membrane fractions isolated from normal TA muscle. Two experiments were performed: one with plakoglobin antibody and one with a-1-syntrophin antibody. Plakoglobin, β-dystroglycan, and the insulin receptor colocalize at costameres on skeletal muscle membrane. Confocal and STED images of TA muscle cross sections stained with the indicated antibodies. Scale bars, 5 µm (Confocal) and 2 µm (STED). n= three independent experiments indicating colocalization of these proteins. STED analysis is an annotated image in which all three proteins are detected using the spots module of lmaris software (white, red and green spheres). The double and triple co-occurrence spots are presented in yellow and blue, correspondingly. Proximity ligation assay (PLA) was performed on TA cross sections with β-dystroglycan and insulin receptor antibodies or β-dystroglycan antibody alone. Red fluorescent dots indicate areas of β-dystroglycan-insulin receptor association.

Article Snippet: Moreover, a built-in Imaris plugin spot colocalization algorithm (requiring ImarisXT module) was used to identify triple spots centers with maximum distance of 250nm.

Techniques: Affinity Purification, Plasmid Preparation, Isolation, Mass Spectrometry, Nickel Column, Western Blot, Size-exclusion Chromatography, SDS Page, Immunoprecipitation, Membrane, Purification, Staining, Software, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Novel signaling hub of insulin receptor, dystrophin glycoprotein complex and plakoglobin regulates muscle size

doi: 10.1101/517789

Figure Lengend Snippet: Plakoglobin knockdown with plakoglobin shRNA (shJUP) promotes DGC-insulin receptor dissociation. TA mouse museles were transfected with shLacz or shJUP (contralateral limbs}, and purified membranes were separated on glycerol gradients and analyzed by SDS-PAGE and immunoblot. Red rectangle marks fractions that in shLacz expressing muscle contain plakoglobin complex with glycosylated-β-dystroglycan. n= three independent experiments. Mice were injected with saline or glucose (1mg/gr body weight) and membrane fractions were isolated from transfected TA museles (contralateral limbs) and analyzed by glycerol gradient, and immunoblotting. Red rectangle marks fractions that in shLacz expressing musele contain plakoglobin complex with glycosylated-β-dystroglycan. n= three independent experiments. Syntrophin was immunoprecipitated from purified membrane fractions of museles expressing shLacz or shJUP from mice injected with saline or glucose (1mg/gr body weight). Precipitates were analyzed by immunoblotting. Right: densitometric measurements of presented blots. Graph depicts ratio of each protein to syntrophin. n= two independent experiments. Soluble fractions of TA museles expressing shLacz or shJUP from mice injected with saline or glucose were analyzed by SDS-PAGE and immunobloting. n= two independent experiments (representative blots are shown). Downregulation of plakoglobin induces musele atrophy. Mean weights of TA museles expressing shJUP are plotted as the percentage of shLacz control museles. Data are represented as mean ± SEM. n = 14 mice; * P< 0.00005 by one-tailed t-test. n= two independent experiments. Cross-sectional areas of 521 fibers transfected with shJUP (also express GFP, green bars) vs. 521 non-transfected fibers (black bars) in the same musele. n= 4 mice. Median is presented. Representative confocal images of cross sections of museles expressing shLacz or shJUP (also express GFP}, stained with anti-insulin receptor and anti-β-dystroglycan. Scale bar, 20 µm. n= two independent experiments from two different mice. Insulin receptor and β-dystroglycan colocalization is markedly reduced in muscle fibers expressing shJUP. Confocal and STED images of cross sections ofTA museles expressing shLacz or shJUP stained with the indicated antibodies. Scale bars, 5 µm (Confocal) and 2 µm (STED). n= two independent experiments. STED analysis is an annotated image in which the two proteins are detected using the spots module of lmaris software (white and red spheres). The double co-occurrence spots are presented in blue. Insulin receptor and β-dystroglycan association is markedly reduced in musele fibers expressing shJUP. Proximity ligation assay (PLA) was performed on cross sections of TA museles expressing shLacz or shJUP stained with β-dystroglycan and insulin receptor antibodies. Red fluorescent dots indicate areas of β-dystroglycan-insulin receptor association.

Article Snippet: Moreover, a built-in Imaris plugin spot colocalization algorithm (requiring ImarisXT module) was used to identify triple spots centers with maximum distance of 250nm.

Techniques: Knockdown, shRNA, Transfection, Purification, SDS Page, Western Blot, Expressing, Injection, Saline, Membrane, Isolation, Immunoprecipitation, Control, One-tailed Test, Staining, Software, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Novel signaling hub of insulin receptor, dystrophin glycoprotein complex and plakoglobin regulates muscle size

doi: 10.1101/517789

Figure Lengend Snippet: Mean body and TA weights of MKR mice at 8 weeks of age are presented as percentage of WT littermates. Body weights, n = 5 mice; TA weights, n = 12 mice; * P < 0.05, by one-tailed t-test. Data are represented as mean ± SEM. n= two independent experiments. Cross-sections of TA museles from MKR mice are smaller than WT. Left: laminin staining in white. Scale bar, 50 µm. Right: Cross-sectional areas of 834 fibers from WT (black bars) and MKR (grey bars) mice are presented. n= 3 mice. Median is presented. Insulin signaling in MKR mice museles is abolished. Left: soluble fractions of museles from WT and diabetic MKR mice injected with saline or glucose (1mg/gr body weight) were analyzed by SDS-PAGE and immunoblot. n= two independent experiments. Each lane represents one musele from one mouse. Right: densitometric measurement of presented blots is depicted by graph and densitometry values. In MKR mice musele, DGC-insulin receptor association is reduced. Insulin receptor immunoprecipitation from membrane fractions of WT and MKR mice museles injected with saline or glucose (1mg/gr body weight). n= two independent experiments. High MW protein peak fractions isolated by size-exelusion chromatography from WT and MKR mice museles were analyzed by SDS-PAGE and silver staining in three independent experiments (a representative is shown). Fractions in (e) were analyzed by immunoblotting. Membrane-cytoskeleton fractions from WT and MKR mice TA museles were analyzed by glycerol gradient fractionation. Red rectangle marks fractions that in WT mouse musele contain plakoglobin complex with glycosylated-β-dystroglycan. n= two independent experiments. Isolated membranes from WT and MKR mice museles were analyzed by glycerol gradient fractionation. n= two independent experiments. Red rectangle marks fractions that in WT mouse musele contain plakoglobin complex with glycosylated-β-dystroglycan. Top: insoluble fractions of TA museles from WT and MKR mice were analyzed by immunoblotting in two independent experiments (a representative is shown). Each lane represents one musele from one mouse. Bottom: densitometric measurement of presented blots. Top: Cross sections of WT and MKR mice TA museles were stained with plakoglobin, β-dystroglycan and insulin receptor antibodies. Scale bar, 20 µm. n= three independent experiments (representative confocal images are shown). Bottom: fluorescent intensity was quantified using Imaris software, and data from three independent experiments in depicted in a graph. Plakoglobin, insulin receptor, and β-dystroglycan colocalization is markedly reduced in museles from MKR mice. Confocal and STED images of cross sections of TA museles from WT and MKR mice stained with the indicated antibodies. Scale bars, 5 µm (Confocal) and 2 µm (STED). n= two independent experiments. STED analysis is an annotated image in which all three proteins are detected using the spots module of Imaris software (white, red and green spheres). The double and triple co-occurrence spots are presented in yellow and blue, correspondingly. Insulin receptor and β-dystroglycan association is markedly reduced in MKR mice museles. Proximity ligation assay (PLA) was performed on cross sections of TA museles from WT or MKR mice stained with β-dystroglycan and insulin receptor antibodies. Red fluorescent dots (left) and blue dot analysis (right) indicate areas of β-dystroglycan-insulin receptor association.

Article Snippet: Moreover, a built-in Imaris plugin spot colocalization algorithm (requiring ImarisXT module) was used to identify triple spots centers with maximum distance of 250nm.

Techniques: One-tailed Test, Staining, Injection, Saline, SDS Page, Western Blot, Immunoprecipitation, Membrane, Isolation, Chromatography, Silver Staining, Fractionation, Software, Proximity Ligation Assay