Proteomic resolution of IGFN1 complexes reveals a functional interaction with the actin nucleating protein COBL

Figures

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  Fig. 1. Differentiating IGFN1 knockout myoblasts have a higher G:F actin ratio. A) Representative Western blot (n = 8) against actin using 7 day differentiated cells. Protein extracts from C2C12, IGFN1 knockout (KO19) and KO19 stably transfected with IGFN1_v1 (KO19+IGFN1_v1) cell lines were processed to obtain globular (G) or filamentous (F) actin fractions and loaded as indicated. B) G:F actin ratio of C2C12, KO19 and KO19+IGFN1_v1, as labelled. One-way ANOVA showed a significant effect of cell type at the p < 0.05 level [F (2, 22) = 4.665, p = 0.02]. Post-hoc comparisons using the Tukey HSD test revealed a significant difference between the mean G:F ratio of C2C12 and KO19 cells (p < 0.05). C) Representative Western blot (n = 5) of total actin from C2C12, KO19 and KO19+IGFN1_v1. Quantifications of n = 5 blots shown on the histogram. Note that all cell lines show similar levels of total actin.

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  Fig. 2. Proteomics results. A) SMART (http://smart.embl-heidelberg.de) predicted domain composition of mouse IGFN1_v1 (UniProtKBreference protein sequence Q3KNY0-04) with the globular domains spanning the two fragments used as baits for pull-downs (IGFN1(d1-d3) and IGFN1(d6-d11)) indicated by the black lines. B) Western blots from immobilized his-tagged recombinant proteins detected with anti-his antibodies. C) STRING-Db output of the putative IGFN1 interactome obtained from all unique hits present in supp Table 1. Lines between nodes represent interactions found in the STRING database, the number of interactions is higher than would be expected from a random set of proteins (PPI enrichment p-value = 0.00983). Functional enrichments in the cytoskeleton and the proteasome are highlighted in blue and red, respectively (FDR = 0.033 and 0.0038 respectively).

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  Fig. 3. COBL is a Z-disc associated protein in mouse skeletal muscle. A) Above, intron/exon structure of the mouse Cobl gene with exons numbered according to the predicted ENSEMBL Cobl-201 (ENSMUST00000046755.13). ENSEMBL Cobl-201 matches the sequence from the mouse cDNA amplified from a skeletal muscle library using the indicated primers (arrow tips). No shorter alternative COBL cDNAs were obtained. The mouse skeletal muscle cDNA predicts a protein of 1337 AA identical to UniProtKB Q5NBX1. Below, the mouse COBL protein with domains annotated as follows: Cobl, ubiquitin-like fold domain (IPR019025); Pro-rich, indicates a proline rich region (compositional bias); WH2, Wiskott-Aldrich homology 2 domain (IPR003124). The antigen used for anti-Cobl antibodies is indicated by the red bar. B) Western blot against COBL using a Gastrocnemius muscle and a 7-day differentiated C2C12 cell extracts confirms expression of COBL in mouse skeletal muscle tissue and in the C2C12 myoblast cell line. The asterisk indicates cross reactivity. C) Longitudinal cross-sections of mouse gastrocnemius muscle. Sections are from mice electroporated with a KY-TdTomato vector to identify the Z-disc (red) and incubated…

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  Fig. 4. IGFN1_v1 interacts with COBL and shifts from nuclear to cytoplasmic localization in the presence of COBL in COS7 cells. A) IGFN1_v1 and COBL coimmunoprecipitate in a pull-down interaction assay. Representative blots (n = 3) are shown from pulldowns of cell extracts from COS7 cells co-transfected with either pDEST47-IGFN1_v1-V5 and pDEST47-COBL-tdTomato or with pDEST47-V5 and pDEST47-COBL-tdTomato to control for non-specific binding between the two tags. Pull-downs were performed against either anti-V5 IgG agarose beads or IgG Agarose beads as a control for non-specific protein binding to the beads. Note that COBL-tdTomato is specifically pulled down together with IGFN1_v1-V5 only. B) Western blot of untransfected (1) and transfected COS7 cells with COBL-tdTomato (2 and 3) probed with anti-COBL antibodies and GAPDH as loading control. No endogenous expression of COBL is detected in COS cells. N.S. denotes cross reactivity. C) Representative images of COS7 cells co-transfected with either pDEST47-IGFN1_v1-GFP and pDEST47-COBL-tdTomato (top row) or with pDEST47-IGFN1_v1-GFP and pDEST47-tdTomato (bottom row). IGFN1_v1-GFP is generally found in the nucleus when expressed on its own or…

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  Fig. 5. COBL protein expression throughout in vitro differentiation. A) (Top) Representative Western blots from wildtype C2C12 (C2C12), and IGFN1 knockout (KO19) cells at the following time points: proliferating (P), fully confluent (D0) in differentiation medium for one (D1) or seven (D7) days. Two independent samples are shown for each cell line/timepoint from a total of 4 repeats. (Bottom) Quantification of mean relative COBL expression. Values were normalised to GAPDH expression on a sample by sample basis (n = 4). One-way ANOVA revealed no significant differences between relative expression (F [7,24] = 0.581, p = 0.764). B) Quantifications of Cobl transcript expression. The histograms show the fold change at day 0 (D0) and after 7 days of differentiation (D7). Note that the statistical testing is done on the average Ct levels and therefore the standard deviation has not been added to figure. C) C) Cycloheximide treatment of transfected COS7 cells. (Top) Representative Western Blots for COBL-tdTomato in COS7 cells co-transfected with either COBL-tdTomato and GFP, or COBL-tdTomato and IGFN1-GFP (n = 2). Lysates were collected from untransfected control cells (UT) and transfected cells…

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  Fig. 6. IGFN1 inhibits COBL-mediated ruffle formation. A) Representative images of transfections of COS7 cells. Transfected constructs are indicated on the left with the font in green or red matching the corresponding fluorescent signal on the images. Phalloidin has been used conjugated to green or red fluorescence as indicated. Ruffles are identified by white arrows. Note that ruffles are detected in cells transfected with COBL-tdtomato alone or co-transfected with maxGFP, but no ruffles are detected in cells co-transfected with COBL-tdtomato and IGFN1_v1-GFP. B) A statistically significant effect of the transfected construct on ruffle percentage was demonstrated by one way ANOVA [F (5,12) = 117.64 p < 0.001], post-hoc comparisons (Tukey HSD) revealed that ruffle percentage was significantly higher in cells transfected with COBL-tdTomato or COBL-tdTomato + GFP compared with all other conditions except one another (*p < 0.01).

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  Fig. 7. COBL overexpression increases myoblast fusion. A) Representative images of C2C12 cells transfected with either pDEST47-COBL-tdTomato or pDEST47-tdTomato and differentiated for 7 days. Scale bar represents 50 μm. B) Mean number of nuclei within a pDEST47-COBL-tdTomato or pDEST47-tdTomato expressing myotube (minimum of 3 nuclei to exclude non-fusing cells). The mean number of nuclei was significantly higher in pDEST47-COBL-tdTomato expressing myotubes than in pDEST47-tdTomato expressing myotubes (n ≥ 50, p < 0.01). A minimum of 4 independent transfections were performed per condition.

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  Fig. 8. Generation and characterization of COBL knockout C2C12-derived cell lines. A) Agarose gel showing results from PCR amplifications. Amplicons were obtained with primers flanking exon 3. Amplification of Actn1 to confirm DNA integrity is also shown (Actn1). Amplicons were obtained from 24 individually selected clones, en masse selection and control C2C12 cells, as indicated. B) Western blot of control C2C12 cells, COBL knockout cells selected en masse and knockout clones KO15, KO18, and KO23. Total loss of COBL is shown for clones KO15, KO18, and KO23. C) Representative images of 7 days differentiated myotubes from control C2C12 cells and COBL knockout clones KO15, KO18 and KO23 stained with anti-α-actinin (green) and DAPI (blue). White squares show amplified insets for C2C12, KO18 and KO23 with no DAPI to show detail. Note α-actinin striations in the control C2C12 cells and α-actinin aggregation pattern in KO18 and KO23. Scale bar represents 75 μm. D) Representative images of 7 days differentiated myotubes from KO18 (T18B5 and T18A7) and KO23 (T23B7 and T23G5) derived cell lines stably expressing pDEST47-COBL-tdTomato and stained with anti-α-actinin (green) and DAPI (blue). Scale…