Figure 1.
MURF2 expression during differentiation of C2C12 cells.
(A) Schematic structure of the MURF2 isoforms of skeletal muscle cells and locations of the epitopes recognized by various antibodies (names are framed). (B) Same amounts of C2C12 lysates obtained at various time of differentiation (Diff.) were used for Western blots analyses and probed with the indicated antibodies.
Figure 2.
Parallel localization of MURF2 isoforms with LC3, p62 and NBR1.
Plasmids expressing the indicated fluorescent proteins were used for double transfection experiments in C2C12 myoblasts. The mCherry (red) and the GFP (green) fluorescence were directly observed by confocal microscopy. Scale Bar: 10 µm.
Figure 3.
MURF2A and MURF2B colocalize with autophagic proteins.
Triple co-transfections were performed in C2C12 myoblasts with combinations of various plasmids encoding the mentioned tagged proteins. Direct observation of fluorescent proteins was performed by confocal microscopy. Arrows show colocalization of the proteins on vesicles and aggregates. Scale Bar: 10 µm.
Figure 4.
A LIR sequence mediates interaction of MURF2B with LC3.
(A,B) Predicted hydrophobicity and aa sequences of the human C-terminus of MURF2A (A, Cter MURF2A) and of the Alternative C-terminus of MURF2B (B, Alter MURF2B). LIR sequences of various proteins and consensus sequence are aligned with the MURF2B C-terminus. (C) The MURF2B LIR domain interacts with LC3. C2C12 myoblasts were transfected with various pairs of vectors encoding GFP-LC3, GST, GST- MURF2A C-terminus domain (GST- Cter MURF2A) or the GST- MURF2B Alternative C-terminus domain (GST-Alter MURF2B). One day after transfection, cells were differentiated for 24h day in 1% HS medium then cultured for 2h 30 min in HBSS medium containing 0.1% BSA, 50 µM MG132 and 200 nM Baf. Immunoprecipitation were performed with GFP-Trap magnetic beads and 400 µg of extracts in RIPA buffer and subjected to SDS-PAGE (IP: anti-GFP). Exogenous proteins were detected with anti-GFP and anti-GST antibodies by Western blot (WB). For each transfection, 20 µg protein extract was used to reveal the expression of exogenous proteins (Extracts). (D) The Alternative C-terminus domain of MURF2B interacts with vesicles decorated with LC3 and the C-terminus domains of the MURF2 isoforms are not needed for MURF2/p62 interactions. Immunofluorescence experiments were performed with C2C12 cells transfected with GST-Alter MURF2B and GFP-LC3 vectors and stained with anti-GST antibody (red). GFP-LC3 was observed directly (green) with confocal microscope. Co-localizations are highlighted by arrows. (E) C2C12 cells were also transfected with the GFP-MURF2Δ and mCherry-p62 vectors and observed by confocal microscopy. Arrows show vesicles displaying both exogenous proteins. Enlarged views reveal curved structures, labelled with GFP-MURF2Δ, in close contact with p62 vesicles (empty arrowheads). Scale Bars: 10 µm.
Figure 5.
During myogenic differentiation, various MURF2 complexes are formed.
(A) MURF2A and MURF2B co-localize in C2C12 myoblasts. C2C12 cells were transfected with mCherry-MURF2A. After 24h, endogenous MURF2B was detected using MURF2B antibody (green) and fluorescent MURF2A was observed directly (red). The magnified region reveals that MURF2A and MURF2B are in close contact (arrows). Scale Bars: 10 µm. (B) MURF2A and MURF2B are complexed with LC3 and p62. C2C12 cells transfected with GFP-LC3 or GFP-p62 were kept undifferentiated (Diff. 0h) or differentiated for 24h (Diff. 24h) in absence (-) or in presence (+) of 200 nM Baf for 4 h before cell lysis in RIPA buffer. Western blots were performed with precipitated proteins obtained using magnetic beads (Bio-Adembeads pAG) incubated with 300 µg extract complemented (+) or not (-) with anti-GFP antibody. Western blots (WB) were probed with the indicated antibodies. Asterisks indicate unspecific signals.
Figure 6.
MURF2A and MURF2B are needed for autophagosome formation.
(A) Expression of MURF2, p62 and LC3 during C2C12 differentiation. Representative immunoblots of C2C12 lysates probed with MURF2, p62, LC3, Troponin T and GAPDH antibodies. Lysates were obtained at various times of differentiation (Diff.). (B) Analysis of autophagy and polyubiquitination during differentiation. Representative immunoblots of C2C12 lysates obtained at various times of differentiation (Diff.) in absence (-) or in presence (+) of inhibitors (50 µM MG132 or 200 nM Baf) applied during 4h. Western blots were probed with the antibodies mentioned. (C) Quantification of the ratio of LC3-II/LC3-I proteins during differentiation. LC3-II and LC3-I detected with 3 independent Western blot analyses were quantified. Differences in LC3-II/LC3-I ratio were evaluated with the Kruskal-Wallis test (analysis of variance). Histograms show medians and quartiles of LC3-II/LC3-I expression during differentiation of C2C12 cells. Compared to undifferentiated cells (Diff. 0), non significant (ns) and significant (*) differences were observed (P<0.05). (D) Autophagic flux increases after 48h of differentiation. Stable C2C12 cells expressing mCherry-GFP-LC3 were induced to differentiate for the indicated times and fluorescent LC3 vesicles observed directly by confocal microscopy. Magnified regions show yellow autophagosomes (arrowheads) and red autolysosomes (arrows). Scale Bar: 10 µm.
Figure 7.
Analysis of autophagy in C2C12 and MURF2 RNAi cells.
(A) Endogenous p62 was detected in C2C12 cells and MURF2 RNAi myoblasts (Diff. 0h) and in cells differentiated for 48h. Cells were immunostained with anti-p62 antibody and analyzed by confocal microscopy. Scale Bar: 10 µm. (B) Quantification of the p62 puncta detected in C2C12 and MURF2 RNAi myoblasts. Data were obtained from independent imunofluorescence experiments and various numbers of cells were analyzed : 8 for C2C12 cells, 6 for RNAi MURF2A cells, 5 for RNAi MURF2B cells and 13 for RNAi MURF2 cells. Differences were evaluated by the Kruskal-Wallis test. Medians and quartiles are shown in the histograms. Compared to C2C12 myoblasts, the MURF2 RNAi cells showed significant differences (*, P<0.05). (C) Quantification of the p62 puncta in the C2C12 and MURF2 RNAi 2 after 48h of differentiation. Data were obtained from independent imunofluorescence experiments, and various numbers of cells were analyzed: 12 for C2C12 cells, 24 for RNAi MURF2A cells, 9 for RNAi MURF2B cells and 12 for RNAi MURF2 cells. Medians and quartiles ranges are shown in the histograms. Multiple comparaisons of median of C2C12 and MURF2 RNAi cells by the Kruskal-Wallis test showed significant differences (*, P<0.05). (D) Representative immunoblot analyses of endogenous proteins expressed during differentiation of C2C12 and MURF2 RNAi cells. Lysates were obtained from myoblasts (Diff. 0) and after 24h of differentiation. Western blots were probed with the indicated antibodies. (E) Quantification of MURF2A protein in C2C12 and MURF2 RNAi cells after 0h and 24h of differentiation. For each time point of differentiation, data were obtained from 3 independent western blot experiments. Medians and quartiles ranges are shown in the histograms. Multiple comparaisons of median of C2C12 and MURF2 RNAi cells by the Kruskal-Wallis test showed significant differences (*, P<0.05). (F) Quantification of MURF2B protein in the C2C12 and MURF2 RNAi cells after 0h and 24h of differentiation. For each time point of differentiation, data were obtained from 2 independent western blot experiments. Medians and quartiles ranges are shown in the histograms. Multiple comparaisons of median of C2C12 and MURF2 RNAi cells by the Kruskal-Wallis test showed significant differences (*, P<0.05). (G) Endogenous p62 quantification in undifferentiated (Diff. 0h) and differentiated (Diff. 24h) C2C12 and MURF2 RNAi cells. Cells were lysed with RIPA buffer and analyzed by Western blots with anti-p62 antibody. For each cell line, the amounts of p62 detected after differentiation were normalized with p62 present in undifferentiated cells. Data were obtained from several independent Western blot experiments: 6 for C2C12 cells (Diff. 0h and 24h), 3 for RNAi MURF2A (Diff. 0h), 2 for RNAi MURF2A (Diff. 24h) and 3 for RNAi MURF2B (Diff. 0h and 24h). Differences were evaluated with the Kruskal-Wallis test. Medians and quartiles are shown in the histograms. Compared to undifferentiated cells (Diff. 0h), non significant differences (ns) and significant differences (*) were observed (P<0.05).
Figure 8.
MURF2A activates the 20S proteasome and is implicated in p62 degradation.
(A) Undifferentiated stable C2C12 and RNAi cell lines expressing the mCherry-GFP-LC3 protein were grown in 10% FCS and observed directly by confocal microscopy. In magnified regions, the yellow autophagosomes are indicated by empty arrowheads. (B) The same cell lines were kept in HBSS medium for 45 min and observed directly by confocal microscopy. Magnified regions show in C2C12 cells yellow autophagosomes (empty arrowheads) and red autolysosomes (arrows). In RNAi cells unconventional autophagic structures are indicated by arrowheads. Scale Bar: 10 µm.
Figure 9.
(A) Representative immunoblots of C2C12 lysates probed with poly ubiquitin, the α5 subunit of the 20S proteasome, myogenin, Troponin T and GAPDH antibodies.
Lysates were obtained at various times of differentiation (Diff.). (B) Activity of the 20S proteasome in C2C12 cells. Proteolytic 20S proteasome chymotrypsin-like activity of the indicated undifferentiated (0h) and differentiated (48h) C2C12 cells was determined measuring the amidomethylcoumarin (AMC) generated as the cleavage product of the fluorogenic substrat succinyl-leucine-leucine-valine-tyrosine-7 amido-4-methyl-coumarin. Differences were evaluated by the Kruskal-Wallis test. Medians and quartiles are shown in the histograms. Compared to C2C12 myoblasts (Diff. 0h), myotubes (Diff. 48h) showed significant differences (*, P<0.05). (C) Activity of the 20S proteasome in various RNAi cell lines. The proteolytic 20S proteasome chymotrypsin-like activity was determined for undifferentiated (0h) and differentiated (48h) cells as previously mentioned for C2C12 cells. Compared to C2C12 and RNAi MURF2A, RNAi MURF2B cells showed significant increase of their 20S proteasome activity (*, P<0.05). (D) Effect of UPS and autophagic inhibition on p62 expression in C2C12 and MURF2 RNAi cells. After 24h of differentiation in 1% horse serum medium (1% HS), cells were shifted to fresh differentiation medium (1% HS) or treated for 2h 30min with fresh differentiation medium complemented with 50 µM MG132 and 200 nM Baf (1% HS+MG132+Baf) before lysis. Western blots were probed with anti p62, anti mono and poly ubiquitin and anti GAPDH antibodies. (E) p62 is degraded by UPS in differentiated C2C12 cells. C2C12 cells were transfected with vectors encoding GFP or GFP-p62 under the CMV promoter. 24h after transfection, cells were split into two groups, induced to differentiate for 24h, then shifted to fresh differentiation medium containing (+) or not (-) 10 µM MG132 for 12h. Extracts were used for Western blot analyses and probed with the indicated antibodies. Rabbit anti mono and poly ubiquitin antibody was used.
Figure 10.
Confocal analyses of p62, UPS and MURF2 proteins in differentiated C2C12 cells.
C2C12 cells were transfected with plasmids expressing the fluorescent proteins as indicated and differentiated for 24h. Confocal observations: fluorescent proteins were detected directly and the 20S proteasome subunit revealed by specific antibody for the α5 subunit of the 20S proteasome. Scale Bars: 10 µm.