Figure 1.
Perinuclear clustering of mitochondria is induced by the C-terminal domain of hDMPK A.
(A,B) C2C12 myoblasts transiently expressing YFP-hDMPK A or C fusion proteins or mock-transfected cells were stained with a cytochrome c oxidase antibody to visualize mitochondria. Typical examples of classes of mitochondrial morphology are shown in (A). Mitochondrial distribution was classified as fragmented, clustered or elongated. Frequencies of appearance are listed as percentage of the total number of cells expressing MOM-associated hDMPK A (n = 3, ∼50 cells analyzed per experiment). Around 40% of YFP-hDMPK A-expressing cells showed a cytosolic expression. These cells contained mitochondria with a typically elongated shape, but were disregarded in the analysis. (C) Schematic representation of YFP constructs used for transfection expression with protein domains, mutations and 3′ UTR indicated. (D) C2C12 myoblasts expressing YFP fusion proteins were stained with a cytochrome c oxidase antibody to visualize their ability to induce clustering of mitochondria. (E) Expression of constructs with altered 3′ UTRs demonstrated that the hDMPK A 3′ UTR was not involved in mitochondrial clustering. (F) Co-expression of YFP-hDMPK A and hDMPK C-HA in N2A cells resulted in mitochondrial clustering, whereas cells only expressing hDMPK C-HA exhibited normal, elongated mitochondria. Bars, 10 µm.
Figure 2.
An intact microtubular cytoskeleton enhances hDMPK A–induced perinuclear mitochondrial clustering.
DMPK KO myoblasts were transduced with YFP-hDMPK A or C-expressing adenoviruses in the presence of cytochalasin D (A) or nocodazole (B). F-actin was visualized by fluorescent phalloidin. The microtubular cytoskeleton was stained with an anti-tubulin antibody. Disruption of the actin cytoskeleton did not affect localization of mitochondria. Depolymerization of microtubules decreased mitochondrial clustering in YFP-hDMPK A-expressing cells, but mitochondria still appeared fragmented. The distribution of mitochondria in YFP-hDMPK C-transduced cells was unaffected by nocodazole treatment. Bars, 10 µm. (C) Quantification of mitochondrial clustering. The number of transduced cells that contain clustered mitochondria are expressed as percentage of the total amount of cells expressing hDMPK A at the MOM, with or without treatment of cytochalasin D or nocodazole (images shown in A and B; n = 3, ∼100 cells per experiment, P = 0.01). (D) Effect of nocodazole wash-out. Quantification of the percentage transduced cells with clustered mitochondria after a 12–16 hours treatment with nocodazole, followed by a 8 hours wash-out (n = 3, ∼30 cells per experiment, P<0.05).
Figure 3.
Mitochondrial clustering is a rapid process and occurs already at low hDMPK A expression.
KO myoblasts were transduced with YFP-hDMPK fusion proteins (top panels), while mitochondria stained with MitoTracker Red (bottom panels). Images were collected every three minutes. The time of first appearance of YFP signal was set at t = 0; other time points are indicated in the top panels. (A) YFP-hDMPK A was first detected in the cytoplasm. Soon YFP-hDMPK–decorated mitochondria appeared which then started to cluster, eventually resulting in severely aggregated mitochondria surrounding the nucleus. (B) YFP-hDMPK C directly appeared on mitochondria which maintained their elongated, reticular structure. (C) In N2A cells, YFP-hDMPK A expression emerged in a similar fashion as in KO myoblasts, except that mitochondrial clustering occurred almost immediately and cytosolic staining was less pronounced.
Figure 4.
Mitochondrial clustering is accompanied by aberrant ultrastructural morphology.
N2A cells were transfected with YFP-hDMPK A or C isoforms and ultrastructure was analyzed by electron microscopy. (A) YFP-hDMPK A–expressing cells contained fragmented, clustered mitochondria near the nucleus. Cristae structure was often partly lost together with electron density of the matrix (arrowhead) and mitophagy was observed (arrow). In contrast, other areas of the cell were completely devoid of mitochondria (B). (C) Mitochondrial morphology was normal in hDMPK C–expressing cells. A fragment of the nucleus is included here for orientation. Note the proper cristae structure and loose distribution of mitochondria and endoplasmic reticulum. Bars, 1 µm. (D) Lysates from myoblasts expressing YFP-hDMPK A or C for 8, 16 or 24 hours were used for western blotting with a LC3b antibody and showed an increased LC3b conversion following YFP-hDMPK A expression. Cells cultured under normal conditions and nutrient-starved cells, cultured in Earle's Balanced Salt Solution (EBSS) for 2 hours, were used as negative and positive controls, respectively. DMPK expression was verified using a DMPK antibody. Tubulin (T) antibody staining was used as loading control.
Figure 5.
Human DMPK A expression affects mitochondrial function and cell viability.
(A) C2C12 myoblasts were grown under different culture conditions and transfected with YFP-hDMPK A or C. The fraction of viable YFP positive cells was determined after 20 hours. When supplied with galactose and pyruvate, YFP-hDMPK A-expressing cells showed a significantly lower viability than YFP-hDMPK C-expressing cells (P<0.01, n = 3, >100 cells analyzed per experiment). (B) The MMP was determined in HeLa cells expressing YFP-hDMPK A or C. YFP-hDMPK A–expressing cells without clustered mitochondria showed a clear MMP signal (upper panels). No TMRM signal was found in YFP-hDMPK A–expressing cells with clustered mitochondria (middle panels, asterisks). YFP-hDMPK C–expressing cells demonstrated a clear mitochondrial signal (lower panels). Bars, 10 µm. (C) The MMP in YFP-hDMPK A–expressing cells was almost completely abolished and significantly lower than in YFP-hDMPK C and non-transfected (NT) cells (P<0.001, n = 3, >35 cells per experiment).
Figure 6.
Expression of hDMPK A induces apoptosis.
(A) The effect of apoptosis-inhibitor z-vad-fmk was tested on cell survival of DMPK KO myoblasts transduced with YFP-hDMPK A or C–expressing adenoviruses. YFP-positive cells were counted after 16, 24, and 48 hours. Z-vad-fmk greatly reduced cell death of YFP-hDMPK A–expressing cells, but had no effect when YFP-hDMPK C was expressed. Z-vad-fmk was applied immediately after transduction and maintained present for the remaining time of the experiment. Values at 16 hours were set at 100% (P<0.05, n = 3, >90 cells counted per experiment). (B) YFP-hDMPK A and C–expressing cells were stained for cytochrome c (cyt c). Cells expressing YFP-hDMPK A displayed a diffuse, cytosolic cytochrome c staining when mitochondria were clustered (upper panels). A clear mitochondrial staining was present when mitochondria appeared fragmented (middle panels). A discrete, mitochondrial cytochrome c staining was also observed in YFP-hDMPK C–expressing cells. Bars, 10 µm.