Vimentin is one of the intermediate filaments that functions in structural support, signal transduction and organelle positioning of a cell. In the present study, we report the contribution of vimentin in mitochondrial morphology and organization. Using subcellular fractionation, immunoprecipitation and fluorescence microscopy analyses, we found that vimentin was associated with mitochondria. Knockdown of vimentin resulted in mitochondrial fragmentation, swelling and disorganization. We further demonstrated that the vimentin cytoskeleton co-localized and interacted with mitochondria to a greater extent than other cytoskeletal components known to support mitochondria. Our results also suggest that vimentin could participate in the mitochondrial association of microtubules. As mitochondrial morphologies determine mitochondrial function, our findings revealed a potentially important relationship between the vimentin-based intermediate filaments and the regulation of mitochondria.
Mitochondria are important organelles that mediate energy metabolism, cell signalling and homoeostasis in eukaryotic cells . These organelles form a complex tubular branching network in a healthy cell . Accumulating evidence indicates that specific changes in mitochondrial morphologies are required during animal development [3,4], while disturbance in the mitochondrial dynamics and organization result in the dysfunction of mitochondria which leads to fatal consequences including disorders in respiration, neurodegeneration and tumorigenesis [3–8]. As mitochondrial morphology and organization determine the functions of mitochondria, characterizing their regulation is important in understanding the diversity of its biological roles.
As mitochondria are composed of outer limiting membranes and complex networks of internal membranes, the highly curved morphology of these organelles is maintained and stabilized by specific mechanisms, such as the attachment to a rigid cellular structure, for example, the cytoskeleton . The cytoskeleton is mainly composed of the interconnected networks of actin, microtubules and intermediate filaments, which extend throughout the cytoplasm of the entire cell [10,11]. Apart from structural support, the cytoskeleton functions in cell motility, intracellular trafficking and organelle positioning in a cell . Traditionally, microtubules are recognized as the key elements that associate with mitochondria on the basis of their cellular co-localization reported in previous microscopy studies [2,9,10,13,14]. Disturbance in the integrity of microtubules by microtubule-disturbing drugs triggers mitochondrial fragmentation, suggesting that microtubules are important in mitochondrial support [13,15]. Growing evidence also indicates the contribution of intermediate filaments in the support of mitochondria . For example, a fluorescence microscopy study has shown that the signal of vimentin intermediate filaments overlaps with mitochondria and microtubules . Desmin intermediate filaments have also been suggested to contribute to the distribution and function of mitochondria, as the knockdown of desmin in mouse cardiac muscle cells results in the disturbance of the mitochondrial organization and the respiration rate . In the present study, we have identified a novel interaction of mitochondria with vimentin intermediate filaments, which supports the morphology, organization and function of mitochondria. Vimentin interacts and co-localizes with mitochondria to a greater extent than other cytoskeleton components such as microtubules and actin filaments, suggesting that vimentin is critical to mitochondrial support.
MATERIALS AND METHODS
COS-7 cells, HeLa cells and NIH 3T3 cells (from American Type Culture Collection) were cultured in DMEM (Dulbecco's minimum essential medium) supplemented with 10% heat-inactivated FBS (fetal bovine serum), 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco), at 37 °C under an atmosphere of 5% CO2/95% air. Human immortalized brown adipocyte PAZ-6 cells were cultured as described previously . Cells were seeded on tissue culture plates for 36 h until the cell density reached 70% confluency before being subjected to each experiment.
siRNA (small interfering RNA) transfection
Endogenous vimentin and β-tubulin expression was reduced by introducing Stealth™ RNAi (RNA interference) specific to vimentin and β-tubulin (Invitrogen) respectively into COS-7 cells by Lipofectamine™ RNAiMAX (Invitrogen). The RNAi negative control was from the Stealth™ RNAi negative control kit (Invitrogen). Cells were cultured for a further 3 days after transfection for each experiment.
Subcellular fractionation was performed as previously described . Briefly, cells were harvested and homogenized in MS buffer [210 mM mannitol, 70 mM sucrose, 5 mM Tris/HCl (pH 7.5), 1 mM EDTA and 1% Complete™ protease inhibitor cocktail (Roche)] by passing the cell mixture through a 22-gauge needle at 4 °C. The homogenate was centrifuged at 1300 g for 10 min at 4 °C to pellet the nuclei and the unlysed cells. The soluble cytosolic fraction and membrane fractions of the ER (endoplasmic reticulum) and mitochondria were purified from the supernatant by stepwise sucrose-density-gradient centrifugation. The supernatant was suspended in 1 ml of ice-cold MS buffer and laid on the top of a 1.0, 1.2 and 1.5 M sucrose buffer gradient before being centrifuged at 10000 rev./min for 30 min at 4 °C (SW60Ti rotor, Beckman). The soluble cytosolic fraction was collected from the top MS buffer fraction. The gradient-purified ER-containing and mitochondria-enriched fractions were collected at the MS buffer/1.0 M and 1.2/1.5 M sucrose buffer interphases respectively, were washed with MS buffer and dissolved in 0.5% SDS lysis buffer on ice for 30 min.
Immunoprecipitation of mitochondria by magnetic beads
Sheep anti-mouse IgG Dynabeads (M-280; Invitrogen) were bound to mouse anti-human mitochondria monoclonal antibody recognizing the surface of the intact mitochondria (Chemicon). Briefly, 10 μl of Dynabeads were washed with 200 μl of MS buffer, and incubated with 1 μl of antibody in 200 μl of MS buffer with 1% BSA for 3 h at 4 °C on a rotating wheel. The beads were washed with MS buffer (at 4 °C) to remove unbound antibody, and incubated in 200 μl of MS buffer with 10 μg of COS-7 total cell lysate and protease inhibitor cocktail for 3 h at 4 °C on a rotating wheel, and then washed with MS buffer. Mitochondrial membrane bound to the beads was subjected to Western blot analysis.
Western blot analysis
Approx. 3 μg of protein per lane was separated on SDS/PAGE (12% gel) and transferred on to a Hybond ECL® (enhanced chemiluminescence) membrane (Amersham Biosciences). After blocking, the membrane was incubated overnight at 4 °C with primary antibody (1:1000 dilution) followed by a further 1 h incubation in the corresponding horseradish peroxidaseconjugated secondary antibody (Bio-Rad) at a 1:5000 dilution, and the signal was detected with the ECL® Western blotting detection system (Amersham Biosciences). Primary antibodies used were: anti-(α-tubulin), anti-(Cox IV) (cytochrome c oxidase complex IV) and anti-(golgin-97) antibody from Molecular Probes; anti-(β-tubulin), anti-(β-actin), anti-LDH (lactate dehydrogenase) and anti-calnexin antibody from Sigma; anti-Bak (Bcl-2 antagonist/killer) antibody from PharMingen; anti-vimentin, anti-(cytochrome c) and anti-PCNA (proliferating-cell nuclear antigen) antibody from Santa Cruz Biotechnology. For signal quantification, developed X-ray films of the Western blot analysis were scanned, and images of the signal were stored in a 24 bit grey-level depth. The signal region was determined by Canny edge detection algorithm for image segmentation, as previously described . The strength of signal was extracted by calculating the difference between the average intensity of the identified area and its corresponding background.
Cells were grown to 70% confluency on a coverslip. Mitochondria and the nuclei were stained with 50 nM MitoTracker Red CMXRos and 250 ng/ml Hoechst 33342 (Molecular Probes) respectively in the cell culture medium at 37 °C with 5% CO2 for 20 min, and then the cells were washed twice with PBS and fixed with 3.7% (w/v) paraformaldehyde for another 20 min. For staining of F-actin (filamentous actin), cells were washed and fixed, permeabilized with 0.1% Triton X-100 for 15 min, and stained with Alexa Fluor® 488 Phalloidin (Molecular Probes) with a dilution of 1 unit in 200 μl of PBS with 1% BSA for 30 min. Vimentin and microtubules were stained with anti-vimentin and anti-(α-tubulin)/anti-(β-tubulin) primary antibodies and conjugated with green fluorescent Alexa Fluor® 488 anti-mouse IgG secondary antibodies (Molecular Probes). Slides were mounted using the ProLong antifade kit (Molecular Probes).
COS-7 and NIH 3T3 cells served as the principal model in this microscopy study due to their spread morphology during which the distribution of cytoskeletal filaments within the cell can be easily visualized. Cell images were captured with a monochromatic camera AxioCam HRM (Carl Zeiss) or a monochromatic CoolSNAP FX camera (Roper Scientific) on an Axiovert 200M microscope using a 63× N.A 1.4 Plan-Apochromat objective (Carl Zeiss). Fluorescence signals were analysed using AxioVision 4 software (Carl Zeiss) and ImageJ NIH (National Institutes of Health) software version 1.37 (http://rsb.info.nih.gov/ij/).
RESULTS AND DISCUSSION
Protein analysis of vimentin in the mitochondrial fraction of mammalian cells
In order to investigate the mechanism regulating mitochondrial morphology and organization, we initially studied the mitochondrial association of microtubules by subcellular fractionation. Sucrose-density-gradient centrifugation was performed to purify mitochondria from the immortalized brown adipocyte PAZ-6 cells for Western blot analysis of microtubules. Microtubules are composed of a heterodimer of α- and β-tubulin, and the heterodimers form the filamentous tube-shaped protein polymers in an eukaryotic cell . Surprisingly, both of the core microtubule components α-tubulin and β-tubulin were mainly found in the cytosol and only trace amounts of α- and β-tubulin were observed in the purified mitochondrial fraction in PAZ-6 cells (Figure 1). The presence of the mitochondrial outer- membrane protein Bak and inner-membrane protein Cox IV in the mitochondrial fraction indicates that the purified mitochondria were intact. It is possible that the isolation of mitochondria could disturb the integrity of microtubule filaments, and that might disrupt their association with microtubules.
Mitochondrial localization of vimentin in PAZ-6 cells
By screening various cytoskeletal members, we found that the actin cytoskeleton was not specifically localized in the mitochondrial fraction, as the actin marker β-actin was mainly localized in the cytosolic fraction (Figure 1). Interestingly, our results showed that the intermediate filament vimentin was distinctively localized in the mitochondrial fraction, whereas a moderate amount of vimentin was observed in the cytosolic fraction and a barely detectable amount of vimentin protein was found in the ER-containing fraction in PAZ-6 cells (Figure 1). This specific mitochondrial association of vimentin was also observed in various cells lines including the human cervical carcinoma HeLa cells, HEK (human embryonic kidney)-293T cells, COS-7 cells, the mouse myoblast C2C12 cells, NRK (normal rat kidney) cells and the Mustela putoris furo (ferret) normal astrocytes CRL 1656 cells (results not shown), suggesting that this is a general phenomenon in the mammalian system.
Interaction of vimentin with mitochondria
Vimentin has been reported to interact with several organelles such as the nucleus, Golgi apparatus, endosomes and lysosomes [23–25], but its interaction with mitochondria remains to be determined . A previous microscopy study demonstrated that mitochondria overlayed with microtubules and vimentin filaments, but they did not provide direct evidence on the mitochondrial interaction with microtubules and vimentin . To identify this novel direct interaction between vimentin and mitochondria, we performed immunoprecipitation studies to purify intact mitochondria from the total cell lysate of COS-7 cells. By Western blot analysis, we detected the presence of a significant amount of vimentin in the immunoprecipitated mitochondrial protein complex; the quantity was even more than that of the mitochondrial markers, Bak and Cox IV (Figure 2). The above findings indicate that vimentin interacts with mitochondria, and this is also in agreement with the results of subcellular fractionation on the co-localization of vimentin and mitochondria in PAZ-6 cells (Figure 1). Figure 2 shows that both of the microtubule markers, α- and β-tubulin, were detectable in the mitochondrial pulldown complex, but their signals were weak and their sizes were close to the signal contributed by the antibody IgG chain at 52 kDa (Figure 2). The weak interaction between microtubules and mitochondria might be due the isolation of mitochondria; as discussed in the previous section, the isolation process could disturb the integrity of the microtubule filaments.
Mitochondrial association of vimentin in COS-7 cells
Co-localization of vimentin and mitochondria
Immunofluorescence microscopy was performed to verify the mitochondrial association of vimentin, as this is a less invasive approach to study the localization of cellular components as compared with subcellular fractionation and immunoprecipitation. We observed that the tubular mitochondria aligned and co-localized with a significant portion of the vimentin filaments in PAZ-6 cells (see Supplementary Figure 1A at http://www.BiochemJ.org/bj/410/bj4100141add.htm). The fluorescence signals were quantified (see Supplementary Figure 1B at http://www.BiochemJ.org/bj/410/bj4100141add.htm), and the results indicated that the vimentin filaments specifically co-localized with mitochondria.
To corroborate the significance of the mitochondrial association of vimentin, we compared the relative localization of mitochondria, vimentin, microtubules and F-actin by fluorescence microscopy analysis. The mitochondria, vimentin, microtubules and F-actin were co-stained in the mouse embryonic fibroblast NIH 3T3 cells. In agreement with our earlier observation in the PAZ-6 cells, almost all mitochondria aligned with the vimentin filaments in these cells (Figure 3A). On the other hand, we observed that the intensive refined microtubule filaments radiated from the perinuclear region through the cytoplasm to the edges of the cells, and certain portions of these filaments overlapped with the tubular mitochondria (Figure 3B). We observed that F-actin ran longitudinally across the cells (Figure 3C). However, these filaments did not specifically overlap with the tubular mitochondria [Figures 3A(iv) and (v), 3B(iv) and (v), and 3C(iv) and (v)], and the signal quantification results showed that the correlation of mitochondria with vimentin is significantly higher than that with microtubules or F-actin (Figure 3D).
Mitochondrial co-localization of vimentin in NIH 3T3 cells
Mitochondrial morphology and organization depend on the integrity of the vimentin network
As vimentin associates with mitochondria, we investigated the contribution of vimentin to the mitochondrial morphology by disturbing the integrity of the vimentin network through knockdown of vimentin. For the control COS-7 cells transfected with the non-specific siRNA, the extended tubular mitochondria still aligned along the vimentin filaments (Figure 4A). For the vimentin knockdown of COS-7 cells, the mitochondrial network was broken down and underwent mitochondrial fragmentation, swelling and disorganization as indicated by the perinuclear redistribution of mitochondria (Figure 4B). Furthermore, our Western blot analysis suggested that the cytochrome c level in the vimentin-knockdown cells was reduced compared with the control cells (see Supplementary Figure 2 at http://www.BiochemJ.org/bj/410/bj4100141add.htm). As cytochrome c serves as a mitochondrial electron carrier which contributes to the mitochondrial polarization and this is essential for ATP synthesis , reduction of cytochrome c levels might disturb the mitochondrial polarization and ATP generation, implying that the morphology and function of the mitochondria are dependent on the integrity of the vimentin network.
Mitochondrial morphology of vimentin-knockdown COS-7 cells
Traditionally, microtubules have been recognized to support mitochondrial morphology; this is supported by the observation that disturbance in the mitochondrial integrity by microtubule-disturbing drugs such as colcemid, nocodazole and taxol results in mitochondrial fission [13,15]. However, these anti-tubulin agents in fact are the anti-cancer drugs which induce apoptosis [28–30]. As mitochondrial fission is a universal phenomenon in apoptosis , there is a concern as to whether the mitochondrial fission induced by these drugs is the cause or the consequence of apoptosis. Therefore we examined the functional role of microtubules in the mitochondrial support by knockdown of the coremicrotubule element, β-tubulin, so as to disturb the microtubule integrity. Figure 4 shows that the knockdown of β-tubulin could result in certain mitochondrial disorganizations such as mitochondrial swelling; however, extensive mitochondrial fragmentation was not observed (Figures 4C and 4D). Taken together with the vimentin knockdown results, we demonstrated that the morphology of mitochondria depends more primarily on the integrity of vimentin than the microtubule network. In addition, we found that the knockdown of vimentin resulted in a significant reduction of α-tubulin levels in the mitochondrial fraction of COS-7 cells, and the remaining mitochondrial α-tubulin level was almost undetectable after the vimentin knockdown (Figure 5). This suggests that vimentin might contribute to the mitochondrial association of microtubules. It has been reported that microtubules could mediate the mitochondrial morphology and distribution through the microtubule-based cytoskeletal motors such as kinesin and dynein . As microtubules regulate the dynamics of vimentin [32–35], it might be possible that microtubules mediate the mitochondrial morphology, organization and function by interacting with mitochondrial-associated vimentin.
Reduction of mitochondrial microtubules in vimentin-knockdown cells
In the present study, we report the novel observation that vimentin interacts and co-localizes with mitochondria. We also provide functional evidence that depletion of vimentin results in the reorganization and fragmentation of mitochondria. Our results further suggest that vimentin might mediate the interaction of mitochondria with microtubules. This draws our attention to the potential role of vimentin in the regulation of the mitochondrial morphology, organization and function. Further investigation into the molecular mechanism of this process is certainly required.
We thank Dr Vladimir Zilberfarb (Institut Cochin de Génétique Moléculaire, Paris, France) for the PAZ-6 cell line and Mr Chun Wai Ma (Hong Kong University of Science and Technology, Kowloon, Hong Kong) for the Western blot signal quantification analysis.