Abstract
Mitofusins (Mfns) mediate the fusion of mitochondrial membranes. However, little is known about how Mfns are regulated to control mitochondrial fusion, which is a multistep process requiring tethering and docking of the outer membranes of two mitochondria. In this study, we report that guanine nucleotide binding protein-β subunit 2 (Gβ2), a WD40 repeats protein and a member of the β-subunits of the heterotrimeric G proteins, has a crucial function in mitochondrial fusion. Gβ2 was found to be enriched on the surface of mitochondria and interacted with mitofusin 1 (Mfn1) specifically. Gβ2 also regulated the mobility of Mfn1 on the surface of the mitochondrial membrane and affected the mitochondrial fusion. Depletion of endogenous Gβ2 resulted in mitochondrial fragmentation, which could be rescued by exogenous Gβ2. These findings have thus uncovered a novel role of Gβ2 in regulating mitochondrial fusion through its interaction with Mfn1.
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Introduction
Mitochondria are dynamic organelles undergoing constant fusion and fission, and dysregulation of mitochondrial dynamics has been linked to altered mitochondrial physiology, abnormal mitochondrial DNA maintenance, apoptosis1,2 and neuromuscular diseases3,4. These two opposing processes are regulated by two members of the dynamin family: mitofusins (Mfns) and dynamin-related guanosine triphosphatases (GTPases) (Drp1)5,6. Proper control of mitochondrial fission and fusion is vital to both the integrity of the mitochondrial morphology and their functions7,8,9,10,11. Several WD40 proteins, such as Mdv1 and Caf4, are found to interact with Dnm1, a major mediator for mitochondrial fission in yeast, to determine the localization of Dnm1 on the mitochondrial surface12 and modulate the process of fission, at least in the yeast system13. Mfn 1/2, two mitochondrial GTPases in the dynamin family, mediate the fusion of mitochondrial membrane. During these processes, Mfn1 and Mfn2 act in a trans-manner leading to a closer apposition of the membranes for completing the fusion14. Removal of the GTPase domain of Mfn1 is still able to induce the aggregation of mitochondria without complete fusion14,15, suggesting that domains other than GTPase activity are required for mitochondrial docking or tethering. We reasoned that there may be other factors that act to coordinate Mfns for mitochondrial fusion.
In this study, we report that guanine nucleotide binding protein-β subunit 2 (Gβ2), a member of the β-subunits of the heterotrimeric G proteins and a WD40 protein, has a crucial function for mitochondrial fusion. Gβ2 is partially found on the surface of mitochondria and physically interacts with Mfn1. Depletion of Gβ2 perturbs mitochondrial morphology and decrease mitochondrial fusion rate. Gβ2 acts by limiting the membrane mobility of Mfn1 at the mitochondrial surface to regulate the process of mitochondrial fusion. These findings demonstrated that Gβ2 interacts with mfn1 to regulate mitochondrial fusion.
Results
Gβ2 localizes to the surface of mitochondria
To identify potential proteins regulating Mfns, we used a yeast two hybrid assay to identify potential molecules, which may interact with Mfn 1 or 2 by using the full-length Mfn1 and Mfn 2, or different domains of Mfn1 as baits. As a result, we obtained 91 positive clones, 3 of which encoded Gβ2 when the fragment of aa241–387 was used as a bait. Gβ2 interaction with Mfn1 is specific, as reconstitution of AD (active domain)-Gβ2 (in the fish vector) with BD (binding domain)-Mfn1 (in the bait vector), but not with BD-Mfn2, gave a strong positive result (see Supplementary Fig. S1a).
Structural analysis has revealed that the Gβ subunits are WD40 repeat proteins with a toroidal structure containing seven-bladed β-propellers16. The unique toriodal β-propeller structure defines surfaces for interactions with numerous receptors, Gα and many other effectors17. Despite accumulating evidence that has shown Gβγ dimers exert its functions on various effectors as the Gα proteins do18,19,20, the role of Gβ2 in mitochondrial dynamics is largely unknown. Thus, we first examined if Gβ2 and Mfns are co-localized in mitochondria by immunostaining. Indeed, our data revealed that endogenous Gβ2 was localized in mitochondria in HeLa (Fig. 1a) and SH-SY5Y neuroblastoma cells (Supplementary Fig. S1b). In contrast, a specific antibody to Gβ1, most similar to Gβ2 in amino acids identity among all Gβ subunits, showed diffused staining in HeLa cells (Fig. 1b). Confocal microscopic analysis showed that Gβ2 appeared to have a patchy distribution along the mitochondrial tubular structure, and this observation was substantiated by three-dimensional analysis using the scanned confocal images (Fig. 1a). To further ascertain that Gβ2 is indeed localized in mitochondria, we performed a gradient fractionation assay. HeLa cells were homogenized with a Dounce homogenizer and the cell lysates were separated into the crude mitochondrial fraction, 10,000×g supernatant (C2) containing plasma membrane, the microsomal fraction, as well as cytosolic proteins. The crude mitochondrial fraction and C2 were further fractionated by Percoll density gradient centrifugation to obtain pure mitochondrial fraction (M), plasma membrane fraction (P) and the cytosol (C1). The purity of the mitochondrial fractions was tested by loading equal amount of proteins onto a gel validated by using mitochondrial-specific markers such as VDAC1 and Mfn1, and by specific markers for endoplasmic reticulum (p62 (ref. 21)), Golgi (GM130 (ref. 22)). As shown in Figure 1c, Gβ2, but not Gβ1, was significantly enriched in the purified mitochondrial fraction compared with other fractions (enrichment 48.5%). Gγ2 was also enriched in the purified mitochondrial fraction, whereas Gγ1 was not detectable. Gα12 and Gαi1 were present in mitochondria, which were consistent with previous reports23,24, and other membrane fractions. To determine the precise localization of Gβ2 on the outer membrane, we incubated intact mitochondria isolated from HeLa cells with protease K. Our data showed that Gβ2, similar to Tom20 and Mfn1 that are mitochondrial outer membrane proteins, was sensitive to protease K (Fig. 1d). Furthermore, Gβ2 was resistant to NaHCO3 treatment, suggesting that Gβ2 does not adhere to the outer membrane of the mitochondria nonspecifically25. Semiquantitative PCR analysis showed that Gβ2 is expressed at high levels in both HeLa and SH-SY5Y cells (Fig. 1e–g, Supplementary Fig. S1c–e).
Gβ2 physically interacts with Mfn1
We next determined whether there is indeed a physical interaction between Gβ2 and Mfn1. Endogenous Gβ2 was coimmunoprecipitated with Mfn1 but not with normal mouse immunoglobulin-G from HeLa cell lysate. Importantly, depletion of Gβ2 in the cells by short hairpin RNA (shRNA) abolished the interaction, whereas Gβ2 could be co-immunoprecipitated with Mfn1 after Gβ2 was reintroduced into the shRNA cells, and vice versa, when Mfn1 was used as the bait (Fig. 2a,b). However, we failed to detect an interaction between Gβ2 and Mfn2, Gβ2 and Drp1, or Gβ1 and Mfn1 (Supplementary Fig. S2a–c) or between Gβ2 and Gα12, which is reportedly localized in mitochondria24. There was a weak interaction of Gβ2 with Gαi1, although the significance of this interaction remains to be determined. To validate our findings that Gβ2 interacted directly with Mfn1, we performed two independent fluorescence resonance energy transfer (FRET) analyses on HeLa cells co-transfected with Mfn1-CFP and Gβ2-YFP. In the cells, the expressed Mfn1-CFP was specifically localized in mitochondria, whereas a majority of YFP-Gβ2 was found in mitochondria (Supplementary Fig. S2d). In this assay, the fluorescent intensity of the donor CFP was measured before and after the fluorescence of the acceptor YFP was photobleached (quenched). As shown in Figure 2c, the fluorescent intensity of CFP was remarkably increased after the fluorescence of YFP was completely quenched by a laser beam. This observation suggested that there was a direct interaction between Mfn1-CFP and Gβ2-YFP in the cells. The energy transfer efficiency from CFP to YFP obtained by using the equation described26 was 33.9%. In contrast, energy transfer efficiency from Mfn1-CFP to Gβ1-YFP was significantly lower (only ~10%) (Fig. 2d). Taken together, our data demonstrate that Mfn1 and Gβ2 have a physical interaction and that the interaction is specific. We next determined the structural domains responsible for the interaction of Gβ2 and Mfn1. Co-immunoprecipitation of truncated mutants of Mfn1 and Flag-tagged Gβ2 in HeLa cells revealed that, consistent with the yeast two hybrid assay, the domain of Mfn1 from aa241 to 350 was necessary for the interaction between Mfn1 and Gβ2 (Fig. 3a,b). Also, the Mfn1 mutant lacking the interaction domain had diminished FRET (Supplementary Fig. S2e). Importantly, we found that Mfn1 lacking the interacting domain from aa241–350 failed to induce mitochondrial aggregation compared with that of wild-type Mfn1 (Fig. 3c), whereas GTPase dead mutants Mfn1 T109A and K88T were able to induce mitochondrial aggregation (Supplementary Fig. S2f). Our results thus identify that, in addition to the heptad repeat region (HR2) domain of Mfn115, the domain between the GTPase domain and the HR1 domain is also important for mitochondrial fusion by its interaction with Gβ2. On the other hand, we generated a series of Flag-tagged Gβ2 with various truncations of WD repeats and transfected these constructs into HeLa cells (Fig. 3d), and found that they failed to induce mitochondrial aggregation (Fig. 3e). Co-immunoprecipitation revealed that the deletion of four WD40 repeats of Gβ2 completely abolished its interaction with Mfn1 (Fig. 3f), suggesting that the toroidal structure needs to be intact for their interaction and subsequent mitochondrial fusion.
Gβ2 requires Mfn1 for its effect on mitochondrial dynamics
We then addressed the biological significance of the interaction between Gβ2 and Mfn1. As shown in Figure 4a, Gβ2 induced mitochondrial aggregation in both wild-type and Mfn2−/− MEF cells, in which Mfn1 is expressed. However, ectopic expression of Gβ2 failed to induce mitochondrial aggregation in Mfn1−/− cells (Mfn1−/− or Mfn1&2−/− MEF cells). These data support our notion that Gβ2 indeed requires Mfn1 for regulating mitochondrial fusion. A previous study has shown27 that mitochondria are spheres or ovals and maintain a certain degree of fusion in Mfn2−/− MEF cells compared with that of Mfn1−/− MEF cells. Interestingly, Gβ2 shRNA could further fragment mitochondria, resulting in the size of mitochondria similar to that in Mfn1−/− MEFs. In contrast, it had little effect on the mitochondrial morphology in Mfn1−/− MEF cells (Fig. 4b,c). It is also interesting to note that Gβ2 localizes at the specific submitochondrial locations where mitochondria appear to have the tendency of branching along mitochondrial tubular structure (see Fig. 1a). Collectively, these data suggest that Gβ2 regulates the mitochondrial fusion, which requires Mfn1.
Gβ2 regulates the mobility of Mfn1 on the mitochondria
It is possible that Gβ2 regulates mitochondrial fusion by affecting the distribution of Mfn1 on the microregion of mitochondrial surface and thereby the membrane mobility of Mfn1. To test this hypothesis, we measured the Mfn1 membrane mobility in the presence or absence of Gβ2 using a FRAP assay. This technique provides a semiquantitative measurement of the movement of the mitochondrial proteins by assessing the depth of bleaching and rate of redistribution of mitochondrial outer membrane-localized yellow/green fluorescence protein (Mfn1-YFP was used in the assay (Supplementary Fig. S3a)) into regions of irreversible fluorophore photobleach28. Intriguingly, downregulation of Gβ2 by shRNA in HeLa cells caused an increase of Mfn1 mobility, whereas overexpression of Gβ2 caused a significant decrease in the mobility of Mfn1 (Fig. 4d,e). In addition, Gβ2 shRNA failed to increase the mobility of Mfn1 mutants (Δ241–350 and Δ1–387) that lack the interacting domain (Fig. 4f). This finding further supports our notion that their interaction is critical for the mobility of Mfn1 on the outer membrane surface of mitochondria. In contrast, the mobility of Mfn2 showed little change when Gβ2 was downregulated in HeLa cells (Supplementary Fig. S3b,c). Moreover, it was evidenced that Gβ2 failed to localize at the mitochondria in MEF Mfn1−/− cells (Supplementary Fig. S3d) unless Mfn1 was reintroduced into these cells (J.Z., unpublished observation). A fractionation assay further showed that Gβ2 levels were significantly reduced in Mfn1−/− cells (Supplementary Fig. S3e). These observations suggest that the interaction between Gβ2 and Mfn1 may help to regulate their submitochondrial distribution and the mobility of Mfn1, which may be important for determining the fusion or branching at particular submitochondrial regions.
Depletion of Gβ2 decreases mitochondrial fusion
To determine whether endogenous Gβ2 has a regulatory function in mitochondrial fusion, we used Gβ2 shRNA to deplete endogenous Gβ2 (Supplementary Fig. S4a). Interestingly, loss of Gβ2 resulted in a notable change in mitochondrial networking as indicated by mito-green fluorescent protein (GFP) labelling. The mitochondria became small tubules and spheres around the nuclear region, most likely due to impaired mitochondrial fusion (Fig. 5a). The effect of Gβ2 on mitochondria was specific, as reintroduction of Gβ2 into these cells significantly rescued the mitochondrial phenotypes (Fig. 5b,c). Mitochondrial dynamics was analysed by using an assay of visualization and quantification of mitochondrial fusion, based on confocal imaging of cells expressing a mitochondrial matrix-targeted photoactivable GFP (PAGFP) dilution rate29. Compared with the fast decrease of mito-PAGFP fluorescence intensity of activated mitochondria in normal HeLa cells observed 30 min after photoactivation, little change or no decrease in mito-PAGFP fluorescence within photoactivated mitochondria in HeLa cells with Gβ2 shRNA was seen (Fig. 5d–f), reflecting slower mitochondrial fusion rate. The result thus indicates that mitochondrial fusion was blocked by Gβ2 shRNA. In addition, overexpression of Flag-Gβ2 significantly caused aggregation of mitochondria around the nucleus in HeLa (Supplementary Fig. S4b, upper panel) and SH-SY5Y cells (not shown), as revealed by confocal microscopy. To further determine that Gβ2 in mitochondria regulates mitochondrial fusion, we constructed mitochondrial targeted Gβ2 using a specific mitochondrial outer membrane targeting signal peptide from Bcl-xL30. Overexpression of mitochondrial targeted Gβ2 induced profound mitochondrial aggregation at the perinuclear region in HeLa cells. In contrast, mitochondrial targeted Gγ2 or GFP protein using the same mitochondrial targeting signal had no effect on mitochondrial morphology (Supplementary Fig. S4b, middle and lower panel). Electron microscopy analysis further confirmed that the mitochondria clustered and aggregated around the perinuclear region when Gβ2 was ectopically expressed (Supplementary Fig. S4c).
The effects of Gβ2 on mitochondria are specific
We next determined if the effects of Gβ2 on mitochondria are specific. Overexpression of Gβ2 had little impact on the morphology and distribution of other cellular organelles such as Golgi, lysosomes or endoplasmic reticulum in HeLa cells (Fig. 6a). To determine the effect of Gβ2 on mitochondrial physiology and function, we measured mitochondrial membrane potential in the HeLa cells overexpressing Gβ2 by using TMRM (tetramethylrhodamine, methyl ester) staining and found that there was no significant difference from the parental cells (see Supplementary Fig. S4d). Furthermore, Gβ2 did not seem to have any effect on the cell cycle (see Supplementary Fig. S4e,f).
The surprising finding that Gβ2 has such a regulatory role in controlling mitochondrial fusion prompted us to test if other Gβ subunits and Gα or Gγ also have a role in mitochondria. We found that Gβ1 induced a small fraction (10%) of cells with mitochondrial aggregation, whereas other Gβ proteins failed to alter mitochondrial morphology (Fig. 6b). RACK1, a protein also named Gβ2-like protein 1 with WD40 repeat domains, could not induce mitochondrial aggregation in the cells (Fig. 6b). Gγ2 or its mutant form (C68S), which fails to localize at the membrane, did neither affect mitochondrial morphology nor the distribution of Gβ2 (Fig. 6c,d). Ectopic expression of Gα proteins, such as Gαi1 or Gαs, failed to induce the alteration of mitochondrial morphology or restore dysfunctioned network of mitochondria induced by Gβ2 knockdown (Fig. 7a). Mitochondrial aggregation was evident when cells were co-transfected with Gβ2 and Gγ2, or Gβ2 plus Gαil and Gγ2 (Fig. 7a,b), although the overall percentage of transfected cells with aggregated mitochondria was slightly reduced compared with that in cells expressing Gβ2 alone or Gβ2 together with Gαi1 (Fig. 7c). Gβ2 lacking the Gγ interacting domain is still able to induce the mitochondrial aggregation; even when it is coexpressed with Gγ2. Moreover, ectopic expression of Gγ2 failed to restore fragmented mitochondria induced by Gβ2 knockdown (Fig. 7d–f). Taken together, we demonstrate that the effect of Gβ2 on mitochondria is unique and Gγ2 has limited effect on the regulatory role of Gβ2 on mitochondrial fusion.
Discussion
Our results have demonstrated a novel role of Gβ2 in Mfn1-dependent mitochondrial fusion. We found that mitochondrial Gβ2 interacts with Mfn1 and regulates the mobility of Mfn1. It is possible that Gβ2, as a scaffold protein31, may function to mediate the clustering of Mfn1 molecules at the microregion of the mitochondrial outer membrane. As a result, Gβ2 may modulate the distribution and activity of Mfn1, which could be important in mitochondrial fusion or branching at specific submitochondrial sites. Intriguingly, several WD40 proteins were found to interact with Dnm1 (Drp1 homologue in yeast), a key protein in mediating fission, for mitochondrial fission13,32 and Caf4 regulates the polarized distribution of Dnm112. Thus, it is of great interest to note that distinct proteins with WD40 repeat domains are involved in both mitochondrial fusion and fission by regulating the submitochondrial distribution of proteins for these processes.
On the plasma membrane, Gβγ interacts with Gα and subsequently transduces signals to the downstream effectors. We found that G-protein-coupled receptor activators (ET1, PGE2) or antagonists (PTX), which are known to affect the dissociation/association of Gα and Gβγ, did not have any effect on mitochondrial networking or Gβ2-induced changes in mitochondrial networking (J.Z., unpublished observations). These data do not support that the activation of G-protein-coupled receptor signalling pathway or the translocation of Gβγ from the plasma membrane onto mitochondria are important for the altered mitochondrial networking observed. The combinatorial expression of Gαi1 and Gγ2 proteins has limited effects on Gβ2-induced mitochondrial aggregation (see Fig. 7). Mutant Gγ2-C68S, which fails to be modified by lipid and fails to localize to mitochondria, also did not affect mitochondrial morphology induced by enforced expression of Gβ2. Recently, it has been reported that Gα12 and Gαi1 are found to localize on the surface of mitochondria23,24. However, their role in mitochondria is largely unknown. We detected a weak interaction between Gβ2 with Gαi, but not with Gα12, although both are found in mitochondria. More studies are needed to further dissect the significant role of G proteins in regulating mitochondrial phenotypes and the interplay between mitochondria and other cellular events.
Previous studies have also shown that Gβγ interacts with dynein and tubulin33 to regulate microtubule reorganization and cell polarity18,19. Indeed, we have found that Gβ2 was able to interact with tubulin in co-immunoprecipitation analysis (data not shown). A Gβ protein was also found to interact with dynein intermediate chain, a member in the dynein family31. The interaction of Gβ2 with microtubules or motor proteins may provide an additional force for bringing mitochondria in proximity for mitochondrial tethering and docking, leading to an initiation of fusion.
Methods
Plasmid construction and mutagenesis
The human Mfn1 cDNA obtained from Dr Margaret Fuller (Stanford University, USA) was subcloned into a plasmid vector pECFP-N1 (Clontech) using EcoRI and BamHI. The Gβ2 gene was cloned by PCR from a human fetal liver cDNA library, and the Gβ2 cDNA was inserted into plasmid vectors pFLAG-CMV-4 (Sigma) and pEYFP-C1 (Clontech) to generate pFLAG-Gβ2 and pEYFP-Gβ2 using HindIII and EcoRI, respectively. The cDNA fragment encoding the Bcl-xL mitochondrial target peptide was amplified by PCR and fused to the C terminal of Gβ2 in pFLAG-Gβ2, named as Flag-Gβ2-M. The GFP10-GNG2 and pEGFP-GNB1 plasmids were from Dr Terence E. Hébert (McGill University, Canada). The Gγ2 cDNA was subcloned to pFLAG-CMV-4 and the mutant (C68S) of GFP10-Gγ2 was generated by site-directed mutagenesis. Flag-Gγ2-M was generated as described above. The GNB1 cDNA was subcloned to pEYFP-C1 and pFLAG-CMV-4 using HindIII and KpnI, respectively. The cDNA fragment encoding the mitochondrial matrix-localized signal peptide of TRX2 was subcloned to pEYFP-C1 and pEGFP-C1 using EcoRI and BamHI. The QuickChange site-directed mutagenesis kit was purchased from Stratagene. The GNB2 RNAi target sequences: CCTGGATGACAACCAAATC were designed by company (RuiBo) and subcloned to the RNAi-Ready pSIREN-RetroQ-DsRed-Express Vector (Clontech).
Antibodies and reagents
Antibodies to Gβ1, Gβ2, Gγ2, Gα12, Gαi1, RACK1, c-Myc, VDAC1 and LAMP3 were purchased from Santa Cruz, and the concentrations of them are 200 μg ml−1. Antibodies to Flag and α-tubulin were from Sigma, and the concentrations of both are 1 mg ml−1. Antibodies to Tom20, Tim23, GM130, Transferrin receptor and Calnexin were from Becton Dickinson, and the concentrations of them are 1 mg ml−1. Antibodies to Mfn1 and Mfn2 monoclonal were from Novus, the concentrations of both are 500 μg ml−1. The antibodies were diluted 1:100 of the original concentration for immunostaining and diluted 1:1,000–1:5,000 for western blotting. Mitotracker Red and Mitotracker Deep Red were purchased from Invitrogen.
Cell culture and transfections
Mfn1 and Mfn2 knockout, and Mfn1 and Mfn2 double knockout MEF cells were kind gifts from Dr David Chan (California Institute of Technology). HeLa and SH-SY5Y cells (American Type Culture Collection) were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100 U ml−1 penicillin and 100 U ml−1 streptomycin at 37 °C in a 5% CO2 incubator. Cells were transfected with plasmid DNAs by using a phosphate calcium method.
Confocal microscopy
HeLa and SH-SY5Y cells cultured on gelatin-coated coverslips were fixed with 3.7% paraformaldehyde, followed by permeabilization in 0.5% Triton X-100. Cells were incubated with primary antibodies in phosphate-buffered saline (PBS) supplemented with 5% bovine serum albumin, followed by incubation with appropriate secondary antibodies. To visualize mitochondria in living cells, cells were incubated with either MitoTracker Deep Red 633 or Red CMXRos (Invitrogen) at 37 °C for 30 min and washed twice with PBS. Images were visualized under an LSM 510 confocal microscope (Carl Zeiss), equipped with 63×/1.45 oil objective and processed using LSM Browser (Carl Zeiss) and Photoshop (Adobe).
Mitochondria fractionation
The mitochondrial membrane was purified on discontinuous sucrose gradients as previously described34, with some modifications. Briefly, cells were washed twice in PBS and resuspended in 1.5 ml buffer A (20 mM Tricine, 250 mM sucrose) containing protease inhibitors for 20 min on ice and then subjected to homogenization. After 20 strokes, the cell homogenate was collected and 100 μl of the samples were taken out as whole cell lysate (W). The remaining cell homogenate was centrifuged at 1,000×g for 8 min at 4 °C, and the supernatant was as the postnuclear solution (PNS). PNS (100 μl) was centrifuged at 10,000×g, and the supernant was collected as C2. PNS (2 ml) was layered on a Percoll gradient (30%) and centrifuged at 30,000×g for 30 min at 4 °C, a white band (1–2 ml) at 2 cm in Percoll gradient just below the PNS was collected as the plasma membrane fraction (P). P was continuingly centrifuged at 100,000×g for 1 h at 4 °C to remove the percoll. Another PNS (2 ml) was centrifuged at 10,000×g, and the pellet was washed twice with Buffer B (0.225 M mannitol, 1 mM EGTA, 25 mM HEPES, pH 7.4) and resuspended in 0.5 ml Buffer B. The suspension was layered to a Percoll density gradient (2 ml 80%, 4.5 ml 52% and 4.25 ml 26%) and centrifuged at 100,000×g for 1 h at 4 °C. The band between the Percoll 26% and 52% gradient was collected as the pure mitochondrial fractionation (M). C1 was the supernatant obtained after the cell homogenate was centrifuged at 250,000×g for 1 h at 4 °C.
Mitochondria membrane motility assay
The mitochondria membrane motility was measured as previous described28. Briefly, circular region of interests (ROIs), 2.5 mm in diameter, were imaged (a-Plan-FLUAR 100×/1.45 oil objective; Carl Zeiss) before and after photobleaching (20 iterations of 514-nm laser (488-nm laser for GFP) set to 100%) of 0.5-mm circular ROIs located in the centre of imaging area. Totally, 150 images (1 scan every 33 ms) were collected and the fluorescence intensity was digitalized in imaged ROIs with an LSM 510 software (Zeiss MicroImaging).
FRET assay
All FRET observations were performed under a Leica DM IRE2 confocal laser scanning microscope, after cells were transfected for 24 h. The donor (CFP) was excited at 458 nm, and its fluorescence was detected in a wavelength of 478–498 nm (CFP channel), whereas the excitation at 514 nm and the emission at 545±15 nm were used for detecting the acceptor (YFP) (YFP channel). FRET was detected at the excitation of 458 nm and the emission of 545±15 nm (FRET channel). Fluorescence images of the transfected cells were taken at the CFP-, YFP- and FRET channels sequentially. Dequenching of the donor fluorescence by photobleaching of the acceptor YFP was performed by illuminating the transfected cells at the YFP excitation wavelength (514 nm) for 250 iterations, and then the CFP-Mfn1 images were taken at the same focal plane. The FRET efficiency was calculated using the equation: E=1−(FDA/FD), where FDA and FD are the fluorescence intensity of CFP in the cells expressing both donor and acceptor, and donor alone (acceptor was quenched), respectively26.
Immunoprecipitation
Cells were collected and washed twice with PBS, and then resuspended in lysis buffer (0.75 M aminocaproic acid, 50 mM Bis-Tris, pH 7.0, 1.5% n-dodecyl-b-d-maltopyranoside, 1 mM phenylmethyl sulphonyl fluoride, 1 mg ml−1 leutpeptin and 1 mg ml−1 pepstatin) for 30 min on ice, and, then subjected to centrifugation at 72,000 g for 20 min at 4 °C. The primary monoclonal antibody (2 μg) was added into the supernatant containing 500 μg proteins, and incubated at 4 °C overnight. After incubation with Protein G-agarose (Santa Cruz Biotech) for 2 h at 4 °C, the beads were washed three times with the lysis buffer and boiled with 6× loading buffer for 5 min. The proteins were then separated by SDS–polyacrylamide gel electrophoresis, and western blotting was performed with proper antibodies as indicated.
Mitochondrial membrane potential assay
The mitochondrial membrane potential was measured. Briefly, HeLa cells were transfected with an empty vector and pFlag-Gβ2. After 24 h, the cells were collected and stained with 200 nM TMRM (Invitrogen) for 15 min at room temperature, and then kept at 4 °C for measurement after washing once with PBS. For each sample, 50,000 cells were scored after excitation with a 488 nm argon laser and emission through the phycoerythrin filter (575 nm) of a flow cytometer (Becton Dickinson). The percentile of cells above and below the threshold TMRM fluorescence signal was determined with Cellquest software.
Cell cycle analysis
Cells were collected and washed and then resuspended in PBS. The cell suspension containing 1×106 cells in 500 μl was fixed by adding 5 ml cold (−20 °C) 75% ethanol at 4 °C overnight. The cells were centrifuged at 1,000×g for 10 min at 4 °C and washed twice with 5 ml PBS plus 1% bovine serum albumin, and resuspended in 400 μl PBS supplemented with 1% bovine serum albumin, 50 μl 500 μg ml−1 propidium iodide and 50 μl boiled RNase A (10 mg ml−1). After incubating at 37 °C for 30 min, the cells were analysed by FACS using an FL2 histogram at 620 nm (Becton Dickinson).
Photoactivation assay
Cells were grown in two-well chambers for confocal microscopy. Images were captured with a microscope (model LSM 510; Carl Zeiss MicroImaging) using a 63×1.4 NA Apochromat objective (Carl Zeiss MicroImaging). Light (405- or 413-nm) was used for photoactivation of PAGFP. ROIs were selected and series of z-sections from the top to the cell bottom with intervals between sections set to 0.5–0.75 μm were irradiated with 405- or 413-nm light. The same intervals between optical sections were used for imaging.
Additional information
How to cite this article: Zhang, J. et al. G-protein β2 subunit interacts with mitofusin 1 to regulate mitochondrial fusion. Nat. Commun. 1:101 doi: 10.1038/ncomms1099 (2010).
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Acknowledgements
We thank Drs Aimin Zhou from Cleveland State University, Shencai Lin from Xiamen University, Heping Cheng from Peking University and Junjie Hu from Nankai University for their constructive suggestions and critical reading of the manuscript. We wish to thank Professor Xun Shen for his advice on the FRET assay. We also thank all laboratory members for their discussions and comments. We would like to appreciate Dr Margerate Fuller (Stanford University) for the Mfn plasmids, and Dr David Chan (California Institute of Technology) for Mfn knockout cell lines. The Chen Laboratory is supported by a key project from the Nature Science Foundation of China (no. 30630038, 90713006) and the National Proprietary Basic Research Program (973 program project, Grant nos. 2007CB914800, 2011CB910903).
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J.Z. performed all the experiments. W.L. helped with the immunoprecipitation assay, and J.L. helped with constructs. W.X helped with electron microscopy experiment, and X.S. assisted with mitochondrial fractionation assay. L.L and C.J helped in data discussion. Q.C. oversaw the project, designed the experiments and wrote the manuscript.
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Zhang, J., Liu, W., Liu, J. et al. G-protein β2 subunit interacts with mitofusin 1 to regulate mitochondrial fusion. Nat Commun 1, 101 (2010). https://doi.org/10.1038/ncomms1099
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DOI: https://doi.org/10.1038/ncomms1099
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