Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins - PubMed (original) (raw)
Mitochondrial fusion in human cells is efficient, requires the inner membrane potential, and is mediated by mitofusins
Frédéric Legros et al. Mol Biol Cell. 2002 Dec.
Abstract
Mitochondrial fusion remains a largely unknown process despite its observation by live microscopy and the identification of few implicated proteins. Using green and red fluorescent proteins targeted to the mitochondrial matrix, we show that mitochondrial fusion in human cells is efficient and achieves complete mixing of matrix contents within 12 h. This process is maintained in the absence of a functional respiratory chain, despite disruption of microtubules or after significant reduction of cellular ATP levels. In contrast, mitochondrial fusion is completely inhibited by protonophores that dissipate the inner membrane potential. This inhibition, which results in rapid fragmentation of mitochondrial filaments, is reversible: small and punctate mitochondria fuse to reform elongated and interconnected ones upon withdrawal of protonophores. Expression of wild-type or dominant-negative dynamin-related protein 1 showed that fragmentation is due to dynamin-related protein 1-mediated mitochondrial division. On the other hand, expression of mitofusin 1 (Mfn1), one of the human Fzo homologues, increased mitochondrial length and interconnectivity. This process, but not Mfn1 targeting, was dependent on the inner membrane potential, indicating that overexpressed Mfn1 stimulates fusion. These results show that human mitochondria represent a single cellular compartment whose exchanges and interconnectivity are dynamically regulated by the balance between continuous fusion and fission reactions.
Figures
Figure 1
Mitochondria exchange soluble matrix proteins by fusion. Differently labeled HeLa cells (A–C) or 143B-ρ0 cells (D–F) were coplated and fused with PEG in the presence of cycloheximide and fixed after further 2 h (A), 4 h (B–E), or 16 h (F). C and E are enlargements of the areas boxed in B and D, respectively. Asterisks indicate the position of polykaryon nuclei and arrowheads indicate the position of single-labeled mitochondria. The presence of double-labeled mitochondria (yellow) demonstrates the exchange of fluorescent matrix proteins by fusion. (A–E) Presence of mitochondria with a single label, even in proximity to differently labeled mitochondria, indicates that mitochondrial fusion events are not completed within 2 h (A) or 4 h (B–E). The different distribution of mtGFP and mtRFP within the double-labeled mitochondrial compartment (B) indicates that their diffusion is still ongoing. (F) Homogeneous double labeling of mitochondria indicates that fusion and diffusion have reached equilibrium 16 h after cytoplasmic fusion. Bars, 30 μm.
Figure 2
Mitochondrial fusion achieves complete mixing of matrix contents. Heterokaryons obtained as described in Figure 1 were fixed at different times after PEG-mediated cytoplasmic fusion. For each time point, the percentage of area with double-labeled mitochondria was estimated in 20–30 polykaryons. The mean values and the SEs are plotted as a function of time. The percentage of double labeling increases with time and is predicted to reach 100% 10–12 h after cytoplasmic fusion. The kinetics of fusion is similar for respiration-competent HeLa cells (squares, r = 0.95) and for respiration-deficient 143B-ρ0 cells (triangles, r = 0.98).
Figure 3
Inhibitor-mediated modulation of cellular ATP and of the ΔΨm. (A) HeLa cells treated with the indicated drugs for 45 min were trypsinized and subjected to measurement of ATP content and of ΔΨm-dependent fluorescence of JC-1 aggregates. Values were normalized to equal amounts of cellular protein and are expressed as percentage of control. Mean values and SEs are derived from three independent experiments. All treatments lead to a variable reduction of cellular ATP, but only cccp induces a significant reduction of the ΔΨm-dependent fluorescence of JC-1 aggregates. (B) HeLa cells treated with the indicated drugs for 3 h were fixed and visualized by phase contrast microscopy. Cellular morphology seems normal upon treatment with oligomycin or cccp, but cells retract upon addition of deoxyglucose. Cells have detached from the substrate after combined treatment with oligomycin and deoxyglucose, and none of these treatments provokes the apoptotic morphology induced by staurosporine.
Figure 4
ΔΨm is necessary for mitochondrial fusion. Differently labeled 143B cells (A) or HeLa cells (B) were coplated and fused in the presence of 10 μM cccp (A) or 2.5 μM oligomycin (B) and cells were fixed 4 h after cell fusion. Drugs were added to the culture medium 30 min before PEG-mediated cell fusion and kept throughout the experiment. Enlargements of the boxed areas are shown separately. (A) In the presence of cccp, mitochondria loose their tubular morphology and appear as punctate structures. Despite thorough intermixing (overlay, box1) and high mobility (overlay, box 2), mitochondria remain single labeled. Postfixation labeling of the plasma membrane with WGA confirms polykaryon formation and demonstrates the absence of intermitochondrial fusion. (B) Mitochondrial filaments are shorter or fragmented in the presence of oligomycin. The appearance of double-labeled mitochondria (overlay, box) demonstrates mitochondrial fusion. Bars, 30 μm.
Figure 5
Inhibition of mitochondrial fusion by dissipation of ΔΨm triggers fragmentation of mitochondrial filaments. Cells expressing mtRFP were treated for 4 h with the indicated drugs, fixed, and incubated with antibodies against tubulin. Treatment with cccp leads to mitochondrial fragmentation but not to disruption of microtubules. Microtubule depolymerization with nocodazole provokes disorganization of the mitochondrial network, but not to fragmentation of tubular mitochondria.
Figure 6
Inhibition of fusion triggers Drp1-mediated mitochondrial fragmentation and is reversed upon repolarization of the inner membrane. (A and B) HeLa cells were cotransfected with plasmids expressing mtGFP and either wild-type Drp1 (A) or dominant-negative Drp1K38A (B) as described in MATERIALS AND METHODS. After 36 h, cells were fixed under control conditions or after a 4-h treatment with cccp or oligomycin. (A) Expression of wild-type Drp1 does not modify mitochondrial morphology and dynamics: mitochondria remain tubular under control conditions and fragment upon addition of cccp or oligomycin. (B) Expression of dominant-negative Drp1K38A prevents drug-induced fragmentation of mitochondrial filaments. (C) HeLa cells expressing mtRFP were treated for 4 h with cccp to fragment mitochondria. Cells were then transferred to cccp-free medium (control) or to cccp-free medium containing nocodazole. Fragmented mitochondria fuse and reassemble into filaments in the presence (control) and absence (nocodazole) of microtubules.
Figure 7
Inner membrane potential is required for the stimulation of mitochondrial fusion by excess mitofusin 1. (A) Control mitochondrial morphology in mtGFP-transfected HeLa cells and in untransfected COS7 cells (Hsp60). (B) Transfection of myc-tagged Mfn1 (mMfn1) induces the appearance of highly elongated and frequently branched mitochondria. Long mitochondrial filaments grow out of perinuclear bundles of mitochondria and mitochondria become clustered at high expression levels (arrowhead). (C) On cotransfection, myc-tagged Mfn1 and untagged Mfn2 colocalize on elongated and branched mitochondria as well as on perinuclear mitochondrial clusters. (D) Myc-tagged Mfn1 is targeted to mitochondria in the presence of cccp, clusters mitochondria at high expression levels (arrowheads), but does not increase mitochondrial length and interconnectivity. The treatment with cccp started before expression of exogenous Mfn1 (9 h after transfection) and lasted for 31 h. (E) Elongated and branched mitochondria are fragmented in Mfn1-transfected cells treated with cccp. The treatment with cccp started after overexpression of exogenous Mfn1 (36 h after transfection) and lasted for 4 h.
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