Mitochondrial Fusion in Human Cells Is Efficient, Requires the Inner Membrane Potential, and Is Mediated by Mitofusins

TLDR

Mitochondrial fusion remains largely unknown despite its observation by live microscopy and the identification of only a few implicated proteins. Human mitochondrial fusion is highly efficient, achieving complete matrix mixing within 12 h, persists without a functional respiratory chain or microtubules, is blocked by loss of inner membrane potential (reversible upon restoration), and is regulated by DRP1‑mediated fission and Mfn1‑driven fusion that requires the membrane potential, underscoring that mitochondria form a single, dynamically balanced compartment.

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.