Activated Mitofusin 2 Signals Mitochondrial Fusion, Interferes with Bax Activation, and Reduces Susceptibility to Radical Induced Depolarization

Abstract

Mitochondrial fusion in higher eukaryotes requires at least two essential GTPases, Mitofusin 1 and Mitofusin 2 (Mfn2). We have created an activated mutant of Mfn2, which shows increased rates of nucleotide exchange and decreased rates of hydrolysis relative to wild type Mfn2. Mitochondrial fusion is stimulated dramatically within heterokaryons expressing this mutant, demonstrating that hydrolysis is not requisite for the fusion event, and supporting a role for Mfn2 as a signaling GTPase. Although steady-state mitochondrial fusion required the conserved intermembrane space tryptophan residue, this requirement was overcome within the context of the hydrolysis-deficient mutant. Furthermore, the punctate localization of Mfn2 is lost in the dominant active mutants, indicating that these sites are functionally controlled by changes in the nucleotide state of Mfn2. Upon staurosporine-stimulated cell death, activated Bax is recruited to the Mfn2-containing puncta; however, Bax activation and cytochrome c release are inhibited in the presence of the dominant active mutants of Mfn2. The dominant active form of Mfn2 also protected the mitochondria against free radicalinduced permeability transition. In contrast to staurosporine-induced outer membrane permeability transition, pore opening induced through the introduction of free radicals was dependent upon the conserved intermembrane space residue. This is the first evidence that Mfn2 is a signaling GTPase regulating mitochondrial fusion and that the nucleotidedependent activation of Mfn2 concomitantly protects the organelle from permeability transition. The data provide new insights into the critical relationship between mitochondrial membrane dynamics and programmed cell death.

Mitochondrial dynamics in the regulation of neuronal cell death

“…This would suggest that Mfn1 plays a more essential role in mediating fusion than Mfn2 or they may regulate fusion signaled from different pathways. While both Mfn1 and Mfn2 are important for mitochondrial fusion, a hydrolysis deficient form of Mfn2 was shown to stimulate mitochondrial fusion in cultured cells, and protected cells from apoptosis induced by staurosporine [79], which indicated additional regulatory functions for Mfn2. These suggest that apart from the fusion role, Mfn2 may also have additional functions that may impact the survival of cells.…”

“…Fusion is thought to protect mitochondrial function by allowing rapid mixing of membranes, mitochondrial DNA and soluble contents that may be damaged by localized deficits of substrates as a result of cellular stress [35]. Recently, several studies indicated that during cell death mitochondrial fusion is inhibited and activation of the fusion machinery could slow down the rate of cell death [29,69,79]. There are, however, some very significant differences in the action of the fusion proteins in the regulation of cell death.…”

“…Interestingly, Drp1, Bax and Mfn2 have been shown to colocalize into discrete foci during apoptosis, suggesting that Mfn2 may be sequestered and inhibited during apoptosis [33]. Increased Mfn2 activity can inhibit Bax activation and cytochrome c release [69,79], whereas the lack of Mfn2 activity sensitize cells to apoptotic stimuli [69]. In neuronal system, Barsoum and colleagues demonstrated that overexpression of Mfn1 can protect against NO induced neuronal cell death.…”

A cut short to death: Parl and Opa1 in the regulation of mitochondrial morphology and apoptosis

“…The notion that mitochondrial fission is a step towards death is reinforced by the ability of overexpressed pro-fusion Mfn1 and Mfn2 to block apoptosis induced by stimuli that recruit the mitochondrial pathway, perhaps by interfering with Bax activation. 56,57 An open question is why increased fission accelerates cell death. A unifying model proposed by Shore and co-workers 58 implies cristae remodelling downstream of Drp1 activation, which in turn depends on mitochondrial Ca 2 þ uptake induced by the BH3-only molecule BIK.…”

Life cell quantification of mitochondrial membrane potential at the single organelle level

“…Continuous illumination of TMRM can trigger singlet oxygen ( 1 O 2 ) generation and thereby lead to photodynamic damage, opening of the mitochondrial permeability transition pore (PTP), and mitochondrial depolarization (11,(21)(22)(23)(24). Additionally, TMRM photobleaching during image acquisi- ORIGINAL ARTICLE tion might reduce the fluorescence signal in a Dw-independent manner.…”