Biophysical Properties of Mitochondria Undergoing Fusion

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Emerging evidence shows the importance of genes controlling mitochondrial fusion in physiology and their deregulation in neurodegenerative and metabolic disorders. However, apart from ΔΨm, the biophysical properties of the fusion-competent mitochondria remain elusive. To evaluate the conditions of contact formation and fusion, we used organelle-targeted fluorescent proteins, including photoactivatable GFP, which allow tracking of individual mitochondria. In H9c2 cells, almost every mitochondrion is aligned with microtubules that provide the primary tracks for mitochondrial movement. Our results show that ∼90% of fusion events involve moving mitochondria. However, fusion occurs irrespective of mitochondrial speed. Furthermore, ∼80% of fusion events involve the tip portion of the mitochondrion, whereas only ∼50% of these events involve organelle side. Nocodazol, a microtubule disrupting agent that inhibits mitochondrial movements decreases the fusion frequency and changes mitochondrial fusion sites. Strikingly, 80-90% of the physical contacts between adjacent mitochondria did not result in fusion events. To evaluate whether the fusion efficacy depends on the spacing between the outer and inner mitochondrial membranes we used drugs that alter the matrix volume. Valinomycin, a K+ ionophore that induces matrix swelling evoked a decrease in mitochondrial motility leading to fewer contacts among mitochondria but the number of fusion events was maintained, indicating an increase in fusion efficacy. This change occurred at a low valinomycin concentration (0.25nM) that did not affect ΔΨm or Opa1 cleavage. Nigericin (0.5μM), a K+/H+ ionophore that induces shrinkage of the matrix elicited fusion inhibition and mitochondrial aggregation. Importantly, no motility inhibition or Opa1 cleavage occurred at the same time and the ΔΨm was increased. These results suggest that mitochondrial fusion is facilitated by mitochondrial motility, the key determinant of the inter-mitochondrial encounter numbers and preferentially involves the front-tip of the moving organelle. In addition, fusion efficacy depends on the mitochondrial matrix volume.