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Technological improvements in microscopy and the development of mitochondria-specific imaging molecular tools have illuminated the dynamic rearrangements of these essential organelles. These rearrangements are mainly the result of two opposing processes: mitochondrial fusion and mitochondrial fission. Consistent with this, in addition to mitochondrial motility, these two processes are major factors determining the overall degree of continuity of the mitochondrial network, as well as the average size of mitochondria within the cell. In this chapter, we detail the use of advanced confocal microscopy and mitochondrial matrix-targeted photoactivatable green fluorescent protein (mito-PAGFP) for the investigation of mitochondrial dynamics. We focus on direct visualization and quantification of mitochondrial fusion and mitochondrial network complexity in living mammalian cells. These assays were instrumental in important recent discoveries within the field of mitochondrial biology, including the role of mitochondrial fusion in the activation of mitochondrial steps in apoptosis, participation of Bcl-2 family proteins in mitochondrial morphogenesis, and stress-induced mitochondrial hyperfusion. We present some basic directions that should be helpful in designing mito-PAGFP-based experiments. Furthermore, since analyses of mitochondrial fusion using mito-PAGFP-based assays rely on time-lapse imaging, critical parameters of time-lapse microscopy and cell preparation are also discussed.

MCL-1, an anti-apoptotic BCL-2 family member that is essential for the survival of multiple cell lineages, is also among the most highly amplified genes in cancer. Although MCL-1 is known to oppose cell death, precisely how it functions to promote survival of normal and malignant cells is poorly understood. Here, we report that different forms of MCL-1 reside in distinct mitochondrial locations and exhibit separable functions. On the outer mitochondrial membrane, an MCL-1 isoform acts like other anti-apoptotic BCL-2 molecules to antagonize apoptosis, whereas an amino-terminally truncated isoform of MCL-1 that is imported into the mitochondrial matrix is necessary to facilitate normal mitochondrial fusion, ATP production, membrane potential, respiration, cristae ultrastructure and maintenance of oligomeric ATP synthase. Our results provide insight into how the surprisingly diverse salutary functions of MCL-1 may control the survival of both normal and cancer cells.

The Bcl-2 family member Bax translocates from the cytosol to mitochondria, where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by prosurvival Bcl-2 proteins, but how they inhibit Bax remains controversial because they neither colocalize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-x(L) in detergents and cell-free MOMP activity but unexpectedly induce Bax accumulation on mitochondria. Fluorescence loss in photobleaching (FLIP) reveals constant retrotranslocation of WT Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on prosurvival Bcl-2 family proteins, and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-x(L) inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In mammals, fusion of the mitochondrial outer membrane is controlled by two DRPs, MFN1 and MFN2, that function in place of a single outer membrane DRP, Fzo1 in yeast. We addressed the significance of two mammalian outer membrane fusion DRPs using an in vitro mammalian mitochondrial fusion assay. We demonstrate that heterotypic MFN1-MFN2 trans complexes possess greater efficacy in fusion as compared to homotypic MFN1 or MFN2 complexes. In addition, we show that the soluble form of the proapoptotic Bcl2 protein, Bax, positively regulates mitochondrial fusion exclusively through homotypic MFN2 trans complexes. Together, these data demonstrate functional and regulatory distinctions between MFN1 and MFN2 and provide insight into their unique physiological roles., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Damage to mitochondria can lead to the depolarization of the inner mitochondrial membrane, thereby sensitizing impaired mitochondria for selective elimination by autophagy. However, fusion of uncoupled mitochondria with polarized mitochondria can compensate for damage, reverse membrane depolarization, and obviate mitophagy. Parkin, an E3 ubiquitin ligase that is mutated in monogenic forms of Parkinson's disease, was recently found to induce selective autophagy of damaged mitochondria. Here we show that ubiquitination of mitofusins Mfn1 and Mfn2, large GTPases that mediate mitochondrial fusion, is induced by Parkin upon membrane depolarization and leads to their degradation in a proteasome- and p97-dependent manner. p97, a AAA+ ATPase, accumulates on mitochondria upon uncoupling of Parkin-expressing cells, and both p97 and proteasome activity are required for Parkin-mediated mitophagy. After mitochondrial fission upon depolarization, Parkin prevents or delays refusion of mitochondria, likely by the elimination of mitofusins. Inhibition of Drp1-mediated mitochondrial fission, the proteasome, or p97 prevents Parkin-induced mitophagy.

The cytoplasmic dynamin-related guanosine triphosphatase Drp1 is recruited to mitochondria and mediates mitochondrial fission. Although the mitochondrial outer membrane (MOM) protein Fis1 is thought to be a Drp1 receptor, this has not been confirmed. To analyze the mechanism of Drp1 recruitment, we manipulated the expression of mitochondrial fission and fusion proteins and demonstrated that (a) mitochondrial fission factor (Mff) knockdown released the Drp1 foci from the MOM accompanied by network extension, whereas Mff overexpression stimulated mitochondrial recruitment of Drp1 accompanied by mitochondrial fission; (b) Mff-dependent mitochondrial fission proceeded independent of Fis1; (c) a Mff mutant with the plasma membrane-targeted CAAX motif directed Drp1 to the target membrane; (d) Mff and Drp1 physically interacted in vitro and in vivo; (e) exogenous stimuli-induced mitochondrial fission and apoptosis were compromised by knockdown of Drp1 and Mff but not Fis1; and (f) conditional knockout of Fis1 in colon carcinoma cells revealed that it is dispensable for mitochondrial fission. Thus, Mff functions as an essential factor in mitochondrial recruitment of Drp1.

PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

Bcl-2 family proteins are potent regulators of programmed cell death. Although their intracellular localization to mitochondria and the endoplasmic reticulum has focused research on these organelles, how they function remains unknown. Two members of the Bcl-2 family, Bax and Bak, change intracellular location early in the promotion of apoptosis to concentrate in focal clusters at sites of mitochondrial division. Here we report that in healthy cells Bax or Bak is required for normal fusion of mitochondria into elongated tubules. Bax seems to induce mitochondrial fusion by activating assembly of the large GTPase Mfn2 and changing its submitochondrial distribution and membrane mobility-properties that correlate with different GTP-bound states of Mfn2. Our results show that Bax and Bak regulate mitochondrial dynamics in healthy cells and indicate that Bcl-2 family members may also regulate apoptosis through organelle morphogenesis machineries.

Summary: The Bcl-2 family member Bax translocates from the cytosol to mitochondria, where it oligomerizes and permeabilizes the mitochondrial outer membrane to promote apoptosis. Bax activity is counteracted by prosurvival Bcl-2 proteins, but how they inhibit Bax remains controversial because they neither colocalize nor form stable complexes with Bax. We constrained Bax in its native cytosolic conformation within cells using intramolecular disulfide tethers. Bax tethers disrupt interaction with Bcl-xL in detergents and cell-free MOMP activity but unexpectedly induce Bax accumulation on mitochondria. Fluorescence loss in photobleaching (FLIP) reveals constant retrotranslocation of WT Bax, but not tethered Bax, from the mitochondria into the cytoplasm of healthy cells. Bax retrotranslocation depends on prosurvival Bcl-2 family proteins, and inhibition of retrotranslocation correlates with Bax accumulation on the mitochondria. We propose that Bcl-xL inhibits and maintains Bax in the cytosol by constant retrotranslocation of mitochondrial Bax. [ABSTRACT FROM AUTHOR]