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Mitochondrial Dynamics in Development and Disease
Summary: David Chan studies how the dynamic fusion and fission of mitochondria within a cell are controlled, how they affect a cell's physiology, and how they play a role in human disease.
Mitochondria are dynamic organelles that continually undergo fusion, fission, and trafficking (Movie 1). These fundamental cell biological processes control mitochondrial shape, number, size, distribution, and physiology. We study the molecular basis for these dynamic properties and their impact on development and disease.
Molecular Mechanisms of Mitochondrial Dynamics
Mitochondrial fusion. Mitochondrial fusion is a membrane-remodeling process that coordinately merges the outer and inner membranes between two mitochondria. It requires three large GTPases: the mitofusins Mfn1 and Mfn2, and the dynamin-related protein OPA1 (Figure 1). Mitofusins are embedded in the mitochondrial outer membrane, whereas OPA1 is associated with the inner membrane. One of our main efforts is to understand how mitofusins and OPA1 mediate fusion of the mitochondrial outer and inner membranes. We showed previously that mitofusins are needed in trans (on adjacent mitochondria) to promote fusion. Our crystallographic studies of Mfn1 suggested that mitofusins are involved in tethering of mitochondria during an early phase of fusion (Figure 2). Current studies are focused on how the mitofusin structure is modified to force full membrane merger. We have shown that mitofusins and OPA1 act at distinct steps during mitochondrial fusion: mitofusins are essential for outer membrane fusion, whereas OPA1 is required for inner membrane fusion. Future studies will use biochemical and structural approaches to understand the underlying mechanism.
Mitochondrial fission. Mitochondrial fission requires the recruitment and assembly of the dynamin-related GTPase Dnm1/Drp1, which constricts the diameter of mitochondria (Figure 1). We are examining how Dnm1/Drp1 is recruited to the mitochondrial surface. The resolution of this issue is important, because Drp1 recruitment in mammalian cells is an early apoptotic event necessary for the efficient execution of death pathways. Using affinity purification and mass spectrometry, we identified Caf4 as a new component of the yeast mitochondrial fission machinery. We discovered that the mitochondrial outer membrane protein Fis1 uses the molecular adapters Mdv1 and Caf4 to mediate Dnm1/Drp1 recruitment to mitochondria. We have reconstituted portions of these fission complexes in vitro and have used x-ray crystallography to determine the structure of Fis1 in complex with either Mdv1 or Caf4. To complement these structural studies, we have yeast and mammalian cellular systems in place to conduct functional experiments addressing mechanism. Our long-term goals are to understand the atomic structure of the fission complexes, to determine how they are activated for fission, and to identify currently unknown members of the mammalian fission machinery. Because the endpoint of fission apparatus assembly is activation of Dnm1/Drp1 activity, our studies may provide general insights into dynamin-family proteins, which have membrane-shaping properties that are involved in diverse intracellular trafficking events.
Cellular Functions of Mitochondrial Dynamics
At the cellular level, a major issue is why cells maintain their mitochondria in a dynamic state. One clearly established reason is that a dynamic equilibrium between fusion and fission regulates the morphology of mitochondria. Cells do not, however, require high levels of fusion and fission to maintain tubular mitochondria, suggesting that there are additional reasons why mitochondria are such dynamic organelles.
We found that mitochondrial fusion is necessary to maintain uniform function within a population of mitochondria. Fusion-deficient cells have greatly diminished respiratory capacity and reduced cell growth. In addition, the mitochondrial population shows heterogeneous properties, including wide variations in membrane potential. Based on these observations, we proposed that mitochondria do not function well as autonomous organelles and that the dynamic property of mitochondria is inherently important for organelle integrity. In normal cells, high rates of fusion and fission enable mitochondria to cooperate with each other through continual exchange of contents. Individual mitochondria can stochastically lose essential components, but such defects are short-lived, because mitochondrial fusion will restore the missing components from neighboring mitochondria. In cells lacking mitochondrial fusion, such restoration of activity cannot occur, and defective mitochondria accumulate. We have recently discovered that, in the absence of fusion, a large population of mitochondria lack mitochondrial DNA (mtDNA) nucleoids. Therefore, mitochondrial fusion is essential to allow defective mitochondria a pathway to recover mtDNA. This defect explains the respiratory and membrane potential aberrations found in fusion-deficient cells.
Although the machineries mediating mitochondrial fusion and fission are being elucidated, little is known about how mitochondrial dynamics is regulated. An understanding of this regulation is important, given the dramatic changes in mitochondrial dynamics that occur with cellular stress, activity, and apoptosis. We are using biochemical and genetic approaches to identify molecules that regulate the activity of the mitochondrial fusion and fission complexes in response to these changes in cellular physiology.
The Role of Mitochondrial Dynamics in Human Disease
Human genetic studies indicate that neurons are particularly sensitive to defects in mitochondrial dynamics. Mutations in the mitochondrial fusion genes Mfn2 and OPA1 cause the neurodegenerative diseases Charcot-Marie-Tooth type 2A and dominant optic atrophy. Both diseases are caused by selective loss of a specific class of neurons. One of our major goals, therefore, is to identify the cellular mechanisms leading to neurodegeneration when mitochondrial fusion is perturbed.
To address this issue, we have generated mice containing conditional null alleles of Mfn1 and Mfn2. Animals lacking Mfn2 have severe cerebellar ataxia. We found that Mfn2 protects against neurodegeneration in both developing and mature Purkinje neurons in the cerebellum. Purkinje cells have extensive dendritic arbors, and normally their dendritic processes are filled with abundant, tubular mitochondria. In the absence of Mfn2, however, Purkinje cells show poor dendritic arborization, few dendritic spines, and ultimately cell degeneration (Figure 3). We have traced this defect to mitochondrial fragmentation, respiratory dysfunction, and improper mitochondrial distribution within the dendrites. The respiratory defect likely results from loss of mtDNA nucleoids from a subset of mitochondria, as discussed above. Similar progressive defects in mutant Purkinje cells are recapitulated in cerebellar cell cultures. These results indicate that dendritic outgrowth is highly dependent on mitochondrial dynamics. With these animal and cell culture models, we can now further dissect the mechanistic link between mitochondrial dynamics and neurodegenerative disease. In related mouse models, we have uncovered an important role of mitochondrial fusion in skeletal muscle function.
The Natural History of mtDNA Mutations and Aging
Although the mitochondrial genome is small, a remarkable number of human disorders are related to mutations in mtDNA. For example, most mitochondrial encephalomyopathies are caused by mutations in mtDNA. A characteristic feature of these diseases is their progressive nature. This age-related progression has been attributed to the stochastic nature of mtDNA inheritance during cell division. As the load of mutant mtDNA increases, the bioenergetic threshold of specific cells is breached, and cellular dysfunction ensues.
Accumulation of mutations in mtDNA may also be involved in aging. Many studies have documented a progressive age-related decline in respiratory function in muscle and brain. For example, aged individuals have clonal mtDNA deletions and respiratory loss in individual substantia nigra neurons, a finding that has implications for Parkinson's disease.
Because aging is associated with accumulation of mutations in mtDNA, mitochondrial dynamics may be a protective factor in the aging process. The merging of mitochondria has major implications for the inheritance of mtDNA mutations, which coexist with wild-type mtDNA. Mitochondrial fusion premixes mutant and wild-type mtDNA prior to segregation of mitochondria during cell division. This premixing buffers the effects of mtDNA mutations within a cell and minimizes the genetic drift that can skew the proportion of mutant mtDNA in daughter cells (Figure 4). In cells that lack mitochondrial fusion, the mitochondria are forced to be autonomous organelles, and they would be more prone to the detrimental effects of mtDNA mutations. We are exploring these issues in mouse and cellular models.
This work is supported in part by grants from the National Institutes of Health and the Ellison Medical Foundation.
Last updated December 18, 2009
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