Prashant Mishra Laboratory

prashant-mishra-lab

Mitochondrial Dynamics and Metabolic Homeostasis

Mitochondria play a central role in cellular metabolism. In addition to their ability to produce ATP from numerous fuel sources, mitochondria also participate in many biochemical pathways ranging from amino acid metabolism to Fe-S cluster assembly to calcium handling (to name a few). In addition to these biochemical processes, mitochondria also exhibit interesting macroscopic behaviors, including fusion (the joining of two organelles into one), fission (or division), active transport along cytoskeletal elements, and mitophagy (or targeted destruction). Together, these gross behaviors are termed mitochondrial dynamics.

Video 1. Mitochondria are dynamic organelles.
Video 1. Mitochondria are dynamic organelles.
In this live cell video, mitochondria are labeled with a fluorescent protein (DsRed) and can be tracked over time. Dynamic behaviors including movement (transport), fusion and fission (division) can be observed. For instance, two mitochondria are labeled with green (using photoactivatable GFP), and fuse with other members of the population. Video courtesy of Anh Pham. Click image to play video.

In this live cell video, mitochondria are labeled with a fluorescent protein (DsRed) and can be tracked over time. Dynamic behaviors including movement (transport), fusion and fission (division) can be observed. For instance, two mitochondria are labeled with green (using photoactivatable GFP), and fuse with other members of the population. Video courtesy of Anh Pham.

On the surface, mitochondrial behaviors are functionally independent from their biochemical roles, and mediated by distinct proteins. However, recent data from us and others suggest that metabolism influences mitochondrial behavior and vice-versa. For instance, culture conditions which enhance the oxidative phosphorylation activity of mitochondria serve to increase fusion rates (Mishra et al., Cell Metabolism 2014) and increase mitophagy rates (Melser et al., Cell Metabolism 2013). Conversely, genetic ablation of mitochondrial fusion results in severe OXPHOS defects (Chen et al., JBC 2005).

Figure 1. Mitochondrial function and behavior are linked.
Prashant Mishra-Laboratory-Figure-1
An overview of mitochondrial metabolism (function) and mitochondrial dynamics (behavior). The processes illustrated are not meant to be exhaustive, but to emphasize the diversity. Although the organellar behaviors are mechanistically distinct from the metabolic functions, recent studies suggest that behavior influences function and vice-versa.
Click image to enlarge.

An overview of mitochondrial metabolism (function) and mitochondrial dynamics (behavior). The processes illustrated are not meant to be exhaustive, but to emphasize the diversity. Although the organellar behaviors are mechanistically distinct from the metabolic functions, recent studies suggest that behavior influences function and vice-versa.

Our laboratory is interested in elucidating the interactions between mitochondrial behavior and function at a global level. We hypothesize that these interactions are a critical part of the organelle’s response to metabolic perturbations, and thus mediate an important aspect of cellular homeostasis. To probe this, we are developing methods to quantitatively measure numerous aspects of behavior and function in a high-throughput manner. We will then measure changes in organelle functions secondary to systematic genetic and metabolic perturbations, thereby constructing a “road map” of the mitochondrial response. This map will provide new insights into the mitochondrion’s ability to control cellular metabolism and therefore promote viability and functionality in the face of a changing metabolic environment.

Mitochondrial Myopathies

Muscle dysfunction and weakness is a common symptom of mitochondrial diseases, caused by mutations within the mitochondrial genome (mtDNA). Mitochondrial myopathies have some unique features, in comparison with other myopathies. In particular, individual muscle fibers are affected in a regional manner, with affected segments surrounded by normal regions of the fiber (Figure 2). Affected regions are classified by a loss of mitochondrial activity, and an accumulation of mutant mitochondria. Interestingly, the mutation does not spread beyond the affected region, suggesting that mechanisms are in place to restrict the defect from disturbing the whole fiber.

Figure 2.
Prashant Mishra-Laboratory-Figure-2
In mitochondrial diseases, myofibers typically exhibit regionalized defects including atrophy and proliferation of mutant mitochondria. Click image to enlarge.

We have recently developed methods to quantify mitochondrial regionalization within muscle fibers (Mishra et al., Cell Metabolism 2015). Satellite cells are muscle-resident stem cell that incorporates into existing myofibers. Using a satellite-cell specific promoter, we have stochastically activated individual myonuclei (in live animals) to label mitochondria in their immediate vicinity (Figure 3). The spread of the mitochondrial signal provides a relative measure of the extent of regionalization, which we term a mitochondrial “domain”. Interestingly, we find that domain size is determined by the type of muscle fiber, as well as the extent of mitochondrial fusion occurring in that fiber. Indeed, mitochondrial fusion appears to promote spreading of the activated signal, thereby extending the length of the domain.

Figure 3.
Prashant Mishra-Laboratory-Figure-3
Stochastic labeling of individual myonuclei demonstrates regionalization of mitochondria. A) Schematic illustrating stochastic activation of a satellite cell, and its expressed gene product. B) A sample volume reconstruction of a mitochondrial domain. Here, an activated satellite cell expresses a mitochondrially-targeted fluorescent protein (Dendra2), shown in green. Myonuclei are labeled in white. C) Mitochondrial domain size varies between fibers of different types: oxidative (type 2A) vs. glycolytic (type 2B) fibers. Click image to enlarge.

We therefore hypothesize that fusion rates play a role in determining progression of mitochondrial disease. Using mouse models of disease, in combination with conditional knockout alleles for pro-fusion proteins (mitofusin 1 and 2), we will test this hypothesis. Data from these studies will suggest whether modulation of organellar dynamics might be beneficial in the treatment of mitochondrial diseases.