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Overview
The primary focus of our lab is to understand the role of mitochondrial dynamics in normal cellular function and human disease (for some general information on this topic, see the reviews listed in the publications section). Mitochondria are remarkably dynamic organelles that undergo continual cycles of fusion and fission (Figure 1). The equilibrium of these two opposing processes determines the overall morphology of mitochondria in cells and has important consequences for mitochondrial function.
Our research falls into several broad areas:
To address these issues, we use a wide range of approaches, including genetics, biochemistry, cell biology, and structural biology.
Figure 1: Mitochondria undergoing fusion. In these still frames from a time-lapse movie, two pairs of mitochondria (labeled blue) approach each other, contact briefly, and fuse. From Chen et al (2003) J Cell Biol 160, 189-200.
I. Cellular and physiological functions of mitochondrial fusion and fission
A typical mammalian cell can have hundreds of mitochondria. However, each mitochondrion is not autonomous, because fusion and fission events mix mitochondrial membranes and contents. As a result, such events have major implications for the function of the mitochondrial population. We are interested in understanding the cellular role of mitochondrial dynamics, and how changes in mitochondrial dynamics can affect the function of vertebrate tissues.
Much of our work focuses on proteins called mitofusins (Mfn1 and Mfn2), which are transmembrane GTPases embedded in the outer membrane of mitochondria (Figure 2). These proteins are essential for fusion of mitochondria. To understand the role of mitochondrial fusion in vertebrates, we have constructed mice deficient in either Mfn1 or Mfn2. We find that mice deficient in either Mfn1 or Mfn2 die in mid-gestation. Mfn2 mutant embryos have a specific and severe disruption of a layer of the placenta called the trophoblast giant cell layer. These findings indicate that mitochondrial fusion is essential for embryonic development. We have also generated conditional alleles of Mfn1 and Mfn2 and are currently using these mouse lines to examine the role of mitochondrial fusion in adult tissues. These studies are relevant to our understanding of several human diseases (see below).
Embryonic fibroblasts lacking Mfn1 or Mfn2 display fragmented mitochondria, a phenotype due to a severe reduction in mitochondrial fusion. Cells lacking both Mfn1 and Mfn2 have completely fragmented mitochondria and show no detectable mitochondrial fusion activity (Figure 3). Our analysis indicates that mitochondrial fusion is important not only for maintenance of mitochondrial morphology, but also for cell growth, mitochondrial membrane potential, and respiration. We are also using RNA interference to disrupt the function of other proteins involved in mitochondrial fusion and fission.Figure 2: Schematic of mitofusin protein in the mitochondrial outer membrane. Note that both the N- and C-termini face the cytosol. HR1 and HR2 indicate two heptad repeat regions, motifs that generally form coiled-coil structures.
Figure 3: Deletion of Mfn1 or Mfn2 results in fragmentation of mitochondria. Wildtype mouse embryonic fibroblasts (left panel) have tubular mitochondria that can be over 10 microns long (arrow). Cells lacking Mfn1 (middle panel) or Mfn2 (right panel) have fragmented mitochondria. This result illustrates the concept that mitochondrial morphology results from a balance between the opposing processes of fusion and fission--in the absence of fusion, fragmentation occurs due to unbalanced fission. From Chen et al (2003) J Cell Biol 160, 189-200.
II. Molecular mechanism of membrane fusion and fission
The best understood membrane fusion proteins are viral envelope proteins and SNARE complexes. Viral envelope proteins, such as gp41 of HIV, reside on the lipid surface of viruses and mediate fusion between the viral and cellular membranes during virus entry (a topic we also study). SNARE complexes mediate a wide range of membrane fusion events between cellular membranes. In both cases, cellular and crystallographic studies have shown that the formation of helical bundles plays a critical role in bringing the merging membranes together. We would like to understand mitochondrial fusion at a similar level of resolution and to determine whether there are common features to these diverse forms of membrane fusion.
Mitofusins are the only conserved mitochondrial outer membrane proteins involved in fusion. Therefore, it is likely that they directly mediate membrane fusion. Consistent with this idea, mitofusins are required on adjacent mitochondria to mediate fusion (Figure 4). In addition, mitofusins form homotypic and heterotypic complexes that are capable of tethering mitochondria (Figure 5). We are trying to determine how tethered mitochondria, mediated by mitofusins, proceeds to full fusion. It should be noted that mitochondrial fusion is likely to be more complicated than most other intracellular membrane fusion events, because four lipid bilayers must be coordinately fused (mitochondria are double-membraned organelles).
We are also exploring the roles of other proteins, such as OPA1, in mitochondrial fusion. In addition, we are using proteomic approaches in yeast cells to identify novel proteins involved in mitochondrial fission.
Figure 4: Mitofusins are required on adjacent mitochondria to mediate membrane fusion. In cell hybrids between wild-type and Mfn-null cells, mitochondria do not fuse. In this experiment, mitochondria from wild-type cells are labeled with GFP, and mitochondria from Mfn-null cells are labeled with dsRed. This lack of fusion suggests that mitofusin complexes act in trans. From Koshiba et al (2004) Science 305, 858-862.
Figure 5: The HR2 region of Mfn1 forms an antiparallel coiled coil that mediates tethering of mitochondria. Using X-ray crystallography, we found that the HR2 region (see Figure 2) forms a dimeric, antiparallel coiled coil. In a trans complex of mitofusin dimers, this structure would mediate tethering of mitochondria. In fact, certain mutants of Mfn1 can trap this tethered state. From Koshiba et al (2004) Science 305, 858-862.
III. Mitochondrial dynamics in human disease
Two inherited human diseases are caused by defects in mitochondrial dynamics. Charcot-Marie-Tooth (CMT) disease is a neurological disorder that affects the peripheral nerves. Patients with CMT experience progressive weakness of the distal limbs and some loss of sensation. A specific type of CMT, termed CMT2A, is caused by mutations in Mfn2 and result from degeneration of axons in peripheral nerves. We are currently analyzing the functional consequences of such disease alleles, and using transgenic and targeted mutagenesis approaches to develop mouse models.
The most common inherited form of optic neuropathy (autosomal dominant optic atrophy) is caused by mutations in OPA1. This mitochondrial protein is localized to the inner membrane space and is essential for mitochondrial fusion.
Finally, an understanding of mitochondrial dynamics will be essential for understanding a large collection of diseases termed mitochondrial encephalomyopathies. Many mitochondrial encephalomyopathies result from mutations in mitochondrial DNA (mtDNA). In mtDNA diseases, tissues maintain their mitochondrial function until pathogenic mtDNA levels exceed a critical threshold. Experiments with cell hybrids indicate that mitochondrial fusion, by enabling cooperation between mitochondria, can protect respiration even when >50% of mtDNAs are mutant. To understand the pathogenesis of mtDNA diseases, it is critical to explore how mitochondria can be functionally distinct and yet cooperate as a population within a cell. We anticipate that our studies with mice lacking mitochondrial fusion will shed light on this group of often devastating diseases.
Postdoctoral positions are available.
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