Molecular mechanisms of mitochondrial membrane dynamics and their relation to cell function

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Research Synopsis

Since the 1940s, the mitochondrion has been known to be the powerhouse of the cell, due to its primary role in generating most cellular energy in the form of ATP. Mitochondria are usually depicted as static, kidney bean shaped organelles dispersed throughout the cytoplasm (such as the cross section electron micrograph shown to the right). However, it is now clear that mitochondria are dynamic organelles, consisting of interconnected tubular networks constantly undergoing rapid directed movements along microtubules combined with multiple and balanced membrane fusion/fission reactions, even in resting cells (click on image to right to watch a movie of mitochondrial movement in a live cell). My lab is interested in determining the protein machinery that both orchestrates and regulates mitochondrial membrane dynamics, particularly fusion. We also aim to understand, on a broader scale, the biological significance of dynamic mitochondria, and the consequences of disrupting this process in relation to human mitochondrial diseases. To do this, we are taking advantage of the power of yeast genetics to specifically identify factors that control membrane fusion. In addition, we using the fruit fly as a model metazoan, to dissect in what cell-biological context a dynamic mitochondrial compartment is required, an area of research that has yet to be pursued in this model organism.

The reason for this dynamic behaviour of mitochondria is not currently known, however, disruption of these mitochondrial dynamics can result in neurodegenerative diseases like dominant optic atrophy, and Charcot-Marie-Tooth type 2A disease. Previous studies have identified only a handful of factors that are involved in regulating mitochondrial membrane dynamics. The purpose of our research is to investigate two key questions in this emerging field.

A) What role do mitochondrial dynamics play for cell and tissue function?

B) What molecular machinery is required to orchestrate and regulate mitochondrial double membrane fusion?

To advance our understanding, we are taking advantage of recent advances in fluorescence technology and genome-wide approaches, with the following specific aims.

1) Fully characterize in the model organism, Drosophila melanogaster, the tissue-specific requirements for mitochondrial dynamics using genetics and mutant fly analyses.

2) Determine the substrate repertoire of Drosophila Rhomboid-7, a mitochondrial protease that regulates mitochondrial dynamics.

3) Identify and characterize genes required specifically for mitochondrial membrane fusion in yeast.

4) Identify genes that are synthetically lethal with yeast mitochondrial fusion factors.

Our introduction into mitochondrial membrane dynamics came from our discovery that a mitochondria-localized rhomboid protease regulated this process. Rhomboids are 7-pass transmembrane (TM) domain proteins that cleave their substrates in the lipid bilayer using a classical serine catalytic-triad mechanism (like trypsin) and represent a new conserved family of intramembrane proteases (see schematic to the right). Rhomboids represent one of the most highly conserved protein families, with members found in almost all organisms from bacteria and archaea to humans. In Drosophila, they initially appeared to be dedicated to regulating the activity of the epidermal growth factor receptor (EGFR). However, our discovery of a novel and conserved subclass of mitochondria-localized rhomboid proteases has shed new light on the range of functions of this protein family. In yeast, the main action of this mitochondrial enzyme is to control (by regulated proteolysis) the action of Mgm1—itself a protein that regulates mitochondrial membrane fusion.

In order to get at the main issues in this emerging field, we are taking a two-model system approach.

In the first instance, we are using the fruit fly, Drosophila melanogaster. We have made a fly that has a mutation in its mitochondrial rhomboid (rhomboid-7). These flies are semi-viable. Homozygous mutant adults only live for three days (normal flies live for about 60 days). In addition, these flies cannot walk or fly, the consequence of a severe neuromuscular defect (click on the two movies to the right to see the difference between a wild type fly and a rhomboid-7 mutant). We are currently characterizing the phenotype of this mutant, by a number of genetic and biochemical approaches. It is our hope that by studying mitochondrial function in a complex metazoan, we will be able to understand better in what cellular context mitochondrial membrane dynamics are required for function.

In a second approach, we are using the yeast, Saccharomyces cerevisiae, to identify novel factors that orchestrate and regulate mitochondrial membrane fusion. By taking advantage of the existing yeast deletion collection, we are both candidate testing, and adopting a high-throughput approach. Genes that are identified in these screens will be further characterized in yeast, and in flies (assuming they are conserved through evolution). In addition to these yeast mating screens that monitor mitochondrial membrane fusion, we are working with the laboratory of Charlie Boone at the CCBR. A powerful strategy to discover the function of genes is to identify other genes that when also mutated result in cell death. This is referred to as synthetic lethality. His lab has pioneered a technique to identify synthetic lethal interactions of the entire yeast genome (called SGA-synthetic genetic array). This approach identifies genes that are involved in the same pathway or biochemical process. We will be doing SGA screens to look for synthetic lethal genes of the mitochondrial rhomboid (RBD1) and of its substrate (MGM1).

Compartments of the mitochondrion


Mitochondrial movement in a mouse cell - (courtesy of D. Chan)
(click to play movie)

Drosophila rhomboid-1protease
with its physiological substrate Spitz (in green) that when cleaved is released from the membrane. The proposed serine-type catalytic triad is shown in red.

Wild type flies
(click to play movie)

Rhomboid-7 mutant flies
(click to play movie)

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