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Essay 11.3

Mitochondrial Dynamics: When Form Meets Function

Iain Scott & David C. Logan, School of Biology, University of St. Andrews, St. Andrews, UK

September, 2006

Introduction

Mitochondria are vitally important eukaryotic organelles, and they were recognized over fifty years ago as the site of oxidative energy metabolism (Kennedy and Lehninger 1949). They synthesize the majority of respiratory ATP in plants, animals, and fungi. In addition to this crucial role, mitochondria are involved in the production of many novel compounds, such as phospholipids, nucleotides, and several amino acids. In fact, mitochondria are essential to eukaryotic life—although some organisms have the capability to respire anaerobically, they can only survive when mitochondria are present to manufacture these unique metabolites.

Mitochondria cannot be created de novo, meaning that any new mitochondrion must be formed from the division of an existing organelle. In addition to division, mitochondria also undergo fusion where two or more individual organelles join to produce a single mitochondrion. Mitochondrial fission and fusion are the primary processes controlling mitochondrial form and together control mitochondrial size and number. Traditionally, the mitochondrion has been portrayed as an immobile, oval-shaped body. In reality, mitochondria are very dynamic organelles, capable of changing size and shape in a matter of seconds (Bereiter-Hahn and Voth, 1994) (see Movie 1). Additionally, they undergo short- and long-distance vectorial transport mediated by association with the cytoskeleton. Advances in bio-imaging have allowed scientists to re-evaluate the behavior of mitochondria in vivo, stimulating a surge of interest in determining the genes, proteins, and mechanisms that control mitochondrial shape, size, number, and distribution (collectively termed, mitochondrial dynamics) (see Logan 2006a).

One of the primary goals of this new research area is the identification of genes controlling mitochondrial division and fusion—two processes that underpin mitochondrial form. In budding yeast, (Saccharomyces cerevisiae) several genes have been identified that play a role in mitochondrial division and fusion. One main protagonist in yeast mitochondrial division is the dynamin-related protein, Dnm1p. Dynamin is a GTPase mechano-enzyme involved in vesicle membrane constriction and severance during endocytosis. Dnm1p is believed to function similarly to dynamin but instead acts on mitochondrial membranes. Fusion of mitochondria in S. cerevisiae is controlled by a second GTPase, encoded by FZO1, homologous to the fuzzy onions gene from Drosophila.

Genes Involved In Higher Plant Mitochondrial Dynamics

Two main approaches have been taken to identify genes involved in the control of higher plant mitochondrial dynamics. The first approach relies on the fact that mitochondria in all eukaryotes have a shared ancestry. This means that plant genes with significant homology to genes involved in yeast mitochondrial dynamics are likely to have analogous functions. This gene homology provides researchers with a variety of "reverse-genetics" approaches (called reverse genetics because the gene is identified before the phenotype; cf. forward genetics, see below) to analyze the effect of gene knockouts or knockdowns on mitochondrial dynamics and cell function. Such an approach has provided researchers with several successes to date; for example, two plant dynamin-like homologues, DRP3A and DRP3B, have been shown to be involved in mitochondrial division (Arimura & Tsutsumi 2002; Arimura et al. 2004; Logan et al. 2004). However, this method of identifying genes involved in plant mitochondrial dynamics is ultimately of limited use. The wild-type morphology of plant versus yeast mitochondria is considerably different, as are the mechanisms controlling mitochondrial inheritance. In a typical yeast cell, five to ten mitochondria form a cortical network and mitochondrial inheritance is an active process involving movement of parental mitochondria into the developing bud. In plants, there may be several hundred discrete mitochondria per cell, usually seen as small spherical or sausage-shaped organelles, and it is thought that mitochondrial partitioning (inheritance) into daughter cells during mitosis is, at least in part, due to the stochastic distribution of mitochondria in the parental cell. These morphological and organizational differences suggest differences in the mechanisms and proteins involved in the processes. Indeed, interrogation of the Arabidopsis thaliana genome databases shows that there are no sequence homologues of many genes crucial to yeast mitochondrial morphology and dynamics. For example, while we know that plant mitochondria fuse, there is no Arabidopsis homologue of the important fusion protein, Fzo1p.

These facts suggest that the genes, proteins, and mechanisms controlling plant mitochondrial dynamics, while exhibiting some similarities to yeast, are predominantly distinct. We recently initiated a novel research program designed to identify some of the genes involved in the control of plant mitochondrial morphology and dynamics (Logan et al. 2003). Arabidopsis plants expressing green fluorescent protein (GFP) targeted to the mitochondria (Logan and Leaver 2000) were mutagenized using ethyl methanosulfonate (EMS), and the second (M2) generation was then screened for altered mitochondrial shape, size, number, and distribution using a fluorescence microscope. Six viable mutants with distinct mitochondrial phenotypes were identified from a population of approximately 9500 individuals. Forward genetics (i.e., mutant phenotype identified prior to identification of the mutated gene; cf. reverse genetics, above) is being used in the form of positional cloning to identify the mutant genes.

In the first motley mitochondrial mutant (mmt1) the mitochondrial population is highly heterogeneous, varying in size from one-quarter to four times the average plan area of wild-type mitochondria (cf. Figure 1c,d and 1a,b). In addition, the size distribution of chloroplasts is affected in the mutant—chloroplast plan-areas in the mutant range from 4 to 240 times the plan area in the wild type.

Figure 1   Images captured using either epifluorescence microscopy (left-hand panels) or transmission electron microscopy (right-hand panels) of wild-type and mutant Arabidopsis leaf mitochondria. Epifluorescent micrographs are false-coloured for GFP (green) and chlorophyll (red) fluorescence. a & b, wild type, arrows = mitochondria; * = chloroplast c & d, mmt1 mutant, * = chloroplast e & f, mmt2 mutant, plain arrows = small mitochondria; arrows with circle = large mitochondria; boxes indicate an area magnified to highlight the heterogeneity of mitochondria size within a single cell; * = chloroplast g & h, bmt mutant, arrow = mitochondrion i & j, nmt mutant, arrows = small mitochondria; * = chloroplast k & l, fmt mutant, arrow = large mitochondrial cluster; boxes indicate a region enlarged to highlight a cluster of mitochondria Scale bars in epifluorescent images = 5 μm; in TEMs = 1 μm, except in d where bar = 5 μm. (Reproduced from Logan, D. C., Scott, I., and Tobin, A. K. (2003) Plant Journal 36: 500–509, with permission from Blackwell Publishing.) (Click image to enlarge.)

The second motley mitochondrial mutant (mmt2) contains a highly heterogeneous mitochondrial population similar to mmt1, although in this second mutant the gross chloroplast morphology remains normal (Figure 1e,f). Micrographs taken of the mmt2 mutant with a transmission electron microscope (TEM), in addition to confirming the mitochondrial heterogeneity, demonstrated that the internal structure of the chloroplasts is severely altered (Figure 1f). Chloroplasts in the mmt2 mutant contain a mass of densely packed membranes instead of the normal morphology of granal stacks connected by stromal lamellae, and there are a large number of electron-dense particles within the chloroplasts.

Mitochondria in the big mitochondrial mutant (bmt1) have plan-areas two to four times wild type, and there are about half as many per microscope field-of-view (Figure 1g,h). The network mitochondrial mutant (nmt) is characterized by the presence of long interconnected mitochondrial tubules extending to many tens of micrometers in length (Figure 1i). Examination of leaf tissue of nmt plants under the TEM showed that the aberrant mitochondrial architecture was not maintained in the fixed tissue (Figure 1j). Instead, the mitochondrial tubules fragmented to form organelles as small as 1/16th the plan-area of those in wild-type cells. We have mapping data for the mmt1, mmt2, bmt, and nmt mutants; no previous mitochondrial development genes/mutants have been mapped to the regions containing the mutant loci, nor are there any obvious candidate genes in these regions.

Finally, the friendly mitochondrial mutant (fmt) was identified by the presence of discrete clusters of tens of mitochondria (Figure 1k,l). While only a proportion of the cell′s mitochondrial population form clusters (many maintain a wild-type distribution), the phenotype of this mutant is striking. We used forward- and reverse-genetics to reveal that the mitochondrial phenotype of the fmt mutant was due to a single point mutation in a homologue of the Dictyostelium discoideum cluA gene, which has been shown to be involved in the maintenance of correct mitochondrial distribution (Zhu et al. 1997). We named the Arabidopsis gene, FMT. There are cluA homologues present in all sequenced eukaryotic genomes, but apart from a short tetratricopeptide repeat (TPR) domain that is thought to function in protein-protein interactions, CluA-type proteins have no homology to proteins of known function. Studies are ongoing to determine the role and function of FMT and its homologues in higher plants.

Conclusion

Observations of mitochondrial behavior have been made for over a century (see Kennedy and Lehninger 1949). However, recently developed techniques using green fluorescent protein, enabling the unambiguous visualization of mitochondria in living tissue, have revolutionized research in this area. Such advances in cell biology have been complemented by advances in molecular genetics, which is supported by the Arabidopsis Genome Initiative; together these have enabled a new era of functional genomics research into mitochondrial dynamics. One of the main conclusions that can be reached from recent research on mitochondrial dynamics is the clear interplay between mitochondrial form and function. For example, studies into plant cell death and apoptosis (a morphologically distinct type of programmed cell death occurring in yeast and animals, see Web Essay 11.8) have uncovered the vital role played by mitochondrial dynamics in these processes. As we learn more about mitochondrial dynamics, we can expect to discover many more instances where mitochondrial form and function are integrated (see Logan 2006b for review).

Acknowledgements

Work by the authors is supported by the Biotechnology and Biological Sciences Research Council of the United Kingdom.

References

Arimura, S., and Tsutsumi, N. (2002) A dynamin-like protein (ADL2b), rather than FtsZ, is involved in Arabidopsis mitochondrial division. Proc. Natl. Acad. Sci. USA 99: 5727–5731.

Arimura, S., Aida, G. P., Fujimoto, M., Nakazono, M., and Tsutsumi, N. (2004) Arabidopsis dynamin-like protein 2a (adl2a), like adl2b, is involved in plant mitochondrial division. Plant Cell Physiol. 45: 236–242.

Bereiter-Hahn, J., and Voth, M. (1994) Dynamics of mitochondria in living cells: Shape changes, dislocations, fusion, and fission of mitochondria. Microsc Res Tech. 27: 198–219.

Kennedy, E. P., and Lehninger, A. L. (1949) Oxidation of fatty acids and tricarboxylic acid cycle intermediates by isolated rat liver mitochondria. J. Biol. Chem. 179: 957–972.

Logan, D. C. (2006a) Plant mitochondrial dynamics. Biochem. Biophys. Acta 1763: 430–441.

Logan, D. C. (2006b) The mitochondrial compartment. J. Exp. Bot. 57: 1225–1243.

Logan, D. C., and Leaver, C. J. (2000) Mitochondria-targeted gfp highlights the heterogeneity of mitochondrial shape, size and movement within living plant cells. J Exp Bot. 51: 865–871.

Logan, D. C., Scott, I., and Tobin, A. K. (2003) The genetic control of plant mitochondrial morphology and dynamics. Plant Journal 36: 500–509.

Logan, D. C., Scott, I., and Tobin, A. K. (2004) Adl2a, like adl2b, is involved in the control of higher plant mitochondrial morphology. J Exp Bot. 55: 783–785.

Zhu, Q., Hulen, D., Liu, T., and Clarke, M. (1997) The cluA^- mutant of Dictyostelium identifies a novel class of proteins required for dispersion of mitochondria. Proc. Natl. Acad. Sci. USA 94: 7308-7731.