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RESEARCH INTERESTS
Cell Biology/Developmental Biology
Muscle development in drosophila melanogaster
Role of cytoskeleton in cellular signaling
Muscle protein dynamics
PUBLICATIONS
Research Statement
Muscle cells contain a highly-organized cytoskeleton that both generates and transduces the contractile forces critical for muscle function. Seemingly in contrast to this, the cytoskeleton is not a static structure; instead its components are very dynamic. The Cysteine-Rich Proteins (CRPs) are muscle cytoskeletal proteins that display a dual localization, being found at sites of actin filament anchorage and the nucleus. This subcellular distribution is very provocative, and suggests that CRPs may shuttle between the cytoskeleton and nucleus, perhaps in response to changes in stress on the cytoskeleton. Biochemical studies indicate that CRPs can function as transcriptional co-activators in tissue culture. However, it is not clear if this nuclear activity has any in vivo relevance. Functional inactivation of one CRP family member (CRP3, also known as Muscle LIM protein, or MLP) in the mouse results in dilated cardiomyopathy and subsequent heart failure, underscoring the importance of this protein family. I am dissecting the function of CRPs, using the fruit fly, Drosophila melanogaster, as a model system.
Previous studies from the lab of Mary Beckerle identified two CRP family members in the fly, Mlp60A and Mlp84B. My current work is focused on Mlp84B. This protein also displays the dual localization that is characteristic of the CRPs, being found in both the nuclear and cytoplasmic compartments during muscle development. In mature muscle, Mlp84B is prominent at both the Z-line and the muscle termini (myotendinous junction). I have generated mutations in the mlp84B gene, and found that it is critical for proper muscle function in Drosophila. Although Mlp84B is expressed during embryogenesis in developing muscles, mlp84B mutant larvae hatch and do not exhibit any dramatic muscle defects. Instead, the mutant animals die at the prepual/pupal transition, and display defects in several muscle-driven processes at this time of development. The mlp84B mutants fail to fully contract their body wall muscles, resulting in elongated pupal cases. In addition, they do not display the high degree of muscle contractions that drive the internal morphogenetic movements necessary to transform the prepupal animal into a nasant pupae. Because of this lack of muscle contraction, the animals arrest development and die.
I have examined the muscles of the mlp84B mutants at the cellular level, in order to ascertain the role of Mlp84B in normal muscle. The initial analysis of the CRP3 mutant mice suggested that CRPs may function to maintain the integrity of the cytoskeleton. However, I have not yet detected any cytoskeletal defects in the mlp84B mutants, at any time of development. Moreover, more recent data concerning the CRP3 mutant mouse has concluded that the cytoskeletal defect may be secondary to some other cellular problem. The muscles from the mlp84B mutants are thinner than wildtype, indicating that muscle growth is impaired in these mutants. I propose that Mlp84B, and the other CRPs, have a primary role in post-natal muscle growth, and shuttle from the cytoskeleton to the nucleus in response to strain on the muscle cell. Currently, I am generating Mlp84B proteins that lack the ability to be retained in the nucleus, and testing whether these mutant proteins maintain wildtype function. Also, my research group is determining whether Mlp84B can bind to candidate muscle transcription factors.
A second focus of my research is to following protein dynamics in vivo and in real-time. My research group generated flies that express an Mlp84B-GFP fusion protein, and we have begun to use this as a tool to visualize muscle development in real-time. We have accumulated several fly lines that express other GFP-tagged proteins, and are in the process of comparing the dynamics of these proteins during muscle development. We hope to use FRAP (Flourescence Recovery After Photobleaching) as a way to further characterize the dynamic nature of these muscle proteins. This work is especially exciting, as it will allow us to study muscle development in situ, and also compare protein dynamics in different mutant backgrounds; both of these aspects of the project are limitations in other model systems.
SELECTED PUBLICATIONS
Clark, K. A. and McKearin D. M. 1996. stonewall encodes a nuclear Drosophila protein required for oocyte differentiation. Development 122, 937-949.
Hobert, O., Moerman, D. G., Clark, K. A., Beckerle, M. C., and Ruvkun, G. 1999. A conserved LIM protein that affects muscular adherens junction integrity and mechanosensory function in C. elegans. J Cell Biol. 144, 45-57.
Walker, C.S., Shetty, R.P., Clark, K., Kazuko, S.G., Letsou A., Olivera, B.M., and Bandyopadhyay, P.K. 2001. On a potential global role for vitamin K-dependent gamma-carboxylation in animal systems. Evidence for a gamma-glutamyl carboxylase in Drosophila. J Biol Chem. 276, 7769-7774.
Clark, K.A., McElhinny, A.S., Beckerle, M.C. and Gregorio, C.C. 2002. Striated muscle cytoarchitecture: an intricate web of form and function. Annu Rev Cell Dev Biol. 18, 637-706.
Clark, K.A., McGrail, M. and Beckerle, M.C. 2003. Analysis of PINCH function in Drosophila demonstrates its requirement in integrin-dependent cellular processes. Development. 130, 2611-2621.
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