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Susan M. Parkhurst
Mechanisms of Morphogenesis and Wound Repair in Drosophila
Building tissues and organs during embryogenesis involves a series of exquisite morphogenetic processes including precisely orchestrated tissue contractions, foldings and migrations. Many of the naturally occurring epithelial movements that shape the embryo during morphogenesis are similar to those employed in the wound healing response and to cell behaviors in tumor progression. We are using developmental, genetic, cell and molecular approaches to look at different regulatory mechanisms and pathways required for proper Drosophila embryonic development. Our current efforts are divided between studies of: (1) molecular and cellular mechanisms of single cell and multicellular (tissue) wound repair, and (2) actin and microtubule cytoskeleton dynamics mediated by the Rho1 small GTPase and its effectors.
Mechanisms of single cell and multicellular wound repair. Epithelial wound repair shares many similarities with normal embryo epithelial morphogenesis: both require cell shape changes and cell migrations that are dependent on the actin/microtubule cytoskeletons. We are investigating the cellular and molecular mechanisms of single cell and multicellular wound repair within the context of a whole organism. We are particularly interested in the regulation of the actin cytoskeleton and in the role of the Rho family of small GTPases in these processes.
SINGLE CELL WOUND REPAIR. Many individual cells in our body suffer “cell wounds”, as a natural consequence of daily physical insults. Survival of individual cells upon injury depends on rapid repair of the disruption through a complex series of events highlighted by membrane fusion and cytoskeletal remodeling. We are characterizing the components and pathways involved in single cell repair using early staged uncellularized embryos that mimic large multinucleate single cells. Our in vivo analysis shows that cell wound repair is a dynamic and fast process: actin, myosin and plasma membrane proteins are recruited to the wound (30-60 seconds post-wounding). To examine the signals that trigger wound repair and control the cytoskeleton changes, we investigated the role of the Rho family of small GTPases in these repair processes. We observe that Rho family GTPases are upregulated at the wound edge, but with different spatial and temporal distributions. Our data is generating a more complete picture of the mechanisms utilized in single cell wound repair, as well as providing a system to systematically identify new components required for these repair processes and to investigate crosstalk among these different components.
MULTICELLULAR WOUND REPAIR. In addition to repairing individually damaged cells, wounded epithelial sheets must be able to seal the hole breaching the surface layer to avoid excessive fluid loss and prevent microbial invasion. Late stage cellularized fly embryos seal these wounds through the formation of an actin-based circumferential ring around the hole. This ring appears shortly after wounding of the epithelial layer (within 10 minutes) and constricts to close the exposed hole in 1- 2 hours. Our focus is to characterize the signaling pathways involved in initiating and terminating this process, and to determine the molecular mechanisms underlying wound repair machinery assembly and contraction. We had previously determined a requirement for the Rho1 small GTPase in wounding through in vivo imaging and genetic analysis. We are currently examining a series of fly lines containing specific Rho mutations, allowing us to dissect the specific Rho functions required for each stage of epithelial sheet repair. We are also identifying and characterizing the downstream effectors required for these repair pathways. Together with our studies on single cell wound repair, we aim to generate a more complete picture of how tissues repair themselves in the embryo.
Cytoskeletal Regulation by Rho GTPase and its Effectors. Rho GTPases play a central role in diverse biological processes including reorganization of the actin cytoskeleton (affecting cell shape changes, cell polarity, cell movement, and cytokinesis), microtubule dynamics, changes in gene transcription, chemotaxis, axonal guidance, cell cycle progression, cell adhesion, oncogenic transformation, and epithelial wound repair. Rho GTPases are also the targets of different classes of pathogens in disease-causing bacterial/viral infections. We have found that Drosophila Rho1 is required for maintenance of proper actin and microtubule architecture/dynamics during oogenesis. We have been characterizing three of its downstream effectors, the de novo linear actin nucleation factors Capu (a formin-homology protein) and Spire (a WH2 domain protein), which act downstream of Rho1 to regulate the onset of ooplasmic streaming during oogenesis, and Wash, a new subfamily of Wiskott-Aldrich Syndrome (WAS) family proteins, that activates the Arp2/3 complex to nucleate branched actin filaments and functions to remodel actin structures and elicit changes in cell shape and movement. wash, capu, spire, and Rho1 are all essential in flies and are present together in protein complexes in vivo. In addition to their actin nucleation activity, we find that Wash, Capu and Spire bundle actin, bundle microtubules, and crosslink them. Our results suggest that Wash may be a link between branched actin filaments, associated with Arp2/3, and linear actin filaments, associated with formins and Spire proteins. We are currently exploring how Wash, Spire and Capu work in the cytoplasm to coordinate actin and microtubule cytoskeleton dynamics. We are also investigating the role of Wash in nuclear architecture and/or organization as we find that Wash is present in the nucleus and that its removal by RNAi leads to altered nuclear shape. Our current efforts are focused on identifying and analyzing the components, pathways, and regulatory mechanism(s) underlying these roles.
1982 - BA, Johns Hopkins University
1985 - PhD, Johns Hopkins University
1986 - Postdoc, Imperial Cancer Research Fund (Oxford UK)
1990 - Postdoc, California Institute of Technology