Susan M. Parkhurst

Appointments and Affiliations

 
 
Fred Hutchinson Cancer Research Center
Division of Basic Sciences
Member
University of Washington
College of Arts and Sciences
Biology Department
Affiliate Professor
Professional Headshot of Susan M. Parkhurst

Mailing Address

Fred Hutchinson Cancer Research Center
P.O. Box 19024
1100 Fairview Ave N, A1-162
Seattle, Washington 98109-1024
United States

Contact

Phone: (206) 667-6466
Fax: (206) 667-6497
susanp@fhcrc.org
http://labs.fhcrc.org/parkhurst/

Degrees

Postdoctoral Fellow, California Institute of Technology, Developmental Genetics, 1990.
Postdoctoral Fellow, Imperial Cancer Research Fund, Oxford UK, Developmental Genetics, 1986.
Ph.D., Johns Hopkins University, Developmental Biology, 1985.
B.A., Johns Hopkins University, Biology, 1982.

Research Interests

Regulatory mechanisms governing early Drosophila development

Our lab is interested in the actions of both maternally and zygotically contributed gene products that govern proper embryonic development in Drosophila. Our lab uses developmental, genetic, cell, molecular, and biochemical approaches to look at different regulatory mechanisms and pathways required for proper Drosophila embryonic development. Our current efforts are divided between studies of: (1) actin and microtubule cytoskeletal dynamics mediated by the Rho1 small GTPase, (2) mechanisms of single cell and multi-cellular wound repair, and (3) mechanisms of transcriptional repression and the rules governing cofactor recruitment.

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 microfilament and microtubule architecture during oogenesis. We have also found that the de novo actin nucleation factors Cappuccino (Capu; a formin-homology (FH) protein) and Spire (a WH2 domain protein) act downstream of Rho1 to regulate the onset of ooplasmic streaming. While this streaming event is microtubule-based, actin assembly is required for its timing. In addition to their actin nucleation activity, we have found that Capu and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC, and SpireD). SpireD was recently shown to nucleate actin, but the activity of the other isoforms had not been addressed. We find that SpireD does not have crosslinking activity, while SpireC is a potent crosslinker. We have found that SpireD binds to Capu and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition. Our results suggest that Rho1 regulates the timing of ooplasmic streaming by regulating the microtubule/microfilament crosslinking that occurs at the oocyte cortex: crosslinking antagonizes the formation of the dynamic subcortical microtubule arrays that are required for ooplasmic streaming. Rho1, Capu and Spire appear to be elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments. We are currently using both genetic and molecular techniques to identify other proteins that act either upstream or downstream in the Rho1-Capu-Spire pathway.

Mechanisms of single cell and multicellular wound repair. Epithelial wound repair shares many similarities with the tissue movements that occur during normal embryo epithelial morphogenesis: both require cell shape changes and cell migrations that are dependent on the actin cytoskeleton. We are investigating the cellular and molecular mechanisms of single cell and multicellular wound repair and their ensuing biological manifestations, in the embryo. 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. 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 embryos prior to cellularization, during which the embryos mimic large multinucleate single cells. Our results show that wound closure in these early embryos is a fast process (5-10 minutes total); actin and Rho1 accumulate at the wound site, as well as ER and plasma membrane proteins. In addition to imaging studies, we are conducting a microarray screen to identify genes involved in the transcriptional response to wounds, with the aim of uncovering the start and stop signals to wound repair. Together, these assays will provide us details of the players, mechanism(s), and regulatory pathways of the cell wound repair process.
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 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 a timeframe of about 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.

Transcriptional Repression and Cofactor Recruitment. Transcriptional repression is an important feature of developmental processes where it is necessary for establishing complex patterns of gene expression. A number of different transcriptional repressors present in the early Drosophila embryo have been shown to encode sequence-specific DNA binding transcription factors that function by recruiting co-repressor proteins. One such repressor, Hairy, is a pair rule segmentation gene that is essential for the establishment of reiterated pattern in the Drosophila embryo. Hairy belongs to the Hairy/Enhancer of split/Deadpan (HES) subclass of basic helix-loop-helix (bHLH) transcription factors that function as dedicated repressors. Our earlier work highlighted the requirement of multiple Hairy domains for its proper function, suggesting that Hairy is likely to be involved in multiple protein-protein interactions. Our focus over the past few years has been on characterizing cofactors required for Hairy-mediated repression, and more recently on the identification of its direct downstream targets.
Targets. We have used DamID, a chromatin-profiling method, to perform a global and systematic search for direct transcriptional targets of Drosophila Hairy. Hairy was tethered to E. coli DNA adenine-methyltransferase (Dam) permitting methylation proximal to in vivo binding sites in both Drosophila Kc cells and early embryos. This approach identified 40 novel genomic targets for Hairy in Kc cells. We also adapted DamID profiling such that we could use tightly staged collections of embryos (2-6 hours) and found 20 Hairy targets related to early embryogenesis. As expected of direct targets, all of the putative Hairy target genes tested show Hairy-dependent expression and have conserved consensus C-box (Hairy binding site) containing sequences that are directly bound by Hairy in vitro. The distribution of Hairy targets from both the Kc cell and embryo DamID experiments correspond to Hairy binding sites in vivo on polytene chromosomes. In addition to finding putative targets for Hairy in segmentation, we were excited to find groups of targets suggesting roles for Hairy in cell cycle/growth and morphogenesis, processes that must be coordinately regulated with pattern formation. We are currently using this information to develop different classes of targets to use in biochemical assays, as well as to compare to other developmental transcription networks such as the Drosophila Myc/Max/Mad family of independent, yet mechanistically similar, proteins.
Cofactors. Identification of the Groucho co-repressor solidified the view that Hairy functions as a promoter-bound repressor: an intact bHLH region is required for Hairy to bind to specific DNA sites where it then recruits the Groucho co-repressor protein. Groucho has been proposed to utilize a chromatin remodeling mechanism through its recruitment of histone deacetylase. Recruitment of Groucho, however, does not account for all of Hairy's repressor properties. Over the past few years, we have identified and characterized a number of additional Hairy-interacting proteins/cofactors using both genetic and protein interaction screens, including dCtBP (encoding NAD-dependent acid dehydrogenase activity), dSir2 (encoding NAD-dependent histone deacetylase activity), dgrn (a RING finger protein), dTopors (a RING finger protein), and dNC2alpha (a basal transcriptional repressor). These factors act in a context-dependent manner and are likely to utilize different mechanisms of repression. One of the major questions in the field concerns how and when particular cofactors are recruited. It has been technically challenging to address this question with current methods such as ChIP assays, since cofactor associations may be transient, unstable, or far removed from the DNA binding protein. Similarly, utilizing expression-based microarray analysis is also not easy, due to the difficulty in sorting direct from indirect interactions with such widely recruited cofactors. To circumvent these technical issues and as a first step towards understanding the rules governing Hairy cofactor recruitment, we used the DamID approach to determine if the three best characterized Hairy cofactors, Groucho, dCtBP, and dSir2, are recruited to all or a subset of Hairy targets. Interestingly, we find that Hairy cofactor recruitment is context-dependent. While Groucho is frequently considered to be the primary Hairy cofactor, we find that it is associated with only a minority of Hairy targets. The majority of Hairy targets are associated with the presence of a combination of dCtBP and dSir2. Thus, the DamID chromatin profiling technique is providing a systematic means of obtaining a global view of cofactor recruitment requirements during development.

Memberships

Genetics Society of America
Society for Developmental Biology

Honors and Awards

1995, Leukemia Society of America Scholar,
1992, Pew Scholars in the Biomedical Sciences,
1992, American Cancer Society Junior Faculty Research Award,
1992, Basil O'Conner Starter Scholar Research Award,
1986, Helen Hay Whitney Foundation Postdoctoral Fellowship,

Recent Publications

2014
2013
2012
2011
2010
2009
2008