I am interested in understanding cell migration and metastasis. My postdoctoral training has focused on the cell biology of the Drosophila spectraplakin Short-stop (Shot), a model cytolinker and integrator, in S2 cells. I have determined that Shot functions to cross-link microtubules to the actin cytoskeleton, and in doing so opposes the forces of molecular motors which would otherwise whip and buckle the free microtubules through- out in the cytoplasm. Shot not only cross-links actin and microtubules, but is also an EB1-dependent plus end tracking protein (+Tip). I have determined which residues Shot uses for its interaction with EB1. Continuing this project, I will determine how Shot regulates cell migration and determine the mechanism behind Shot's own regulation. I will expand upon my skill set by incorporating Drosophila genetics and developmental biology, and biochemistry in order to take a comprehensive approach to investigating Shot's role in cell migration. Cell migration requires the dynamics of the actin-microtubule cytoskeleton and the regulation of both cell-matrix and cell-cell adhesion, however, there is a fundamental gap in our knowledge of how these net- works are coordinated. Continued existence of this gap represents an important problem because until it is filled, a comprehensive understanding of the mechanisms that govern metastasis will remain elusive. The long term goal is to determine how adhesion and the cytoskeleton are integrated during cell migration. The objective of this application is to identify how the Shot coordinates these networks during cell migration. Shot is an excellent candidate for this coordination as it can physically cross-link actin and microtubules while playing roles critical to the development of the Drosophila embryo in the regulation and maintenance of both cell-matrix and cell-cell adhesion. Cell migration is a result of a synergistic relationship between these networks, investigating molecules that potentially facilitate this synergism represents the next crucial step in understanding this process. Our central hypothesis is that molecules that coordinate actin-microtubule cross-linking and adhesion function as a nexus in the regulation of cell migration. This hypothesis has been formulated on the basis of data I generated in as well as mounting evidence present in the literature. The rationale for the proposed re- search is that metastasis represents a major cause of mortality in cancer patients. A comprehensive under- standing of cell migration will lead to future therapeutic targets and a decrease in cancer related deaths. This hypothesis will be tested by two specific aims: 1) Determine how Shot mechanistically regulates cell migration;and 2) Determine Shot's regulatory mechanism. To accomplish the first aim, a novel motile Drosophila epithelia cell line amenable to RNAi and high resolution microscopy will be used. This cell line readily forms cell-matrix and cell-cell adhesion in tissue culture and this aspect will be capitalized upon to study Shot's role in the formation and regulation of both. To complement these studies, analysis of Shot's role in cell migration will be carried out in two in vivo models of cell migration, border cell migration and hemocyte migration. In the second aim biochemical and microscopic analysis will be used to determine whether Shot undergoes an intramolecular conformational change, and both cell biology based and developmental biology based assays will be used to elucidate the biological significance of this conformational change. The approach is innovative because it utilizes both cell culture models of cell migration and Drosophila embryogenesis in a complementary manner. The proposed research is significant because it is expected to elucidate the combinatorial affects of adhesion and cytoskeletal dynamics during cell migration. This knowledge has the potential to advance the field of cell motility and putatively uncover therapeutic targets for the prevention of metastasis. The research environment at UNC has been extremely supportive and collaborative. In addition to the continued training and mentoring from Dr. Rogers, I will work closely with Dr. Mark Peifer an expert in Droso- phila genetics and developmental biology and Dr. Kevin Slep an accomplished crystallographer and biochemists to obtain my training goals. Outside of the resources Rogers, Peifer, and Slep labs, I have access to a number of cutting edge microscopes and equipment through a combination of core facilities and departmental facilities. Furthermore, UNC has a number of investigators with expertise I can seek and through campus wide seminars I will have the opportunity to regularly interact with them and receive crucial feedback.
/Public Health Relevance Statement Cell motility, which is the basis of metastasis, requires the integration of the actin and microtubule cytoskeletons as well as the adhesion machinery. Key molecules facilitate this coordination is vital to our comprehensive understanding of cell migration. The spectraplakin family of proteins are unique in that they physically couple actin and microtubules, and regulate both cell-cell and cell-matrix adhesion. The written critiques and criteria scores of individual reviewers are provided in essentially unedited form in the Critique section below. Please note that these critiques and criteria scores were prepared prior to the meeting and may not have been revised subsequent to any discussions at the review meeting. The Resume and Summary of Discussion section above summarizes the final opinions of the committee.
|Girdler, Gemma C; Applewhite, Derek A; Perry, Wick M G et al. (2016) The Gas2 family protein Pigs is a microtubule +TIP that affects cytoskeleton organisation. J Cell Sci 129:121-34|
|Applewhite, Derek A; Grode, Kyle D; Duncan, Mara C et al. (2013) The actin-microtubule cross-linking activity of Drosophila Short stop is regulated by intramolecular inhibition. Mol Biol Cell 24:2885-93|