Jocelyn McDonald - US grants

Regulated cell migration is an important component of many biological processes, including wound repair and development, and one that goes away in cancer, resulting in tumor invasion and metastasis. The border cells, a small group of migratory cells in the Drosophila ovary, are an ideal system for dissecting the molecular mechanisms of cell migration. Although several genes convert border cells from a stationary to migratory state, only a few genes are known to initiate migration after this step. The goal of this proposal is to identify new genes directly involved in promoting border cell migration. Two lethal mutations, 17E1 and 27C1, were identified in a screen for mutations that cause border cell migration defects in mosaic clones. These two mutations dominantly interact, such that females heterozygous for both mutations exhibit defective border cell migration. Since the expression of several border cell markers is normal in each of the mutants, the genes disrupted by 17E1 and 27C1 may directly control border cell migration rather than specify border cell identity. I will conduct a detailed analysis of the roles of 17E1 and 27C1 in cell migration by examining their effect on the distribution of border cell markers and on the migration of other cell types. The genes will be mapped and cloned using standard molecular and genetic approaches. Finally, a genetic screen will be conducted to identify deficiencies that dominantly interact with 27C1 to disrupt border cell migration.

DESCRIPTION (provided by applicant): Cell migration is essential for many aspects of normal embryonic development as well as in wound healing in the adult. Failure of cells to migrate contributes to birth defects and difficulties in wound healing, whereas unregulated cell migration triggers tumor metastasis in cancer. Therefore, it is of fundamental importance to understand how cells migrate, both in normal processes and in diseases such as cancer. The long-term goal of our research is to identify critical molecules that control guided cell migration within a living organism. To this end, we study the simple migration of the border cells, a group of follicle cells in the Drosophila ovary. These cells develop from a continuous, polarized epithelium from which they break away and invade the surrounding germline. Thus, border cells are an excellent model for identifying the genetic and molecular mechanisms that control tumor invasion and metastasis. Although some of the proteins that regulate border cell migration have been identified, questions remain. For example, little is known about what regulates detachment of border cells from the epithelium. In addition, only a few molecular pathways are known to promote the polarized, dynamic protrusions that direct cell migration. To better understand how these and other aspects of border cell migration are regulated, we identified a mutation in par-1 that disrupted border cell migration in mosaic mutant clones. Par-1 is a highly conserved serine-threonine kinase that functions in cell polarity, microtubule stability, and Wnt signaling, but has not been previously implicated in regulating cell migration. In preliminary studies, we found that Par-1 functions in border cells and adjacent follicle cells and that half the border cells mutant for par-1 completely fail to migrate. Thus, Par-1 likely regulates an early step in the migration process. A known downstream target of Par-1, the Wnt signaling pathway component Disheveled (Dsh), regulates cellular protrusions in vertebrates, and we found that Dsh regulates normal border cell migration. Unexpectedly, we identified an allele of patched in a dominant genetic modifier screen of par-1-dependent border cell migration. Patched is the receptor for the Hedgehog (Hh) glycoprotein, suggesting that Hh signaling functions in Par-1-mediated border cell migration. The goal of this proposal is to further investigate the precise functions of Par-1 and its partners. We will use a powerful new live imaging technique, along with genetic, molecular and immunofluorescence techniques to address the following Specific Aims: (1) Determine the role of Par-1 in regulation of normal border cell migration;(2) Investigate the role of the Par-1 target Dsh in border cells;(3) Characterize the role of Patched in Par-1 signaling and border cell migration. These studies are anticipated to provide new insights into the mechanisms of cell migration in a living organism, which will lead to a better understanding and treatment of diseases such as cancer. Project Narrative: Cells move (migrate) in a variety of normal processes, whereas abnormally migrating cells contribute to birth defects, cannot close wounds, and trigger tumor metastasis. Using a simple model system, we found that a protein called Par-1 regulates cell invasion and migration. The goal of this study is to better understand the mechanisms that Par-1 and its protein partners use to regulate normal cell migration, with the hope that they will provide new therapeutic targets in human tumor metastasis and wound healing.

? DESCRIPTION (provided by applicant): Tumor cell invasion, the process by which tumor cells break away from the primary tumor and spread to distant organs, signals a transition to more advanced disease and greatly contributes to the lethality of many cancer types. The migration of single tumor cells has been well described, but mounting evidence suggests that tumor cells also migrate as a collective, or a group of cells that maintain adhesion and communication through cellular junctions. Cells migrating as a collective have been reported to invade more deeply into organs and to disperse into multiple tissue types. One hallmark of the most common malignant brain tumor, glioblastoma (GBM), is the invasion of tumor cells into the brain parenchyma, a characteristic that greatly contributes to the high rate of tumor recurrence after therapy. Our inability to successfully treat GBM is also driven by the presence of a therapeutically resistant population of self-renewing cells termed cancer stem cells (CSCs). CSCs have been shown to have an enhanced ability to migrate compared with non-stem tumor cells (non-CSCs), although the mechanisms by which CSCs invade and the consequences of this invasion have yet to be determined. The translational goal of this project is to conduct mechanistic studies into collective cell migration by CSCs and to determine the impact of this invasion on GBM progression. Based on our preliminary data showing collective cell migration of CSCs, we hypothesize that collective cell invasion is essential for GBM growth and can be disrupted by compromising the molecular processes governing collective cell invasion. To gain insight into these molecular processes, we performed a screen in Drosophila and identified novel genes responsible for collective invasion. Using an integrative approach spanning Drosophila development to human CSC models, we will interrogate this hypothesis by investigating the following aims: 1) that collective invasion requires dynamic cell-cell junctions and adhesion and 2) that disrupting collective cell invasion decreases GBM growth and increases the efficacy of standard-of-care therapy. The long-term goal of this project is to translate the information gained about collective cell migration and invasion of GBM CSCs to inhibition strategies useful as clinical therapies.

Cells move and migrate to new locations in the bodies of developing animals, an important step for the correct formation and function of organs. The proposed research uses a simple genetic model, the fruit fly, to investigate how cells move as organized groups within the animal. The overall goal of these studies is to identify fundamental mechanisms that keep cells together during cell movement, a poorly understood process. Results from these studies are expected to shed light on the way that cells migrate to populate tissues and organs during animal development. A major impact of the proposed project is the training of students and encouragement of underrepresented groups to pursue scientific-intensive careers. Because of the ease of use and available genetic tools, these studies with the fruit fly model are ideally suited for the training of students. The McDonald lab has a history of training undergraduate and graduate students from diverse backgrounds, and will continue to do so in the proposed studies. The investigator will specifically recruit female and underrepresented minority undergraduate students from three local institutions that currently offer limited research opportunities. The project also includes several outreach efforts, based on the proposed research, to a local grade school and high school comprised mainly of underrepresented minorities. These activities together will introduce students to research in genetics and animal cell and developmental biology and encourage them to pursue scientific and technical careers.

Throughout development cells frequently move in groups to form, shape and remodel tissues and organs. Such "collectively" migrating cells adhere strongly to one another but also coordinate movement of the whole group as a single unit. Despite their crucial importance to development, how cells migrate collectively rather than individually remains poorly characterized. Drosophila border cells represent a genetically accessible model for the discovery of fundamental regulatory pathways that underlie collective cell movement. Dynamic cycles of phosphorylation and dephosphorylation regulate the cellular signaling pathways and physical/cytoskeletal dynamics involved in individual and collective cell migration. While the functions of protein kinases are well established, much less is known about the roles of protein phosphatases. Novel preliminary data indicate that protein phosphatase activity is an unanticipated controller of collective cohesion and migration of border cells. Border cells in which phosphatase activity is inhibited round up, break off from the main group and are unable to complete their migration. The central hypothesis of this proposal is that distinct phosphatase complexes maintain a collective mode of migration through dynamic regulation of identifiable substrates. The proposed project combines genetic, live imaging, cell biology and biochemical approaches to determine which phosphatase complexes regulate collective border cell migration. Moreover, these studies will identify phosphatase target proteins responsible for the collective cohesion and migration of border cells. As common mechanisms underlie different collective cell migration processes, the proposed studies utilizing the powerful border cell model will uncover new cellular and molecular pathways regulated by protein phosphatases. Together, this will critically advance knowledge of how organs form and are remodeled by collectives during development of multicellular organisms.

Nontechnical Description:
During the development of embryos, groups of cells (referred to as 'collectives') move together to help form tissues and organs. Movement of these collectives inside the developing embryo is poorly understood. This fellowship will study development of the fruit fly as a model system to understand how cell collectives move within, and interact with, living, intact tissues. The scientific goals of this project are to use new advanced microscopic methods to reveal how collectives move in tissues, and to use light to control cell movements. The PI and a postdoctoral fellow will be trained in these new techniques at the University of California, Santa Barbara, and will then bring the techniques back to the home institution (Kansas State University). These studies will result in new tools that can be used to study how embryos develop into fully formed animals, and to improve our broader understanding of how cell collectives move within tissues.

Technical Description
The migration of multicellular groups, or cell collectives, is vital to the formation and reorganization of tissues during organogenesis. While collective migrations are required for development, it is not well understood how cells stay ordered and migratory despite moving inside densely packed tissues. Drosophila border cells are an excellent genetically tractable model system to address how cell collectives physically and molecularly interact with the native tissue environment. The goal of this fellowship is to uncover mechanisms that underlie interactions between migrating collectives and tissues through the use of cutting-edge live cell imaging of the border cell system. The work will be performed in partnership with reasearchers at the University of California, Santa Barbara, which is a leader in the field of collective cell migration and innovative live cell manipulation and imaging. The PI will employ a combination of Selective Plane Illumination (SPIM) light sheet fluorescent microscopy (LSFM), in vivo biosensors, and optogenetic tools that employ light to manipulate protein function in live cells, will be used at the host site. These techniques will address: (1) how tissue shape and organization impacts border cell collective migration; and (2) how Rap1, a key signaling protein, controls the adhesion strength and dynamics of border cells during their migration inside the tissue. The project will allow the PI to create and use innovative imaging and optogenetic tools to considerably advance our conceptual and mechanistic understanding of how cell collectives migrate in complex native environments. The PI will introduce and promote adoption of this technology at the PI?s home institution, Kansas State University (KSU) by training other investigators on how to use biosensors and optogenetic tools.

The development of animals requires the movement of cells to create and shape tissues and organs in the embryo. Cells often cooperate with each other by moving as a united group, but how this works is unclear. This research uses the simple fruit fly model, along with genetics and live cell imaging, to probe how groups of cells come together, adapt their shapes, and move as one within a whole tissue. This project will define the way that cells move together in the tissues and organs of many animals. A broader impact will be the recruitment of college students from underrepresented groups to perform in-depth research. Workshops for 6th-8th grade and 9th-12th grade girls will use a low-cost ?build your own? microscope (the Foldscope) to learn about nature and science. Lastly, engineering students will design and make a device to alter the tissue and find out the impact on how cell groups move. These activities will help students get excited about biology and inspire them to become scientists or engineers.

Movement is a fundamental property of life, that crosses many levels, from molecules to cells and tissues to organisms. Cell migration is fundamental to how diverse tissues and organs form and are remodeled during animal embryonic development. Migrating cells must sense, respond, and adapt to changes in the immediate tissue environment. Cells that move collectively are further challenged to stay together, communicate, respond, and adapt cooperatively as multicellular units to tissue-scale forces and signals. An integrated understanding of the mechanisms that keep cell collectives moving inside tissues is lacking. The proposed studies will fill this knowledge gap by elucidating the dynamic molecular, cytoskeletal, organellar, cellular, and tissue-level mechanisms that promote collective cell motility in complex environments. Specifically, this project uses the Drosophila border cell collective because it is a tractable model amenable to real-time live cell imaging, genetic tools, and optogenetic cellular manipulation, all within the native developing tissue. Building on previous studies, this project will uncover: (1) how the critical signaling nexus Rap1 coordinates adhesion and the cytoskeleton to keep border cells moving inside the developing tissue; (2) how nuclear deformation, along with connections to the cellular cytoskeleton, helps border cells adapt to changes in the tissue microenvironment and morphology; (3) how alterations in gene expression facilitate movement inside crowded tissues. These studies will uncover conserved mechanisms that drive and regulate collective cell movement within tissues, thus linking principles of cooperativity and adaptability in the movement of cell nuclei, single cells, collectives, tissues, and animals as a whole.

This research award is funded by the Developmental Systems, Cellular Dynamics and Function, and Rules of Life Programs, all within the Biological Sciences Directorate.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.