Daniel P. Kiehart - US grants

Our goal is to determine which cellular movements require myosin for force production and to understand, on the molecular level, the mechanisms of myosin function in vitro and in vivo. Our approach has been to produce and characterize monoclonal, site-specific antibodies to cytoplasmic myosin as probes of myosin function in Acanthamoeba. We will analyze force production by myosin with purified proteins, in cell free model systems and in living cells through microinjection. Acanthamoeba contains two classes of myosin that are morphologically distinct. The role these two myosins play in cell movement is not known. In vitro, we have and will map antibody binding sites by electron microscopy of antibody-myosin complexes and by antibody binding to myosin peptides. We have and will analyze the interaction of antibodies with purified contractile proteins to determine how distinct domains in the myosin molecule contribute to myosin filament formation, actin binding and actin-activated ATPase activity. We have and will apply the antibodies to cell free model systems of contractility. To date we have characterized 23 monoclonal antibodies directed against at least 15 unique sites distributed from the myosin-II head to the tip of its tail. Two antibodies block polymerization, 12 block actomyosin-II ATPase activity and 14 inhibit contraction of gelled extracts of amoeba cytoplasm. Our data shows that regions near the tip of the myosin tail are required for polymerization and that another domain on the myosin tail, close to the myosin head, is essential for ATPase activity and force production. Finally, we have and will probe myosin function in whole cells. Fluorescent antibody staining of fixed cells, electron microscopy of antibody stained frozen thin sections, and the distribution of labeled myosin microinjected into living cells will localize myosin. Most significantly, we will microinject the antibodies into living cells to determine which motilities (locomotion, cytokinesis, etc.) require myosin function. We are encouraged by preliminary microinjection studies on Acanthamoeba, and by our antibody-microinjection studies on myosin function in starfish eggs, which showed that myosin is necessary for cytokinesis but not chromosome movement. By analyzing antibody effects on biochemical function, on contractility in vitro, on the location of myosin in whole cells, and on motility in vivo, we expect to elucidate the molecular mechanism of myosin function in living cells.

DESCRIPTION: Development of the Drosophila embryo is characterized by a wide variety of cellular movements and cell shape changes. This research examines at the molecular level the role of conventional nonmuscle myosin (myosin II) in cell shape changes during development. The investigator has previously shown that myosin II plays an essential role in cytokinesis, cell sheet morphogenesis, and cell locomotion. The basic goals of this renewal application is to continue the studies of myosin II but to focus on how regulation of myosin function sculptures the embryo. Three specific aims are described. First, myosin regulation will be investigated through a genetic analysis of myosin light chain function and by a directed analysis of myosin heavy chain phosphorylation. Second, myosin-associated proteins that have a possible role in regulating myosin function will be identified by biochemical and genetic methods. Third, a continuation of investigations into the role of myosin during embryogenesis. Findings from this project will define cellular elements that regulate myosin function and identify the supramolecular structures that transmit myosin-generated forces to the cortex and cytoplasm during the complex cellular rearrangements in the Drosophila embryo. This basic level of research is relevant to the understanding of how abnormal cell movements during embryonic development or later in the adult contribute to birth defects and cancer.

We hypothesize that the classical genetic and molecular genetic approaches available in Drosophila provide a unique opportunity to investigate the molecular basis of nonmuscle myosin-II. Nonmuscle myosin-II is a highly conserved motor protein that contributes to cytokinesis, the distribution of cell surface receptors, post mitotic cell shape changes for morphogenesis, cellular locomotion and wound healing. Previously, we showed that in fly a single gene, zipper (zip) encodes the nonmuscle myosin-II heavy chain and a single gene, spaghetti squash (sqh) encodes the nonmuscle myosin regulatory light chain. These genes encode polypeptides that are very similar to their mammalian orthologs (the regulatory light chain protein is 81 percent identical and 93 percent similar). Thus, the fly proteins are considerably more similar to their mammalian orthologs than are myosin-IIs from other invertebrate model systems that are amenable to classical genetic or molecular genetic approaches. We have used a variety of methods to establish which cell shape changes require myosin-II function. Here we plan to investigate how myosin functions in living cells by using genetic, molecular, cell biological and protein biochemical approaches to identify proteins that are required for myosin function and then to characterize HOW they function. Our Specific Aims are as follows. 1) We will continue to use simple and powerful F1 screens to recover genes whose products interact with myosin-II to effect cytokinesis and morphogenesis. 2) We will characterize GENETICALLY new myosin alleles and putative interacting loci. 3) We will characterize MOLECULARLY new myosin alleles and putative interacting loci. Finally, 4) we will use a variety of CELL BIOLOGICAL approaches to investigate how these interacting proteins facilitate myosin function. We will evaluate links between a) the molecular defects that characterize myosin mutations; b) the molecular identity of interacting gene products and c) the molecular defects that characterize those alleles of interacting loci that actually modulate the ability of the myosin to perform its functions. Our strategy will be to investigate alterations in movements, the structure of the actin cytoskeleton and the localization of myosin in appropriate mutant animals. We will characterize these phenotypes of various myosin mutation homozygotes, interactor mutation homozygotes, myosin/interactor double homozygotes and if instructive, various other potential combinations. Our goal is to ascertain the basis of myosin function at the cellular and molecular level.

Trauma that results in loss of tissue or tissue integrity constitutes a wound, and initiates a wound healing or repair process. Key features of that process include achieving immediate tissue homeostasis, repelling microorganisms and ultimately, the restoration of normal tissue physiology. Wounding and wound healing in humans is of considerable biological and social significance. Wounds occur by accident, can be inflicted in anger, or occur during surgery, for beneficial medical reasons. The ultimate goal of the work we propose is to understand the basic biology of epithelial response to wounds with the long range goal of contributing to the design of intervention strategies designed to speed healing or prevent excessive scarring. A number of vertebrate, and especially mammalian, animal model systems have been developed to study wounding and wound healing. As a consequence, the molecular players responsible for proper wound healing have begun to be identified. Recently, knockout and transgenic mouse technologies have been applied to the study of wound healing. Nevertheless, mammalian model systems can be used in wounding studies only after careful ethical consideration, are naturally time consuming and are very expensive. Moreover, forward genetic screens are essentially impractical. Here, we propose to pursue molecular/genetic strategies that are uniquely available in Drosophila to investigate the biology of wounding and wound healing. Recently we used mechanical and laser methods to ablate tissue in Drosophila embryos and found that such ablations are followed by rapid and robust healing. Moreover, we found that healing was completely inhibited in embryos homozygous for defects in the structural gene for myosin heavy chain. We propose to characterize wounding and wound healing in Drosophila, test the role of candidate loci in the process of wounding and wound healing, and perform forward genetic screens to recover loci that fail to heal properly. These studies will address directly those aspects of epithelial wound healing that are conserved over the 0.5 to 1 billion years of evolution that separate insects from mammals. Together, they promise to bring the powerful tools of Drosophila genetics and molecular genetics to bear on this important biological problem.

DESCRIPTION (provided by applicant): Quantitative analysis of dorsal closure in Drosophila establishes that this model cell sheet movement depends on the contribution of three distinct biological processes (M.S. Hutson et al 2003 Science 300:145 and references therein). The three processes include contractility in a supra-cellular purse-string at the leading edge of the lateral epidermis; contractility of the amnioserosa; and celt sheet zipping, which in native closure maintains curvature and allows the purse string to maintain curvature and contribute force - favor closure. A fourth process produces tension in the lateral and ventral epidermis that opposes closure. Dorsal closure is robust and resilient: the individual forces that contribute to closure are far in excess of the net, applied force and closure proceeds at near native rates even after the removal of one of the forces that usually contributes. Here we focus on applying the laser-surgical and quantitative-modeling tools that we have developed in order to explore in greater detail the cellular and molecular mechanisms of cell sheet morphogenesis in this model system. By applying these methods to the analysis of mutants that fail to complete closure (mutations in so-called DC genes), we test the following hypotheses. That nonmuscle myosin II provides contractile force for the supra-cellular purse-string, the amnioserosa and the lateral epidermis. That quantitative analysis will reveal how other DC genes contribute to the process. That zipping requires genes whose homologs contribute to focal adhesion and adherens junction formation in vertebrates. And finally, that the relative balances of forces that contribute to dorsal closure are regulated through the function of mechanically gated channels and/or components of focal adhesions or junctional complexes. We speculate that these studies on cell sheet morphogenesis in Drosophila will provide insight into the cellular and molecular basis for the biological processes that coordinate cell shape changes in vertebrate morphogenesis and wound healing.

[unreadable] DESCRIPTION (provided by applicant): We propose a multidisciplinary research program that investigates the function of protein complexes at the heart of auditory reception in the model system, Drosophila melanogaster. The biology of hearing is astonishingly conserved between humans and fly: both the transcription factors that specify the development of organs of auditory reception and the molecular machines that transduce sound into neural signals are highly conserved. In both systems, cellular differentiation for the perception of sound requires elaborate subcellular arrangements of cytoskeletal elements, including actin filaments and microtubules. Mutations in the human and mouse orthologs of ck/myoVIIA and 10A/myoXV demonstrate the key roles these proteins play in auditory reception and vestibular function, but the molecular mechanism(s) by which these motor proteins contribute to these processes remain controversial. We propose an interdisciplinary approach to structure/function analysis of ck/myoVIIA, 28B/myoVIIB and 10A/myoXV (together, the myosin VII subfamily) in Drosophila where we use uniquely powerful molecular genetic tools to relate protein structure-function studies in vitro to structure/function analyses in vivo. We have already shown that ck/myoVIIA mutates to lethality, is required to drive changes in the structure of the actin cytoskeleton during morphogenesis and is required for auditory reception in adult flies. We have demonstrated that 10A/myoXV is essential for fly viability. We propose three specific aims 1) that address the cellular mechanisms of motor protein function; 2) that identify the binding partners with which these motor proteins interact; and 3) elucidate the structure of their cargo binding tails. Our specific aims will inform one another, but do not depend on one another's success. Together, our studies offer unique opportunities to probe the supramolecular protein complexes that require motor protein function and that drive cellular function in auditory reception, vestibular function and morphogenesis. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Dorsal closure in Drosophila is a key model system for cell sheet movements during development and wound healing. During closure two sheets of lateral epidermis advance to close a dorsal opening. Forces for closure are produced by a unique array of actomyosin in the leading edges of each sheet of lateral epidermis and by two distinct arrays of actomyosin in the amnioserosa, which fills the dorsal opening. Supra-cellular actomyosin rich purse strings (PSs) provide tension in the leading edges (leading edge cells oscillate during the bulk of closure, then shorten as they are zipped into the canthi). In the amnioserosa, junctional belts (JBs) and apical medial arrays (AMAs) drive both oscillations in cell shape and then ingression when individual cells apically constrict and drop out of the plane of the amnioserosa. This proposal focuses on how these actomyosin arrays generate force, how force generation is regulated and on identifying the molecular players that characterize the cells that contribute to both processes. We use high-resolution confocal imaging of genetically encoded fluorophore-fusions in living embryos coupled with laser surgical, mechanical jump protocols to interrogate mutant, drug-treated and wildtype specimens. Confocal images are segmented and digitally analyzed. Biophysical reasoning is used to develop quantitative models that describe our results, make testable hypotheses and refine models for further rounds of experiment and analysis. We also implement new candidate and systems approaches to identify the molecular players that characterize the cells that contribute to closure. We seek answers to key extant questions about dorsal closure and morphogenesis in other cell sheets: How are forces produced at the cell and molecular level? How are such forces regulated? Which transcripts/proteins are present in the cells that drive closure? These studies are at the next frontier in understanding morphogenesis in dorsal closure and in the myriad of other systems that require apical constrictions for powering changes in cell sheet morphology.

DESCRIPTION (provided by applicant): Understanding the mechanisms by which cells produce, transmit and sense forces constitutes a next frontier in cellular and developmental biology. Major advances have improved our understanding of the biophysics of chemomechanical force production by motor proteins in vitro. Nevertheless, how such proteins collaborate with the dynamic cytoskeleton in vivo to produce forces that drive cell shape changes for morphogenesis, cytokinesis and cell locomotion is unclear. Moreover, how such forces cause changes in gene expression and signaling also remain challenging unanswered research questions. We propose to construct and characterize a family of genetically encoded myosin force sensors designed to quantitatively map forces in vivo with subcellular resolution. Our approach is based on successful force sensors in vinculin and ¿-actinin. Our focus is on the forces produced by ensembles of nonmuscle myosin II in Drosophila, which are essential for cytokinesis and morphogenesis. Specific Aim 1: We will construct genetically encoded Tension Sensing Modules (TSMs) based on Fluorescence Resonance Energy Transfer (FRET) pairs separated by protein springs. We will systematically test a) different FRET pairs; b) different protein springs; and c) different locations in the myosin motors. These myosin force sensors will be expressed in both fly cell lines and transgenic flies. Specific Aim 2: We will characterize the myosin force sensors (MFSs) in fly cell lines. We will evaluate their ability a) to localize with endogenous myosin; b) to sense tension, using force traction microscopy on flexible substrates; and c) to respond to acute changes in tension induced by drugs or laser surgery. Appropriate control constructs will verify that changes in FRET are due to changes in force across the sensor. Specific Aim 3: We will characterize the myosin force sensors in transgenic flies. We will evaluate their ability a) to rescue myosin function in specimens depleted of endogenous myosin; b) to sense tension in dorsal closure (myosin II) and c) to report acute changes in tension induced by drugs or laser surgery. Specific Aim 4: We will calibrate the myosin force sensors that work in cultured cells and in fly specimens using single-molecule and/or solution strategies in vitro. Our experiments will deliver optimized myosin force sensors and the first measurements of the forces produced by ensembles of myosin in vivo. They will provide new information about the biophysics of cell shape changes in cytokinesis and morphogenesis. Because myosins play key roles in a variety of processes fundamental to human homeostasis and development, we expect these force sensors to have broad impact. Moreover, they will contribute to the resolution of several existing controversies regarding the role of myosins in biology. PUBLIC HEALTH RELEVANCE: Living cells are the fundamental building blocks of all life and respond to and influence their environments in a variety of ways. Here, we focus on how cells generate and respond to mechanical forces by proposing to design and implement genetically encoded tension sensing modules that will allow us monitor when and where cells generate forces as they grow, divide, crawl and change shape. This multidisciplinary research will impact human health because it addresses the basic biological problem of how myosin motor proteins produce forces for development and homeostasis.

This study will test the hypothesis that awarding of tenure represents an opportunity to nurture innovation as well as non-incremental and novel research pursuits. Development of faculty as highly successful researchers and educators is a critical goal of all universities. Considerable time and effort are invested into recruiting and mentoring exceptionally promising junior faculty. Tenure recognizes the most promising faculty and provides long term security and stability to develop their research ideas. However, less attention is focused upon post-tenure faculty development and the formulaic tenure process in most universities discourages risk associated with pursuing innovative research. This project will test a program to effect a refocusing of research direction in newly tenured faculty, with the goal of increasing their desire and ability to engage with significant problems.

This study seeks to leverage achievement of tenure as a pivot point for inducing reflection, with the goal of stimulating newly tenured faculty to engage with novel research directions with increased potential for societal impact. The study will involve development of a unique, interdisciplinary program, the Faculty Springboard, targeting recently tenured associate professors drawn from the sciences and engineering at Duke University to facilitate the exploration of new and potentially groundbreaking research initiatives. The program will consist of: 1) an annual innovation workshop, focusing on community building, networking and brainstorming; 2) professional coaching and mentoring to further develop faculty?s novel research ideas; 3) a follow-up workshop to cement project development. Program delivery and effectiveness will be assessed through a comprehensive evaluation plan. Best practices will be institutionalized to ameliorate societal impact achieved through faculty research.

The anticipated Broader Impact of this research is the expansion of research programs focused on addressing societal challenges. Through developing a faculty network and skillsets in support of creative risk-taking for innovative research, this professional development model has the potential to enhance researchers? abilities to address "wicked problems". By establishing proof of principle at Duke through targeted iterative evaluation, core essential components critical to success of this model program will be identified and disseminated for adaptation by other universities.

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.