The actin cytoskeleton is a large-scale network that fills all cells of the human body. Through its dynamic interaction with myosin motors is it involved in virtually any cellular process and aberrant function of the actomyosin interaction is linked to numerous human diseases including cancers and diseases of the heart and blood. The Research Strategy of the proposed work is to determine the molecular details by which the virtually uncharacterized human hematopoietic myosin-1G undergoes a calmodulin-mediated dimerization to crosslink and bundle actin (Aim 1), to attribute kinetic parameters and actin binding properties to myosin-1G monomers and dimers (Aim 2), and to describe the physiological significance of the myosin-1G monomer-dimer equilibrium at the immunological synapse of lymphoid T-cells (Aim 3). Completion of the proposed Research Strategy will produce critical insights into a novel mode of myosin dimerization that causes the formation of emerging higher-order actin structures that will fundamentally advance our understanding of the interaction between myosin and actin at the molecular level. The candidate Dr. Heissler is currently a postdoctoral fellow at the NIH/NHLBI in the laboratory of Dr. James Sellers. Through this K22 Career Development Award proposal, the candidate seeks to systematically acquire additional mentored research training and career development training at the NIH/NHLBI through a detailed Career Development Plan designed to complement her current skill set. With the continued support of members of her Advisory Committee, the K22 Career Development Award will establish a training framework to initiate the research program in preparation for the candidate's independent career. Central part of the intramural phase of the K22 award will be the candidate's Advisory Committee that will evaluate her progress on the proposed research and career development training as outlined in the detailed Career Development Plan. The scientific training will support the proposed Specific Aims through a combination of specialized course work and hands-on training to complete the proposed innovative Research Strategy. New scientific skills that will be acquired include general training in cell biology techniques to study dynamic aspects of the actomyosin cytoskeleton including training in the in vitro activation of T-cells and the use of the modern genome-editing technique CRISPR for gene knockout. This specialized training in cell biology combined with training in super-resolution microscopy techniques to image single molecules in vitro and in cells with exceptional spatiotemporal resolution will systematically complement the candidate's current interdisciplinary skill set in biochemical and biophysical techniques and allow her to bridge the gap between in vitro single- molecule studies in reconstituted systems of purified proteins and live cells. Importantly, the techniques and approaches developed during the funding period of the award will not only advance our understanding of the interaction between myosin-1G and actin but also allow for the successful completion of the proposed Research Strategy. This will establish the basis of the candidate's first NIH R01 and additional independent funding applications upon her transition to independence. Besides the scientific training, the candidate will experience extensive career and professional training in the intramural phase of this award to master academic challenges in the extramural phase of the award. Training activities such as mentoring and teaching will be complemented with training in management and leadership in form of seminars and workshops. The professional career development training also involves mentoring of a post-baccalaureate student, participation in grant-writing workshops, development of communication skills, career counseling and assessment coaching to prepare the candidate at best for her transition to independence and her long-term goal of becoming a successful independent investigator at a prestigious academic institution.
This proposal will explore the synergistic interaction between myosin-1G and actin at the molecular level. The unique feature of myosin-1G to dimerize and promote the formation of emerging higher-order actin structures will be dissected with a multi-tiered approach of biochemical, biophysical, and cell biological methods combined with super-resolution microscopy techniques. This proposal will bridge the gap between studies of single molecules in reconstituted systems of purified proteins and live cells to advance our knowledge of how the virtually uncharacterized myosin-1G interacts with the actin cytoskeleton in immune cells in health and disease.
