Kenneth A. Myers, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Variations in the configuration and behavior of microtubules within specific regions of the neuron serve as the basis for critical events in axonal and dendritic differentiation. Diseases of the nervous system are characterized by abnormal neuronal morphologies attributable to the improper configuration and malorientation of the microtubule array. Both mitotic and postmitotic cells employ molecular motor proteins to transport vital cargoes according to the needs of the cell, as well as to organize and reconfigure microtubules in response to environmental cues. Questions remain as to which motor proteins are responsible for engineering complex patterns of microtubule organization, and exactly how the balance of motor forces contributes to establishing the neuronal microtubule array. This application proposes to (1), use high resolution imaging to confirm that short microtubules are in transit down the axon (2), test the hypothesis that a small number of mitotic motor proteins are the key players in axonal microtubule transport and (3), determine whether different categories of microtubule transport have specific functional roles in axonal development in response to morphological guidance cues. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The candidate's thesis research was performed in the laboratory of Dr. Peter W. Baas, and was directed towards identifying the mechanisms of microtubule transport by molecular motor proteins. This work has aided in the understanding of molecular motor protein regulation and coordination during neuronal development, and has spurred continuing studies targeting these same motor proteins as they function and malfunction in both neurodegenerative diseases and traumatic spinal cord injury. Research training in the Baas lab involved many diverse molecular and cellular biological techniques, and resulted in the publication of six peer-reviewed articles, two review articles, and two book chapters. The transition from neuronal studies of microtubules and molecular motor proteins to high-resolution imaging in endothelial cells was intuitive. Postdoctoral research studies were directed toward understanding mechanisms controlling endothelial cell branching morphology and vascular development by targeting local regulation of microtubule dynamic instability; specifically, how microtubule dynamics are driven by physical, contact-initiated signals from the extracellular matrix. These studies revealed that during angiogenesis, the formation and extension of cell branches by endothelial cells is directly related to the regulation of their microtubule growth speeds. Moreover, these studies revealed that microtubule growth speeds and endothelial cell branching can be predicted by the stiffness and dimensionality (2D vs. 3D) of the extracellular matrix, and suggest that microtubule regulatory proteins must respond to physical signals from the ECM with regional specificity to drive productive endothelial cell branching. The Career Development Award will provide continued training at NIH/NHLBI and support the goal of transitioning the proposed research plan to an independent laboratory upon the completion of the intramural phase. The Career Development Award will guide focused training at NIH to support the proposed Specific Aims in this application, as well as foster development as a mentor and teacher of science. Specific activities that will be supported during the intramural phase of the Career Development Award will include the formation of a designated Advisory Committee, responsible for evaluating progress of the proposed research plan as well as providing career development advice. Training during the intramural period of the Career Development Award will also involve mentoring of a post-baccalaureate student in experimental, interpretive, and communication skills, experimental training including further development of MatLab-based software and design of micro-fabricated patterns. The candidate's training in experimental design and technique will take place alongside the candidate's training as a teacher and mentor, including teaching experimental technique, data analysis and interpretation, and public presentation of results in the physiology course at the Marine Biological Laboratories. The intramural period of the Career Development Award will also involve the communication and presentation of results obtained from the experiments in the proposed Research Plan at local meetings and public presentations. The experiments in the proposed Research Plan will investigate how the localized regulation of microtubule dynamics is achieved during the process of endothelial cell vascular angiogenesis, a physiological process required for the development and maintenance of human vasculature throughout life. Angiogenesis is critically dependent upon endothelial cell branching, a process driven by signaling cues from the Rac1 and RhoA GTPases that coordinate the organization of the microtubule and acto-myosin cytoskeletons. In addition to these signaling cues, microtubule and acto-myosin organization can be modified by the stiffness and dimensionality of the extracellular matrix. The convergence of signaling cues on the regulation of microtubule dynamics suggests that Rac1 signaling, extracellular matrix signaling, or both, must control specific factors capable of regulating microtubule dynamics during endothelial cell branching. How such regulation is achieved is not known. One targeted regulator of MT dynamics is the MT catastrophe factor, MCAK, which localizes to growing MT ends until signaled to catalyze MT disassembly, thereby enabling spatiotemporal regulation of MT dynamics. During mitosis, MCAK-mediated catalysis of MT catastrophe is phospho-regulated, yet the regulation of cytoplasmic MCAK at growing MT ends, and its roles in mediating EC angiogenesis remain to be elucidated. The studies proposed in this application will use live-cell, high-resolution light microscopy and automated tracking of MT dynamics to first identify spatiotemporal Rac1-mediated regulation of MCAK on MT dynamics and EC branching morphology, and will then determine how cell engagement of 2D and 3D collagen ECMs target and regulate MCAK via myosinII-dependent and -independent pathways to drive productive EC branching morphogenesis and directed migration.

Project Summary/Abstract The coordinated and regulated remodeling of the actin and microtubule (MT) cytoskeleton is required for cell migration for developmental processes and homeostatic maintenance, as well as during the body?s response to external insults and disease states including heart disease and tumor metastasis. During cell migration, actin filaments assemble and become linked to focal adhesion (FA) complexes, while MTs undergo dynamic instability that is locally controlled by MT- associated proteins (MAPs). These two processes enable cells to establish a leading-edge and a trailing-edge, and to migrate with directional persistence. Despite many advances in our understanding of the functional implications of MAPs on MTs and MT-FA interactions, it remains unknown how exactly MT organization is spatially and temporally coordinated with FAs to promote directional cell movement. A recent discovery showing that non-centrosomal MTs are both sufficient and required to drive polarized cell migration has established a paradigm shift, suggesting that non- centrosomal MTs are primed to function in a way that is distinct from MTs nucleated by the centrosome. The finding underscores the need to determine how cytoskeletal proteins identify and regulate non-centrosomal versus centrosomal MT dynamics and effects on polarity and migration. This gap in knowledge impacts our understanding of fundamental processes, including how signaling molecules simultaneously regulate families of proteins to achieve complex tasks, such as guiding persistent cell migration. The small GTPase, Rac1, is a key signaling protein that is spatially controlled to promote FA formation, MT growth, and actin filament assembly, resulting in leading edge advance. Rac1 signaling is complemented by the molecular motor protein, myosin-II, which organizes actin stress fibers, promotes FA maturation, and generates forces that pull the trailing-edge of the cell forward. Thus, Rac1 and myosin-II are spatially and temporally controlled to drive directional cell movement. One targeted MT effector protein, MCAK, is locally inhibited by Rac1 to promote leading-edge MT growth and cell polarity, and MCAKs effects on MT dynamics are sensitive to myosin-II contractility. Despite this knowledge, how Rac1 and myosin-II contribute to the organization of FAs, MTs, and actin is not well understood. Preliminary evidence demonstrates that FA-associated MTs are predominantly of non-centrosomal origin and that Rac1 activity enhances the association of two different families of MAPs, CAMSAPs and septins, which increase non-centrosomal MT growth into FAs. Here, we will test the hypothesis that Rac1 and myosin-II promote the association of CAMSAP and septins with non-centrosomal MTs, which inhibits MCAK-mediated MT disassembly and drives MT-FA interactions. Our approach will incorporate a team of undergraduate researchers using fluorescence microscopy of live endothelial cells to determine: (1) how Rac1 and myosin-II regulate CAMSAP and septin interactions with non-centrosomal MTs, (2) how MCAK disassembly of non-centrosomal MTs controls MT dynamics and FA size, and (3) how septins promote MT growth into FAs. These investigations will provide critical advances to the field of cell migration by functionally linking Rac1 and myosin-II with cytoskeletal effector proteins that control non-centrosomal MT growth into FAs and the regulation of cell migration in health and disease. ! !