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.
Vascular angiogenesis is a critical life-long process required for embryonic vascular development, maturation of the placenta during pregnancy, and wound-healing following injury. Uncontrolled angiogenesis is responsible for diseases such as diabetic retinopathy, and is required for tumor growth and metastasis. Angiogenesis occurs when endothelial cells extend branches in response to signaling cues that guide directional migration and formation of new vasculature. Endothelial cell branching is guided by the dynamics of the microtubule cytoskeleton, yet the mechanisms by which microtubule dynamics and cell branching are regulated remain unknown. This research proposal will explore the regulation of microtubule dynamics during endothelial cell angiogenesis, to determine how endothelial cells modify their branching and migration in response to signaling cues from the surrounding environment.
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