Regulation of Endothelial Cell Branching Morphogenesis via MCAK-targted Control

描述(申请人提供)：候选人的论文研究是在Peter W.Baas博士的实验室进行的，旨在确定分子马达蛋白的微管运输机制。这项工作有助于了解神经元发育过程中分子马达蛋白的调节和协调，并推动了针对这些相同的马达蛋白在神经退行性疾病和创伤性脊髓损伤中的功能和故障的持续研究。BAAS实验室的研究培训涉及许多不同的分子和细胞生物学技术，并发表了六篇同行评议文章、两篇综述文章和两本书的章节。从微管和分子马达蛋白的神经元研究过渡到内皮细胞的高分辨率成像是直观的。博士后研究旨在了解通过局部调节微管动态不稳定性来控制内皮细胞分支形态和血管发育的机制；具体地说，微管动力学是如何由来自细胞外基质的物理接触引发的信号驱动的。这些研究表明，在血管生成过程中，内皮细胞的细胞分支的形成和延伸与其微管生长速度的调节直接相关。此外，这些研究揭示了微管生长速度和内皮细胞分支可以通过细胞外基质的硬度和维度(2D和3D)来预测，并表明微管调节蛋白必须响应来自ECM的具有区域特异性的物理信号来驱动高效的内皮细胞分支。职业发展奖将在NIH/NHLBI提供持续培训，并支持在完成内部阶段后将拟议的研究计划过渡到独立实验室的目标。职业发展奖将指导NIH的重点培训，以支持在这一申请中提出的具体目标，并促进作为科学导师和教师的发展。在职业发展奖内部阶段将支持的具体活动将包括成立一个指定的咨询委员会，负责评价拟议研究计划的进展情况并提供职业发展咨询。在职业发展奖的内部培训期间，还将包括指导毕业后学生的实验、解释和沟通技能，实验培训，包括进一步开发基于MATLAB的软件和微型制造图案的设计。候选人的实验设计和技术培训将与候选人作为教师和导师的培训同时进行，包括教授实验技术、数据分析和解释，以及在海洋生物实验室的生理学课程中公开展示结果。在职业发展奖的内部期间，还将在地方会议和公开陈述中交流和介绍拟议研究计划中的实验结果。拟议的研究计划中的实验将研究微管动力学的局部调节是如何在内皮细胞血管生成过程中实现的，内皮细胞血管生成是人体一生血管系统发育和维持所需的生理过程。血管生成严重依赖于内皮细胞分支，这一过程由协调微管和acto-myosin细胞骨架的rac1和RhoA GTP酶的信号信号驱动。除了这些信号信号外，微管和肌球蛋白组织还可以被细胞外基质的硬度和维度所改变。微管动力学调控中信号信号的趋同表明，rac1信号、细胞外基质信号或两者都必须控制特定的因子。 调节内皮细胞分支过程中的微管动力学。这种监管是如何实现的，目前尚不清楚。MT动力学的一个靶向调节因子是MT灾难因子MCAK，它定位于不断增长的MT末端，直到被信号催化MT分解，从而实现对MT动力学的时空调节。在有丝分裂过程中，MCAK介导的MT突变的催化是受磷酸调节的，但在生长的MT末端细胞质MCAK的调节及其在介导EC血管生成中的作用尚不清楚。这项应用中提出的研究将使用活细胞、高分辨率光学显微镜和MT动力学的自动跟踪来首先确定rac1介导的MCAK对MT动力学和EC分支形态的时空调节，然后将确定2D和3D胶原ECM的细胞参与如何通过肌球蛋白II依赖和独立的途径靶向和调节MCAK，以驱动有效的EC分支形态发生和定向迁移。

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.