Regulation of Endothelial Cell Branching Morphogenesis via MCAK-targted Control

通过 MCAK 靶向控制调节内皮细胞分支形态发生

• 批准号: 8734610
• 负责人: Kenneth Albert Myers
• 金额: $ 24.48万
• 依托单位: UNIVERSITY OF THE SCIENCES PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-23 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8734610
• 关键词: Actins; Advisory Committees; Binding; Biological; Blood Vessels; Book Chapters; Catalysis; Cells; Characteristics; Collagen; Communication; Coupling; Cues; Cytoskeleton; Data; Data Analyses; Development; Diabetic Retinopathy; Dimensions; Disease; Dominant-Negative Mutation; Down-Regulation; Educational process of instructing; Embryo; Endothelial Cells; Environment; Experimental Designs; Extracellular Matrix; Fostering; Frequencies; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Human; Image; Immigration; Injury; Interphase; K-Series Research Career Programs; Laboratories; Life; Link; Location; Maintenance; Marines; Measures; Mediating; Mentored Clinical Scientist Development Award (K08); Mentors; Microtubules; Mitosis; Molecular; Molecular Motors; Morphogenesis; Morphology; Motor; Movement; Myosin ATPase; National Heart, Lung, and Blood Institute; Neoplasm Metastasis; Neurodegenerative Disorders; Neurons; Pathway interactions; Pattern; Peer Review; Phase; Phosphorylation; Physiological Processes; Physiology; Placenta; Plus End of the Microtubule; Pregnancy; Process; Proteins; Publications; Regulation; Research; Research Proposals; Research Training; Resolution; Role; Science; Signal Pathway; Signal Transduction; Site; Software Design; Specificity; Speed; Spinal cord injury; Students; Takeda brand of pioglitazone hydrochloride; Techniques; Testing; Training; United States National Institutes of Health; Vascular Endothelial Cell; Work; Wound Healing; angiogenesis; base; blebbistatin; career development; cell motility; extracellular; genetic regulatory protein; human STK6 protein; light microscopy; meetings; migration; mutant; neuron development; research study; response; skills; small hairpin RNA; spatiotemporal; teacher; tumor growth

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.

描述（由申请人提供）：候选人的论文研究是在博士彼得W。Baas等人的研究成果，并致力于通过分子马达蛋白鉴定微管转运的机制。这项工作有助于理解神经元发育过程中分子马达蛋白的调节和协调，并刺激了针对这些相同马达蛋白的持续研究，因为它们在神经退行性疾病和创伤性脊髓损伤中发挥功能和发生故障。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，并导致发表了6篇同行评审文章，2篇评论文章和2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。博士后研究工作旨在通过靶向微管动态不稳定性的局部调节来了解控制内皮细胞分支形态和血管发育的机制;具体而言，微管动力学如何由来自细胞外基质的物理接触引发的信号驱动。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，这些研究表明，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与3D）来预测，并表明微管调节蛋白必须对来自ECM的物理信号做出区域特异性反应，以驱动富有成效的内皮细胞分支。职业发展奖将在NIH/NHLBI提供持续培训，并支持在完成校内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，以支持本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。在职业发展奖的校内阶段将支助的具体活动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意见。在职业发展奖的校内期间的培训还将涉及在实验，解释和沟通技能，实验培训，包括进一步开发基于MatLab的软件和微制造模式的设计后学士学位学生的辅导。候选人在实验设计和技术方面的培训将与候选人作为教师和导师的培训一起进行，包括教学实验技术，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。职业发展奖的校内期间还将涉及在当地会议和公开演讲中交流和介绍拟议研究计划中的实验结果。拟议研究计划中的实验将研究如何在内皮细胞血管生成过程中实现微管动力学的局部调节，这是一个在整个生命过程中发育和维持人类血管系统所需的生理过程。血管生成严重依赖于内皮细胞分支，这是一个由来自Rac 1和RhoA GTP酶的信号线索驱动的过程，Rac 1和RhoA GTP酶协调微管和肌动蛋白细胞骨架的组织。除了这些信号线索，微管和肌动球蛋白组织可以修改的刚度和尺寸的细胞外基质。微管动力学调节信号线索的会聚表明，Rac 1信号传导、细胞外基质信号传导或两者都必须控制特定的因子， 在内皮细胞分支过程中调节微管动力学。如何实现这种调节尚不清楚。MT动力学的一个有针对性的调节剂是MT突变因子，MCAK，其定位于生长的MT末端，直到发出信号以催化MT分解，从而实现MT动力学的时空调节。在有丝分裂过程中，MCAK介导的MT突变催化是磷酸化调节的，但细胞质MCAK在生长的MT末端的调节及其在介导EC血管生成中的作用仍有待阐明。本申请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，首先确定时空Rac 1介导的MCAK对MT动力学和EC分支形态的调节，然后确定2D和3D胶原ECM的细胞参与如何通过肌球蛋白II依赖性和非依赖性途径靶向和调节MCAK，以驱动生产性EC分支形态发生和定向迁移。