Consequences of myosin-1G dimerization on its regulated interaction with actin

 描述（由申请人提供）： 项目摘要/摘要肌动蛋白细胞骨架是一个充满人体所有细胞的大规模网络。通过其与肌球蛋白马达的动态相互作用，它几乎参与任何细胞过程，并且肌动球蛋白相互作用的异常功能与许多人类疾病有关，包括癌症以及心脏和血液疾病。拟议工作的研究策略是确定几乎未表征的人类造血肌球蛋白-1G经历钙调蛋白介导的二聚化以交联和束肌动蛋白的分子细节（目标1），将动力学参数和肌动蛋白结合特性归因于肌球蛋白-1G单体和二聚体（目标2），并描述淋巴免疫突触中肌球蛋白-1G单体-二聚体平衡的生理意义T 细胞（目标 3）。所提出的研究策略的完成将对肌球蛋白二聚化的新模式产生重要的见解，这种模式会导致新兴的高阶肌动蛋白结构的形成，这将从根本上增进我们对分子水平上肌球蛋白和肌动蛋白之间相互作用的理解。候选人 Heissler 博士目前是 NIH/NHLBI James Sellers 博士实验室的博士后研究员。通过此 K22 职业发展奖提案，候选人寻求通过旨在补充其当前技能的详细职业发展计划，在 NIH/NHLBI 系统地获得额外的指导研究培训和职业发展培训。在顾问委员会成员的持续支持下，K22职业发展奖将建立一个培训框架来启动研究计划，为候选人的独立职业生涯做好准备。 K22 奖校内阶段的核心部分将是候选人的咨询委员会，该委员会将评估她在详细职业发展计划中概述的拟议研究和职业发展培训方面的进展。科学培训将通过专业课程工作和实践培训相结合来支持拟议的具体目标，以完成拟议的创新研究策略。将获得的新科学技能包括细胞生物学技术的一般培训，以研究肌动球蛋白细胞骨架的动态方面，包括体外激活T细胞的培训以及使用现代基因组编辑技术CRISPR进行基因敲除的培训。这种细胞生物学专业培训与超分辨率显微镜技术培训相结合，以卓越的时空分辨率对体外和细胞内的单分子进行成像，将系统地补充候选人目前在生物化学和生物物理技术方面的跨学科技能，并使她能够弥合纯化蛋白质和活细胞重建系统的体外单分子研究之间的差距。重要的是，在该奖项资助期间开发的技术和方法不仅将增进我们对肌球蛋白-1G 和肌动蛋白之间相互作用的理解，而且有助于成功完成拟议的研究策略。这将为候选人过渡到独立后的第一个 NIH R01 和其他独立资助申请奠定基础。除了科学培训之外，候选人还将在该奖项的校内阶段经历广泛的职业和专业培训，以应对该奖项的校外阶段的学术挑战。指导和教学等培训活动将辅之以研讨会和讲习班形式的管理和领导力培训。专业职业发展培训还包括指导学士学位后学生、参加资助写作研讨会、培养沟通技巧、职业咨询和评估辅导，以便为候选人过渡到独立做好最好的准备，并实现成为著名学术机构成功的独立调查员的长期目标。

 DESCRIPTION (provided by applicant): Project Summary/Abstract 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.