Signaling Roles of Intermediate Filament Isoforms in Neural Development

这个项目旨在通过解决生物学中一个悬而未决的问题来推进科学知识：创造不同类型细胞的分子原理是什么？研究集中在一类以细胞类型特有的模式表达的蛋白质，即中间丝(IF)蛋白质。该项目的重点是大脑中的神经元，这些神经元在发育成熟的形状时会转换亚型，包括将长轴突延伸到大脑中的正确位置。这项研究旨在确定神经元在生长过程中为什么会从一种IF亚型切换到另一种IF亚型。该项目还将扩大影响，促进预期的社会成果：1)扩大STEM领域的教育，以支持更有见识和更有科学素养的公众，2)增加STEM工作人员的多样性和包容性。特别是，该实验室的目标是让妇女、残疾人和代表性不足的少数群体充分参与各级科技教育和教育。该项目资助每年夏天为一名代表不足的少数族裔学生提供本科生研究经验。由该项目资助的REU通过研究不同的中间细丝在轴突生长中的作用，提供了实验室研究的实践经验。此外，被选中的REU学生将参加弗吉尼亚大学设立的SRIP(暑期研究实习计划)，该计划为个人和团体在STEM寻求职业生涯提供专业技能培训和指导。SRIP有成功地将参与的本科生研究人员安排到STEM职业生涯的历史。该项目试图发现神经元中从巢蛋白亚型到神经丝亚型的转换如何影响微管细胞骨架的信号，以调节生长锥结构和运动性。PI的实验室最近发现IF蛋白Nestin调节生长锥的形态。此外，Nestin还改变了生长锥体对指导信号Sema3a的反应。它通过结合激酶CDK5和它的底物微管相关蛋白DCX来实现这一点，从而增加DCX的磷酸化。随着神经元的成熟，它们会从巢蛋白亚型转换为神经细丝，神经丝结合DCX而不是CDK5。该项目将检验这一新的假设，即神经元IF通过Sema3a信号下游的DCX调节微管动力学和轴突生长锥体中的捆绑。目标1将发现，如果转换影响轴突微管和生长锥体行为，将如何实验。目标2将发现如果转换影响皮层神经元的Sema3a反应是如何实验的。该项目将在培养的皮质神经元中使用最先进的成像方法，结合不同IF亚型的下调和过度表达，以揭示不同IF亚型在轴突生长期间调节生长锥体微管动力学的机制。在发育过程中，生长锥对细胞外信号的反应不仅受到可用的信号、表面表达的受体、细胞内底物的影响，而且还受到表达的IF亚型的影响。PI建议，神经元IF亚型可以充当无处不在的激酶级联的放大器或阻尼器，以提供局部和细胞类型的效应器反应的特定调节。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

This project aims to advance scientific knowledge by addressing an unanswered question in biology: what are the molecular principles that create different types of cells? The research focuses on a class of proteins which are expressed in strikingly cell-type specific patterns, the intermediate filament (IF) proteins. The project focuses on neurons in the brain which are known to switch IF subtype as they develop their mature shape, including extending long axons to the correct location in the brain. The research aims to determine why neurons switch from one IF subtype to another as they grow. The project will also broaden the impact by contributing to desired societal outcomes: 1) broadening education in STEM fields to support a better informed and more scientifically literate public, and 2) increasing the diversity and inclusivity of the STEM workforce. Especially, the lab aims for full participation of women, persons with disabilities, and underrepresented minorities in STEM at all levels. The project funds research experiences for undergraduates (REU) for one underrepresented minority student during each summer. The REU funded by this project provides hands-on experience in laboratory research by investigating the roles of diverse intermediate filaments in growing axons. In addition, the selected REU student will participate in the institutionally established SRIP (summer research internship program) at the University of Virginia which provides professional skill training and guidance for pursuing a career in STEM in individual and group settings. SRIP has a history of successfully placing participating undergraduate researchers into STEM careers.This project seeks to discover how developmental IF subtype switching from nestin to neurofilaments in neurons affects signaling to the microtubule cytoskeleton to regulate growth cone structure and motility. The PI’s lab recently discovered that the IF protein nestin regulates growth cone morphology. In addition, nestin changes the response of growth cones to the guidance cue Sema3a. It does so by binding the kinase Cdk5 and its substrate, the microtubule associated protein DCX, thereby increasing DCX phosphorylation. As neurons mature, they switch IF subtype from nestin to neurofilaments, which bind DCX but not Cdk5. The project will test the novel hypothesis that neuronal IFs regulate microtubule dynamics and bundling in axonal growth cones via DCX downstream of Sema3a signaling. Aim 1 will discover how experimental IF switching affects axonal microtubules and growth cone behavior. Aim 2 will discover how experimental IF switching affects Sema3a responses in cortical neurons. The project will use state-of-the-art imaging approaches in cultured cortical neurons in combination with downregulation and overexpression of different IF subtypes to uncover the mechanisms by which different IF subtypes modulate microtubule dynamics in growth cones during axon outgrowth. Responsiveness to extracellular cues by growth cones during development is influenced not only by the cue available, the receptors expressed on the surface, the intracellular substrates present, but also by the IF subtypes expressed. The PI proposes that neuronal IF subtypes can act as either amplifiers or dampeners of ubiquitous kinase cascades to provide local and cell-type specific regulation of effector responses.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.