Calcium and Rho GTPase Crosstalk in Growth Cone Motility

在神经发育过程中，长轴突和树突前缘的活动生长锥利用环境信号导航到它们的突触目标。虽然已经确定了许多可扩散的、细胞表面和细胞外基质结合的引导信号及其受体，但对它们激活的下游细胞内信号转导级联却知之甚少。胞质钙离子（Ca2+）是一种对轴突和树突生长具有特别不同影响的细胞内信号。生长锥细胞内Ca2+的短暂升高可以促进、抑制或定向运动，这取决于这些信号的大小、持续时间和分布，以及下游可用的Ca2+依赖靶标。有趣的是，Ca2+进入的模式也被发现决定了一些反应的特异性。Ca2+对神经突生长的不同影响可能是由于生长锥表达的许多细胞骨架调控靶点。重要的是，最近的证据表明，Ca2+信号可能影响一些rho家族的小G蛋白的活性。rho家族gtpase是生长锥中肌动蛋白细胞骨架的有效和多样化的调节剂，可能是Ca2+信号和生长锥运动之间的关键中介。Gomez博士假设通过质膜通道的Ca2+内流与细胞内受体储存的释放调节生长锥局部微域内Ca2+传感器的不同亚群。此外，通过不同通道的Ca2+运动激活的下游信号通路将对生长锥运动产生相反的影响。本研究的目的是首先描述改变一般和特定Ca2+内流和释放途径对神经突生长的影响。接下来，将测量这些Ca2+变化对三种不同Rho GTPase活性的影响，并确定GTPase功能对运动的必要性。Gomez博士研究的长期目标是了解特定的Ca2+信号如何协调控制生长锥寻路的下游目标的空间和时间激活。了解Ca2+对生长锥运动的不同影响的分子机制将支持发育障碍和神经损伤的治疗策略。来自三个不同研究生项目的五名研究生和两名本科生是戈麦斯实验室的现任成员。拟议的项目将直接涉及两名研究生，但所有学生都将获得该奖项的间接支持。学生在实验室训练作为发育神经生物学家使用分子和生物光子为基础的方法。学生在研究、课程作业、期刊俱乐部、实验室会议、研究生项目小组会议和国际会议中的参与反映了这一重点。除了接受培训外，研究生还需要教初级研究生、本科生或某些情况下的高中生。目前有几名学生参加了大学组织的教学项目。戈麦斯博士也是卡内基博士计划（CID）计划委员会的积极成员。如果授予神经科学培训计划，CID将是一个多年的研究计划，旨在丰富和振兴博士生的教育。最后，作为生物光子学集群的一员，Gomez博士与生理、电子和计算机工程等其他部门的实验室合作。这些合作的重点是技术转让，根据该协议，戈麦斯实验室提供成像专业知识以及使用实验室的现代化成像设施。

During neural development motile growth cones at the leading edge of elongating axons and dendrites use environmental cues to navigate to their synaptic targets. While many diffusible, cell surface and extracellular matrix bound guidance cues and their receptors have been identified, relatively little is known of the downstream intracellular signal transduction cascades they activate. One intracellular signal that has particularly diverse effects on axonal and dendritic growth is cytosolic calcium ions (Ca2+). Transient elevations of intracellular Ca2+ in growth cones can promote, inhibit or orient motility depending on the size, duration and distribution of these signals, as well as on the downstream Ca2+-dependent targets available. Interestingly, the mode of Ca2+ entry has also been found to determine the specificity of some responses. The diverse effects of Ca2+ on neurite outgrowth are likely due to the many cytoskeletal regulatory targets expressed by growth cones. Importantly, recent evidence suggests that Ca2+ signaling may influence the activity of some of the Rho-family of small G proteins. Rho-family GTPases are potent and diverse regulators of the actin cytoskeleton in growth cones and could be key intermediaries between Ca2+ signals and growth cone motility. Dr. Gomez hypothesizes that Ca2+ influx through plasma membrane channels versus release from intracellular receptor stores regulate distinct subsets of Ca2+-sensors within local microdomains of growth cones. Further, the downstream signaling pathways activated by Ca2+ movement through different channels will have opposing effects on growth cone motility. The aim of this study is to begin by characterizing the effects of altering general and specific Ca2+ influx and release pathways on neurite outgrowth. Next, the effects of these Ca2+ changes on the activity of three different Rho GTPases will be measured and the necessity of GTPase function for motility determined. The long-term goal of Dr. Gomez's research is to understand how specific Ca2+ signals orchestrate the spatial and temporal activation of downstream targets that control growth cone pathfinding. Knowing the molecular mechanisms through which Ca2+ exerts such varied effects on growth cone motility will support treatment strategies for developmental disorders and neural injury.Five graduate students from three different graduate programs and two undergraduates are current members of the Gomez Lab. The proposed project will directly involve two graduate students, but all students will receive some indirect support from this award. Students in the lab are trained as developmental neurobiologists using molecular and biophotonic based approaches. Student participation in research, as well as coursework, journal clubs, lab meetings, graduate program subgroup meetings, and international meetings reflect this focus. In addition to receiving training, graduate students are expected to teach junior graduate students, undergraduates or in some cases high school students. Several students are currently involved in University organized teaching programs. Dr. Gomez is also an active member of the Carnegie Initiative on the Doctorate (CID) planning committee. If awarded to the Neuroscience Training Program, CID will be a multi-year research program aimed at enriching and invigorating the education of doctoral students. Finally, as a member of the Biophotonics cluster hire, Dr. Gomez collaborates with labs in other departments such as Physiology and Electrical and Computer Engineering. These collaborations focus on technology transfer and as indicated by this agreement the Gomez lab provides imaging expertise as well as access to the lab's modern imaging facilities.