RIG: Postsynaptic Molecular Assembly at the Onset of Neuromuscular Synaptogenesis in vivo

由于胚胎中的通路有限，很少在活细胞中分析突触的启动机制。研究者在果蝇活体胚胎中使用荧光标记生物探针的三维和四维成像，观察到在神经肌肉突触发生之前，丝状突起（肌足）从肌肉细胞向神经元延伸。这种肌足的出现被认为与运动神经元的生长相吻合，这表明突触伙伴在形成永久接触之前相互作用的机制。随着发育的继续，可以观察到来自匹配伴侣的突触前和突触后丝状足的短暂稳定。这个肌群事件是当前项目的重点。研究者将测试“突触成核”假说，该假说认为肌聚簇是一个亚细胞信号室，新生的突触在其中进行协调。利用体内成像和免疫细胞化学，研究人员将研究参与突触伴侣诱导的肌团簇的潜在分子，特别是将研究这些蛋白质如何被招募到肌团簇室。该项目的目标是通过识别突触形成部位的突触后分子参与者，并表征它们被招募的机制，从而更好地了解突触配对和突触发生。曾经被认为是被动参与者的肌肉细胞正在成为主动的突触形成伙伴，肌间的相互作用为剖析导致突触成功形成的分子整合提供了机会。波多黎各马亚圭斯大学生物系超过99%的本科生被确定为科学领域代表性不足的群体的成员，该提案将支持对大量少数民族学生的培训。他们的研究经验将通过获得导师和榜样的机会而得到加强，这将由研究者促进。

The mechanisms that initiate a synapse have rarely been analyzed in living cells due to limited access in embryos. The investigator has previously observed filopodial processes (myopodia) extending from muscle cells toward neurons prior to neuromuscular synaptogenesis, using three- and four-dimensional imaging of fluorescent-labeled bioprobes in live Drosophila embryos. This appearance of myopodia was seen to coincide with motoneuron outgrowth, suggesting a mechanism in which synaptic partners interact before they make permanent contact. As development continues, transient stabilization of pre- and post-synaptic filopodia from matched partners can be observed. This myopodial clustering event is the focus of the current project. The investigator will test the "synaptic nucleation" hypothesis, which states that myopodial clustering serves as a sub cellular signaling compartment where the nascent synapse is coordinated. Using in vivo imaging and immunocytochemistry, the researcher will study the underlying molecules involved in synaptic partner-induced myopodial clustering, and in particular, will investigate how these proteins are recruited to the myopodial cluster compartment. The goal of this project is to gain a better understanding of synaptic matchmaking and synaptogenesis by identifying the postsynaptic molecular players at the site of synapse formation and to characterize the mechanism by which they are recruited. Once considered as passive players, muscle cells are emerging as active synaptogenic partners, and myopodial interactions offer a chance to dissect the molecular integrations that lead to the successful formation of the synapse. With over 99% of the total undergraduate student enrollment in the Department of Biology at the University of Puerto Rico Mayaguez identified as members of underrepresented groups in the sciences, this proposal will support training of a significant number of minority students. Their research experiences will be enhanced through access to mentors and role models which will be facilitated by the investigator.