2011-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643445摘要：神經軸索分枝為擴展並修飾神經系統迴路連結的重要機轉。研究顯示胞外訊息和細胞骨架在此過程中扮演重要角色，但兩者之間的交互關係仍不清楚。微管為細胞骨架的基本要素，由a-和b-tubulin 蛋白聚合而成，負責許多重要的細胞生理功能。胞外訊息分子如Netrin 和Wnts 可藉由調控微管的結構來影響神經的極化和神經生長錐的活動性。這些證據暗示胞外訊息也可能經由類似的機轉來調控軸索分枝。mec-12 和mec-7 是兩個線蟲的基因，分別表現a-和b-tubulin 蛋白。我們初步的研究顯示，mec-12 和mec-7 作用於同一個分子路徑；在mec-12 和mec-7 突變株中，PLM觸覺神經元突觸分枝的生長被阻斷。我們也發現Netrin 和rpm-1 基因的突變有類似的軸索分枝缺陷。在本研究計畫中，我們試圖探討Netrin 和mec-12 或mec-7 之間的交互作用。我們也將探討rpm-1、mec-12 和mec-7 之間的交互作用。我們計畫探討微管結合蛋白和微管切割蛋白在控制軸索分枝中所扮演的角色。我們將利用熱休克啟動基因表現的方式，來探討tubulin 在軸索分枝過程中的作用時序。最後，我們會利用可受光激發而轉變散射光譜的螢光標記，來探討在神經軸索分枝的過程中，微管聚合物的動態變化。本計畫的結果將有助於我們進一步了解胞外訊息和微管動態變化調控神經軸索分枝之分子生物學機轉。<br> Abstract: Axon branching is an important mechanism to elaborate and refine the connectivity ofneuronal circuits. Various lines of evidence suggest that extracellular signals and cytoskeletalelements play critical roles in axon branching, but the mechanistic links between the two arefar from clear. Microtubules are polymers formed by a- and b-tubulin monomers and are anintegral part of the cytoskeleton. In addition to their critical roles in cell division, cell polarity,cell motility and intracellular transport, microtubules had recently emerged as an importantregulator for axon branching. It had been shown that extracellular cues, such as Netrin orWnts, control neuronal polarization or growth cone motility by regulating microtubuleconfigurations. These observations raise the intriguing possibility that extracellular signalingalso controls axon branching by regulating the dynamics of axonal microtubules.The C. elegans genes mec-12 and mec-7 encode the a- and b-tubulin, respectively, andare required for mechanosensation mediated by a group of microtubule-rich sensory neurons,the touch receptor neurons. Our observations suggest that mutations in mec-12 and mec-7disrupt synaptic branch development of the PLM touch neurons, and mec-12 and mec-7 act inthe same genetic pathway. In search of regulators of microtubule cytoskeleton in axonbranching, we found that elimination of the UNC-6/Netrin signaling and mutations in rpm-1,gene that encodes a neuronal E3 ligase, also disrupt PLM branching. Phenotypes caused byperturbation of UNC-6/Netrin or RPM-1 functions are indistinguishable from those of mec-12and mec-7 mutants, suggesting that Netrin signaling and RPM-1 are potential regulators ofmicrotubule cytoskeleton in PLM branching. In this proposal, we first investigate potentialgenetic interactions between mec-12, mec-7 and genes in the UNC-6/Netrin pathway. Wefurther ask whether Netrin acts as an instructional signal to specify the axon branching points.We follow these experiments by testing genetic interactions between mec-12, mec-7 and therpm-1 pathway. We also propose to decipher the roles of microtubule-associated proteins andmicrotubule-severing proteins in axon branching. In the last part of the proposal, we set out todetermine the temporal requirement of mec-12 and mec-7 functions in axon branching byheat-shock modulation of tubulin expression. Finally we propose to use a photoconvertiblefluorescence reporter to directly visualize microtubule dynamics during axon branchformation. Progress in this project should extend our current knowledge of the molecularmechanisms by which extracellular signaling and microtubules dynamics interact to regulateaxon branching.線蟲微管軸索分枝NetrinRPM-1C. elegansmicrotubuleaxon branchingNetrinRPM-1