Cep170 於神經突之非中心體分佈及其促使神經突生長之研究

Cep170 localizes as non-centrosomal puncta along the neurite and promotes neurite elongation

吳佳佳

微管 ； 神經 ； 細胞骨架 ； 微管相關蛋白 ； Cep170 ； microtubule ； neurons ； cytoskeleton ； microtubule-associated-proteins ； Cep170

交通大學生物科技學系學位論文

2019年

碩士

黃兆祺

英文

微管是神經中重要的一種細胞骨架，其參與了多種神經發育的進程。在過去的研究中，運用定量蛋白質體學在小鼠胚胎癌細胞進行神經分化的前後，進行微管相關蛋白質組研究。已經發現構成有絲分裂中心體一部分的中心體蛋白Cep170在神經元分化時的微管上表現出顯著增加。過度表現Cep170導致P19細胞衍生的神經元和小鼠海馬迴神經元中神經突的長度顯著增加，這表明Cep170參與神經內微管的調控。然而，我們仍無法確定Cep170對於神經型態發生過程的準確調控機制為何。在本研究中，我們試圖通過研究Cep170過度表現對微管細胞骨架的影響以及檢測內源性Cep170的定位來回答上述問題。我們發現過度表現Cep170導致分離的海馬神經元中軸突和樹突長度顯著增加。過度表現的Cep170沿著軸突和樹突形成非中心體斑點狀分布，並且在神經突的不同區域中顯示出形態的變化。此外，過度表現的Cep170富含在軸突末端，但在樹突末端並沒有相同的表現。此外，在海馬迴神經元中過度表現Cep170增加了微管聚合速率。我們還嘗試通過免疫熒光染色確定內源性Cep170在神經元中的定位。與過度表現的研究一致，內源性Cep170也沿著神經突形成非中心體斑點狀分布，在軸突末端具有顯著的聚集。我們的研究結果表明，Cep170在有絲分裂後細胞中所形成的非中心體斑點狀分布，與其對微管聚合速率的影響可能是促進神經突伸長的機制。

Microtubule is the major cytoskeleton in neurons and involved in numerous developmental processes. In a previous study, quantitative proteomics has been used to examine microtubule-associated proteomes in mouse embryonal carcinoma cells before and after neuronal differentiation. The centrosome protein Cep170, which made up part of the mitotic centrosome, has been found to exhibit a significant increase on microtubules upon neuronal differentiation. Overexpressing Cep170 caused a significant increase in neurite length in both P19 cell-derived neurons and mouse hippocampal neurons, suggesting that Cep170 is involved in the regulation of neuronal morphogenesis. However, the exact mechanism of Cep170 in controlling the morphogenetic process of neurons remain elusive. In this study, we attempt to answer the aforementioned question by studying the effect of Cep170 overexpression on microtubule cytoskeleton as well as examining the localization of endogenous Cep170. We discovered that overexpressing Cep170 causes a significant increase in both axon and dendrite length in dissociated hippocampal neurons. Overexpressed Cep170 localizes as non-centrosomal puncta along both axons and dendrites, and displays variation in morphology in different regions of the neurite. In addition, overexpressed Cep170 is enriched at axon but not dendrite tips. Furthermore, overexpressing Cep170 in hippocampal neurons increases the microtubule polymerization rate. We also attempted to determine the localization of the endogenous Cep170 in neurons by immunofluorescence staining. Consistent with the overexpression study, endogenous Cep170 also localizes as non-centrosomal puncta along the neurite with a significant enrichment at the axon tip. Our results suggest that Cep170 localizes as non-centrosomal puncta in post-mitotic cells and its effect on microtubule polymerization rate may be the mechanism in promoting neurite elongation.

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