果蠅Spindle-F蛋白質之功能性研究

Abstract

神經網絡需要精確的連接以維持神經系統的正常功能,來去處理動物個體內在的變化以及外來的刺激，神經網絡的重塑發生在發育過程中，目的是重整不確實的連接。修剪(pruning)，是一個神經系統重塑的機制之一，也是一個不會導致細胞死亡而去移除特定的神經細胞突出的自我摧毀過程。它是可以在發育過程以及神經系統的損傷和疾病中廣泛觀察到的一個現象，果蠅周邊神經系統中一群特定的神經細胞，第四樹突型神經(Class IV da neuron)之樹突在果蠅變態的過程中產生大規模的修剪。果蠅具有一個強大的遺傳工具，所以對於去了解在神經修剪過程中的分子機制以及神經受損和疾病的基礎病理概念是很理想的系統。 Ik2 是一個絲氨酸/蘇氨酸的激酶，它的酵素活性對於起始整個修剪過程中樹突的斷裂，決定卵子生成過程中背腹側的體軸決定，以及果蠅背毛(bristle)中肌動蛋白束的結構維持來說很重要。Spindle-F(Spn-F)是一個會與 Ik2 結合的蛋白質，Spn-F 的突變會產生和 Ik2 突變時有相似變異的性狀，分別出現在背毛和卵中，這個可能暗示著 Spn-F 和 Ik2 可能在樹突修剪中共同合作產生並相同的功能。我們實驗室之前發現到 Spn-F 和 Ik2 的突變下，在第四樹突型神經有樹突斷裂(severing)的缺失。然而，我們並不清楚 Ik2 和 Spn-F 是藉由何種機制共同合作來導致樹突的斷裂，因 此，在這份研究中，我們決定去分析 Spn-F 蛋白質中各別區段(domain)的功能，並且期望這些結果可以幫助我們了解神經細胞中樹突修剪的分子機制。 Spn-F 會產生含有三個盤繞型(Coiled-coil )區段的蛋白質，並且在Ｃ端有個高度保留的區段(SCD)。我們分析了 Spn-F 不同區段刪除的突變蛋白，並且發現到在第四樹突型神經細胞中，Spn-F 第三盤繞型區段(CC3)是和Ik2 結合的位置，而Spn-FＣ端高度保留的區段則是對於 Ik2 激酶引起的點狀蛋白(puncta)之分散很重要。並 且移除第三盤繞區域的 Spn-F 蛋白質沒辦法回復第四樹突型神經細胞之樹突斷裂的情形，同時它也會去干擾內生型 Spn-F 的功能進而導致過度表現後產生樹突斷裂的異常。這個現象指出對於樹突修剪的過程中，Spn-F 第三盤繞型區域是很必要的。此外，Spn-FＣ端保留區域缺失的突變蛋白質只會部份地回復樹突修剪的缺失，但是卻在過度表現的情況下產生異常的背毛。這些結果暗示了在果蠅樹突修剪以及背毛發育過程中 Spn-F 的Ｃ端保留區域都很重要，此外，針對 CC3 以及 SCD 區段缺失的 Spn-F 蛋白質都無法回復缺陷背毛的性狀這個結果，也都指出這兩個區段對於果蠅背毛的發育都很重要。 總結來說，在本次的研究結果中我們發現到位在 Spn-F 蛋白質中的兩個區段，Spn-F 第三盤繞型區域和 Spn-FＣ端保留區域不只在第四樹突型神經樹突斷裂的過程以及果蠅背毛的發育都很重要。

The precise connection of neuronal circuitry is essential for proper function of the nervous systems to adapt internal changes and external stimuli of the animals. Neuronalremodeling is often required to refine the imprecise connections during development. Pruning, one of the remodeling mechanisms, is a self-destructive process of removing specific parts of neuronal processes without causing cell death. It is widely observed phenomena in the nervous systems during development, and in neuronal injury and disorders. The dendrites of a subset of Drosophila peripheral sensory neurons, class IV dendritic arborization (da) neurons, will undergo a large-scale dendrite pruning during metamorphosis. It is an ideal system to study the underlying molecular mechanisms of neuronal pruning by taking the powerful genetics of Drosophila, and might get insights of the pathologic basis of neuronal injury and diseases. It was known that Ik2, a Serine/Threonine kinase, and its kinase activity is essentialto initiate dendrite severing during pruning, to determine the dorsoventral axis during oogenesis, and to maintain actin bundles during bristle formation. The mutants of spindle-F (spn-F) ,encoded an Ik2-interacting protein, showed similar phenotypes as ik2 mutants in oocytes and bristles, suggesting that spn-F and ik2 might function together in dendrite pruning. The studies done in our lab demonstrated that spn-F mutants, like ik2 mutants, have dendrite severing defects in class IV da neurons. However, the mechanisms how Ik2 and Spn-F act together and lead to dendrite severing are largely unknown. Thus, in this study, we decided to dissect the function of each domain in Spn-F proteins, and expected these results will help us further understand the mechanisms of dendrite severing in neurons.Spn-F proteins contain three coiled-coil domains (CC1, CC2 and CC3), and one C-terminal conserved domain (SCD). Our analyses on the Spn-F deletion mutant proteins have indicated that the CC3 domain of Spn-F is the Ik2-interacting domain and the SCD domain of Spn-F is crucial for the Spn-F punctate dispersal by Ik2 signals in Drosophila S2 cells and in class IV da neurons. The CC3-deletion mutant of Spn-F proteins not only fail to rescue the dendrite severing phenotypes in class IV da neurons, but also interfere the function of endogenous Spn-F proteins and result in the failure of dendrite severing while over-expression. It indicates that the Ik2-interacting domain CC3 of Spn-F protein plays an essential role in dendrite pruning of class IV da neurons. In addition, the SCD-deletion mutant of Spn-F only partially rescues the dendrite severing phenotypes in class IV da neurons, but causes severely abnormal bristles when over-expression. It suggested that the SCD domain of Spn-F is crucial for dendrite pruning and bristle morphogenesis. Furthermore, the failure of rescuing the abnormal bristles of spn-F mutants by the CC3- and SCD-deletion mutants of Spn-F protein indicated that both domains of Spn-F play important roles in bristle morphogenesis. Taken together, our studies have identified the CC3 and SCD domains of Spn-F proteins play essential roles not only in dendrite pruning of class IV da neurons, but also in bristle morphogenesis.