果蠅Spindle-F蛋白質磷酸化之功能性研究

Functional study of Spindle-F protein phosphorylation in Drosophila

潘柏元(Po-Yuan Pan)

指導教授 ： 李秀香

國立臺灣大學/醫學院/分子醫學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.10745

摘要

一個神經系統要能適當的運作，神經網絡的精確連接在這當中扮演了重要的角色。為了因應內在生理以及外在環境的變化，神經網絡會透過不同的 “神經重整”(neuronal remodeling processes)的方式，在動物體的發育過程中重新塑造整個神經網絡。“神經樹突的修剪”(dendrite pruning)是其中一種神經重整的方式，這一修剪過程會選擇性地移除神經細胞中樹突(dendrite)的部份，但是保留下軸突 (axon)與細胞本體(cell body)，並且不會造成神經細胞的死亡。此一神經樹突的修剪現象廣泛的在脊索動物與非脊索動物的發育過程中可以觀察到。在果蠅的周邊神經系統中，有一群 “第四型樹突神經細胞”(Class IV da neurons)會在果蠅變態的過程中經歷大規模的神經樹突修剪。這是一個很好的模式生物系統來研究神經樹突修剪的分子運作機制。
在先前的研究中已知一個稱之為Ik2的絲胺酸/蘇胺酸激酶(serine/threonine kinase)在神經樹突的斷裂(dendrite severing)過程中扮演了必要的角色。此外，一個包含有捲曲螺旋區段的新蛋白質Spn-F在先前的報導中已知道會與Ik2有交互作用。再者，我們實驗室先前的研究結果顯示Ik2或Spn-F的基因突變會在神經樹突細胞的修剪過程中產生相似的樹突斷裂的缺失，且它們之間的交互作用對於神經樹突細胞的正常斷裂扮演著必要的角色。然而，Ik2-Spn-F 這一複合物是如何來起始神經樹突的斷裂仍是未知的。我們假定Ik2在神經細胞中執行樹突的斷裂是藉由對Spn-F進行磷酸化的修飾來達成的，為此，我們決定研究Spn-F蛋白質的磷酸化(phosphorylation)在果蠅神經樹突的斷裂過程中扮演何種角色。
首先，我們透過以質譜儀為基礎的蛋白質體學研究方法，在Spn-F中找尋到了八個重要的絲胺酸(serine)具有磷酸化的修飾，而這些位置的磷酸化修飾是由Ik2參與其中催化而成的。根據胺基酸結構上的相似度，我們將絲胺酸的密碼子(codon) 改變為丙胺酸(alanine)及天門冬胺酸(aspartatic acid)的密碼子，分別創造出磷酸化缺陷型的Spn-F突變體(spn-F-S8A)以及模擬磷酸化修飾型的Spn-F突變體 (spn-F-S8D)。在我們的西方墨點法(western blot)的實驗中顯示Spn-F-S8A可以抑制Spn-F-wt在有Ik2表現的情況下所產生的蛋白質帶位移(band-shifting)現象。此一結果證實了在Spn-F中，這八個serine所產生的磷酸化修飾確實是Ik2所造成的。接著對於Spn-F磷酸化的分析研究中，我們指出Spn-F-S8A會在果蠅幼蟲(larva stage)的第四型樹突神經細胞的細胞質中形成一個個的小點(puncta)，並在果蠅形成蛹之後五小時可以觀察到Spn-F-S8A可抑制Ik2對Spn-F所產生的均勻散佈效果。另一方面，Spn-F-S8D在果蠅細胞株中或是在果蠅幼蟲的第四型樹塗神經細胞中，它可以模擬Spn-F-wt在有Ik2表現的情況下所產生的均勻散佈的表現性狀。此外，在果蠅細胞株(S2 cell)實驗中，我們發現在有Ik2 共同表現或是在Spn-F-S8D的情況下，Spn-F之間的交互作用會有明顯的減弱。以上這些結果暗示一個可能的分子機制，即Spn-F在神經細胞中一個個的小點的形成是由Spn-F之間互相有交互作用所形成的，而Spn-F的磷酸化修飾會破壞Spn-F之間交互作用，使得Spn-F均勻的散佈於細胞質中。更重要的是，Spn-F-S8A的過度表現沒有辦法完全的來挽救Spn-F 雙套染色體突變(spn-F homozygous mutant)所造成的樹突斷裂的缺失，但Spn-F-S8D可以來挽救這樣的缺失。這也暗示著Spn-F的磷酸化在樹突斷裂的起始過程中扮演著重要的角色。最後，我們也檢視了Spn-F與果蠅中的動力蛋白輕鏈(dynein light chain, Ctp) 之間的交互作用關係，我們發現了Spn-F是用第三個螺旋區段(coiled-coil domain 3) 以及C端的高度保留區段(SCD)來與Ctp進行交互作用的。此外，在我們實驗室其他成員的研究中亦發現了 Ik2是藉由Spn-F來與Ctp連結上的。
總結以上所做的研究，我們的研究結果指出Spn-F的磷酸化在果蠅第四型樹突神經細胞發育過程中的樹突切斷這個階段扮演了關鍵性的角色。此外，我們的研究結果也揭露了Spn-F可能作為Ik2的接應者來幫助Ik2與Ctp進行連結，這也暗示著 Ik2-Spn-F-Ctp這個複合物會一起合作來啟始神經細胞的樹突修剪這一過程。

關鍵字

神經細胞樹突的修剪 ； Ik2 ； Spn-F ； 蛋白質磷酸化 ； ctp

並列摘要

The precise connection of neural circuitry is crucial for the proper function of nervous system. The neural circuitry is reshaped to adapt internal changes and external stimuli during development by neuronal remodeling processes. Dendrite pruning, one of the neuronal remodeling processes, which selectively eliminates dendritic branches and leaves axons and cell bodies intact. Pruning is widely observed in both vertebrates and invertebrates. In the peripheral nervous system of Drosophila, the class IV dendritic arborization (da) neurons undergo a large-scale dendrite pruning during metamorphosis. It is a good model system to study the underlying molecular mechanism of dendrite pruning.
It was known that Ik2, a serine/threonine kinase, plays an essential role in the dendrite severing. In addition, Spn-F, a novel coiled-coil domain containing protein, was reported to interact with Ik2. Furthermore, previous studies in our lab showed that both Ik2 and Spn-F mutants have similar dendrite severing defects, and the interaction between Ik2 and Spn-F plays an essential role in dendrite severing. However, how Ik2- Spn-F complex initiate dendrite severing is still unknown. Here, we hypothesized that Ik2 execute dendrite severing by phosphorylating Spn-F, and investigated the role of Ik2-dependent Spn-F phosphorylation in the dendrite severing.
First, we identified eight important phosphorylation sites on Spn-F that is Ik2- dependent by mass spectrometry-based proteomics approach. Based on structure similarity, we mutated serine into alanine or aspartate by PCR method to produce a phospho-deficient mutant (spn-F-S8A) and a phospho-mimetic mutant (spn-F-S8D), respectively. We demonstrated that the Spn-F-S8A prevent the band-shifting phenotype with Ik2 co-expression. It confirmed that those eight serine sites on Spn-F are certainly Ik2-dependent phosphorylation sites. Next, our analysis on the Spn-F phosphorylation indicated that Spn-F-S8A could form puncta in S2 cells and class IV da neurons at larva stage, and Spn-F-S8A could resist Ik2-dependent dispersal in class IV da neurons at 5 h APF. On the other hand, Spn-F-S8D could mimic Ik2-dependent Spn-F dispersal in S2 cells and neurons at larva stage. Furthermore, Spn-F self-interaction is diminished with Ik2 co-expression or under Spn-F-S8D condition in S2 cells. It suggested a possible mechanism that Spn-F puncta is formed by Spn-F self-interaction, and Spn-F phosphorylation is required to disperse Spn-F in the cytoplasm. Most importantly, overexpression of Spn-F-S8D, not Spn-F-S8A, could fully rescue dendrite severing defects in spn-F homozygous mutants, suggesting that Spn-F phosphorylation is required for dendrite severing. Finally, we also characterized the interaction between Spn-F and Drosophila dynein light chain, Ctp. We identified that both CC3 domain and SCD domains of Spn-F are required to interact with Ctp.
Taken together, our studies indicated that Spn-F phosphorylation plays a critical role in dendrite pruning of Drosophila class IV da neurons. Also, we demonstrated that Spn-F might function as an adaptor of Ik2 to associate with Ctp, suggesting Ik2-Spn-F- Ctp tertiary complexes work together to initiate dendrite severing.

Keywords: dendrite pruning, Ik2/IKKε, Spindle-F (Spn-F), phosphorylation, Drosophila dynein light chain (ctp).

並列關鍵字

dendrite pruning ； Ik2/Ikk-epsilon ； Spindle-F(Spn-F) ； phosphorylation ； Drosophila dynein light chain (ctp)

參考文獻

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