研究激酶GCK-2及PKG-1對線蟲神經纖毛生成及鞭毛內運輸機制之影響調控

Characterization of GCK-2 and PKG-1 affecting ciliogenesis and intraflagellar transport (IFT) in Caenorhabditis elegans

黃欣怡(Hsin-Yi Huang)

指導教授 ： 王歐力

國立清華大學/生命科學院/分子與細胞生物研究所/碩士(2014年)

摘要

纖毛是普遍存在於多種真核生物中的一種特異化細胞構造，其中非運動纖毛(又稱初級纖毛)可藉由膜上的受體蛋白檢測外界的環境與刺激，進而影響細胞的反應，在生理及發育中扮演重要的角色。纖毛蛋白缺失會導致嚴重的纖毛相關疾病(ciliopathy)，例如人類多囊腎臟病(polycystic kidney disease, PKD)、腎消耗病(nephronophthisis, NPHP)或巴德-畢德氏症候群(Bardet-Biedl syndrome)。複雜的纖毛生成(ciliogenesis)主要由物種間高度保留的鞭毛內運輸(intraflagellar transport, IFT)和細胞內多種調控機制負責。儘管在過去幾年的研究中，對纖毛生成及鞭毛內運輸的基礎認識已有大幅度的躍進，但其中複雜的調控機制仍待進一步仔細探討。先前在遺傳篩選及趨化性的實驗中，我們已經成功篩選出影響線蟲頭部化感器功能的兩個激酶GCK-2及PKG-1。在本研究中，我們進一步去探究這兩個激酶在纖毛生成及鞭毛內運輸中所扮演的調控角色。在量測纖毛長度的實驗中，我們發現gck-2突變株的纖毛較長；而鞭毛內運輸的分析結果則顯示gck-2突變株及pkg-1突變株對纖毛內運輸的速度及鞭毛內運輸蛋白間的組裝皆會造成影響。此外，我們觀察到在pkg-1突變株中，OSM-3致動蛋白會不正常地堆積在纖毛遠端。總而言之，我們推論出GCK-2和PKG-1在纖毛生成及鞭毛內運輸的調控機制中可能扮演不同的角色：GCK-2負責調控纖毛長度，而PKG-1則負責穩定在逆向運輸中OSM-3致動蛋白和其他纖毛蛋白的組合。

關鍵字

纖毛生成 ； 鞭毛內運輸 ； 線蟲 ； 激酶 ； 纖毛

並列摘要

Cilia are specialized subcellular organelles which exist in various cell types in most eukaryotic organisms. Non-motile cilia, or primary cilia, are mainly responsible for sensory function and are recognized to have essential roles in physiology and development. Defects in ciliary proteins lead to severe diseases termed ciliopathies, such as polycystic kidney disease (PKD), the most common inherited kidney disease and one of the most life-threatening diseases in the world, or nephronophthisis (NPHP), the most common genetic cause of chronic kidney disease. The conserved bidirectional intraflagellar transport (IFT) is fundamentally involved in ciliogenesis and is coupled with multiple intracellular signaling pathways. Although significant advances have been made in understanding ciliogenesis and IFT in the past few years, details on how the process is regulated remains poorly understood. By genetic screening and chemotaxis assay, we have identified two kinases GCK-2 and PKG-1 which affect the chemo-sensibility of amphid neurons in C. elegans. In this study, we further characterize their regulation roles in ciliogenesis and IFT process by cilia length measurement and IFT motility analysis. Here, we observed that worms lacking GCK-2 tend to grow longer cilia. Later in the IFT analysis, the transport speeds of different IFT-components are affected in the absence of either GCK-2 or PKG-1. And the stable IFT machinery assembly may also be modulated by GCK-2 and PKG-1. In addition, we observed abnormal OSM-3 kinesin accumulation at the distal segment of cilia in pkg-1 mutant strains. Overall, here we reported that GCK-2 and PKG-1 may regulate ciliogenesis and IFT particle transport in different ways: GCK-2 controls cilia lengthening, while PKG-1 affects the association of OSM-3 kinesin to the retrograde IFT machinery.

並列關鍵字

ciliogenesis ； intraflagellar transport ； IFT ； kinase ； C. elegans ； cilia ； PKG-1 ； GCK-2

參考文獻

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