- 學位論文
第一章: 線蟲三聯複合體RAB-3-UNC-10-SYD-2之活性區調控驅動蛋白-3 UNC-104的作用角色 第二章: PKG-1與GCK-2 能調節線蟲感覺神經之纖毛長度與鞭毛內運輸 第三章: 線蟲中似神經絲蛋白TAG-63能促進神經軸突傳遞機制
Chapter 1: “Role of active zone tripartite complex RAB-3-UNC-10-SYD-2 on the regulation of kinesin-3 UNC-104 in C. elegans”. Chapter 2: Cilium length and intraflagellar transport regulation by kinases PKG-1 and GCK-2 in C. elegans sensory neurons.Chapter 3: TAG-63 is a neurofilament-like protein that affects the fast axonal transport machinery in C. elegans.
摘要
Abstract I 神經細胞體中合成的突觸蛋白經由微管依賴馬達沿軸突將蛋白運輸至其各自的目的地。UNC-104是線蟲中KIF1A的同系物，為運輸突觸小泡前體必需物。 它的C終端PH結構區域可以特異地與囊泡膜上的酸性磷脂（PI（4,5）P2）直接結合，並將馬達和貨物連結起來。 然而，我們假設UNC-104之PH域與貨物的聯繫很微弱，並且可能不足以用於捆綁和識別貨物。有趣的是，研究已經顯示RIM / unc-10在C端表現了Liprinα/ syd-2的結合位點，而其N端與Rab3 / RAB-3結合。 因此，我們推斷增加連接RAB-3 / UNC-10 / SYD-2，以提高馬達-貨物的連通性和強度。 為了證明我們提出的連接子存在，我們首先進行即時PCR分析，指出unc-10突變體中SYD-2增加表現量，而其在rab-3突變體中明顯降低。此外CO-IP實驗鑑定出unc-10突變體和rab-3突變體中會降低SYD-2與UNC-104的結合能力。在unc-10和rab-3突變體中，UNC-104集群區域也受到嚴重影響。 SYD-2和UNC-104在線蟲神經元中彼此共定位。然而此共定位在unc-10和rab-3突變體中大大減少。且類似地，UNC-104和SYD-2相互作用的BiFC信號分佈模式也分別在unc-10和rab-3突變體中降低。此外，三種活性區域蛋白(syd-2，unc-10和rab-3) 突變體中，UNC-104運動能力均受到強烈影響。另外對於含有RAB-3的囊泡觀察到類似的運動模式，表明RAB-3運輸需要SYD-2 / UNC-10結合，但SNB-1則無需。同樣地，在軸丘中RAB-3行進距離在syd-2和unc-10突變體中都受到影響，但SNB-1僅在syd-2突變體中受影響。 而從另一種方法，我們試圖了解與SNB-1和UNC-104相比，RAB-3和UNC-104之間額外連接點是否存在，當去除PH結構域時有較低的共定位程度影響。事實上，當去除PH區時，SNB-1和UNC-104之間的共定位有強烈影響，而RAB-3和UNC-104共定位則無。相應地，在syd-2和unc-10突變體動物中，UNC-104（缺失PH結構域）和RAB-3共定位明顯減弱。RAB-3和SNB-1在BiFC分析中也表現出輕微的相互作用，並且這種複合體進一步與體內UNC-104共定位。 從我們的研究中，我們得出結論認為要加強SYD-2與UNC-104的聯繫，需要UNC-10 / RAB-3複合體存在，而進一步增強馬達與貨物的連接性。 Abstract II 過多的人類疾病是來自於纖毛功能障礙，包括多囊性腎病變，Bardet- Biedl和Meckel-Gruber綜合症。幸運地，近幾年來纖毛發育和鞭毛內運輸（IFT）的基礎機制更加清楚明瞭。然而在纖毛機制被解開的同時，也產生了許多出現新的問題，尤其是IFT貨物如何在纖毛基部組成、如何定位纖毛、以及如何調節“IFT列車”。最近有個令人感興趣的研究，兩種線蟲激酶DYF-5和DYF-18的纖毛調節功能被找出來，我們推測應該還能發現許多激酶與磷酸酶。我們因此使用資料探勘的工具，來鑑定線蟲纖毛感覺神經元中特定表達之激酶和磷酸酶。使用廣泛的方法來探討已選定的激酶和磷酸酶對纖毛形成和IFT的影響，例如染劑填充，化學趨化性，IFT組成表達模式，並確定PKG-1和GCK2 為可能的候選蛋白且明顯影響纖毛發育和貨物運輸。 在pkg-1突變中一同被發現到，驅動蛋白2 OSM-3同源二聚體在纖毛末梢大量積累並降低纖毛動力蛋白XBX-1逆行速度，導致異常纖毛形態，且可能伴隨減少纖毛微管蛋白乙酰化。而在gck-2突變體中，OSM-3和IFT-顆粒A（CHE-11）運動性隨著微管蛋白乙酰化的增加而明顯升高，降低KAP-1運動性，證實最近的模型，KAP馬達限制了OSM-3運動。最重要的是，我們觀察到突變體動物都可以利用過度表達PKG-1或GCK-2蛋白來挽救這些缺陷（在纖毛特異性Posm-5啟動子下）。 由於PKG-1和GCK-2與IFT之組成元件的共定位，兩種蛋白在纖毛中都遵循相似的表達模式以及表現運動。PKG-1與cGMP途徑有關，我們分別研究了上游效應物DAF-11和ODR-1的影響，發現與pkg-1突變體具有相似的結果。相反地，GCK-2與mTOR途徑有關，所以我們使用雷帕黴素來抑制該途徑，也發現與gck-2突變體中所見的影響相似。有趣的是，我們還利用組蛋白脫乙酰酶HDA-4和SIR-2.1來了解調節cGMP和mTOR途徑中的作用角色。簡要來說，我們描述以及確定了兩種新穎激酶的獨特功能，來影響秀麗隱桿線蟲的纖毛形成和鞭毛內運輸。 Abstract III 有發現不同的神經疾病包含帕金森病，肌萎縮性側索硬化症，Charcot-Marie-Tooth疾病和Tau蛋白病都與神經絲(NF’s)缺陷有關。神經病理學中已經確定NF的角色與作用，但對於這些疾病在分子層級是如何發展的知識仍甚少，是促進未來藥物設計的重要關鍵。模式生物例如斑馬魚、果蠅、線蟲，都有被利用研究這些疾病的發展，然而在線蟲中是否存在NF同原物仍然不清楚。因此，我們的目標主要是辨識與描繪線蟲假定的似NF蛋白。使用生物資訊工具我們鑑定了與NEFH (BLAST e值為 5E-09) 於三螺旋以及各種磷酸化位點有高度同源性的TAG-63，並採用廣泛技術，例如西方墨點法、穿透式電子顯微鏡、線蟲成像、運動分析來描摹TAG-63在線蟲的角色。 為了鑑定NF同源物，KEGG數據庫提供了來自多種動物的NEFH同源基因。生物資訊工具顯示後，我們注意到一群NEFH直系同源基因在來自一般TAG-63祖先根源。雖然我們沒有辦法檢測到KSP重複序列，但利用Scansite工具我們確定了9個潛在的磷酸化位點。此外，捲曲螺旋預測工具在TAG-63中發現了三個潛在的捲曲螺旋。為了理解TAG-63是否在神經元組織中表達，我們製作了轉錄TAG-63報告能在廣泛的身體，頭部和尾部神經元中表現。最重要的是，TAG-63還具有NF-L的特徵，例如分子量約70kDa，缺乏KSP重複序列和在透射式電子顯微鏡下觀察時呈現絲狀結構。 此外有研究指出，NF’s會影響軸突運輸，我們調查敲除tag-63對突觸小泡運輸的影響。發現TAG-63突變體動物中UNC-104的順行運輸明顯減少，指出TAG-63的激活馬達運動之作用。雖然我們沒有得到UNC-104與貨物SNB-1有類似的影響，但我們測量後發現這種貨物有增加逆行運動。而且tag-63缺陷線蟲的UNC-104速度與流量顯著減少，另一方面，tag-63突變體中SNB-1在細胞體中螢光強度增強，但在靠近HSN神經突觸區域減少。總之，我們在線蟲中鑑定並表徵了似NF蛋白質，並且還證明缺乏這種蛋白會限制軸突運輸效率，表明這種模型生物體可以用於研究有關神經絲的神經疾病。
並列摘要
Abstract I Newly synthesized synaptic proteins in the cell body are transported down the axonal processes to their respective destinations by microtubule-dependent motors. UNC-104, a homologue of KIF1A in C. elegans is required for the transport of synaptic vesicle precursors. Its C-terminal PH domain can specifically and directly bind to acidic phospholipids (PI (4,5)P2 ) located on the vesicular membrane and associate the motor to the cargo. However, we hypothesize that the classical linkage of UNC-104 to the cargo via its PH domain is weak owing to protein-lipid interaction and may not be sufficient for motor-cargo binding and recognition. Interestingly, it has been shown that RIM/UNC-10 reveals a binding site for Liprin-α/SYD-2 at the C-terminus, while its N-terminus binds to Rab3/RAB-3. As it has been shown that SYD-2 is a functional adaptor for UNC-104 and RAB-3 is known to be a synaptic-vesicle specific, membrane-bound protein, we propose the presence of an additional linker RAB-3/UNC-10/SYD-2 that could enhance the motor-cargo connectivity. To prove the existence of this proposed linker, we performed real-time PCR analysis in the first place indicating an increase of SYD-2 expression in unc-10 mutants while its expression in rab-3 mutants is significantly reduced. Further, from Co-IP assays, we identified that SYD-2’s association to UNC-104 was strongly reduced in both unc-10 and rab-3 mutants. Also, the UNC-104 cluster area in sublateral as well as ALM neurons was severely affected in these (unc-10 and rab-3) mutants. SYD-2 and UNC-104 colocalize with each other in C. elegans neurons. However, the colocalization between SYD-2 and UNC-104 largely diminished in unc-10 and rab-3 mutants. Similarly, the distribution patterns of bimolecular fluorescence complementation assay (BiFC) signals revealing UNC-104 and SYD-2 interactions were also reduced in unc-10 and rab-3 mutants respectively. Alongside, UNC-104 motor motility was 5 significantly affected in all the three active zone protein (syd-2, unc-10 and rab-3) mutants. In addition, we also observed the motility pattern of RAB-3-containing vesicles demonstrating that SYD-2/UNC-10 combination are essential for RAB-3 transport but not for SNB-1. In the same way, the distance travelled by RAB-3 particles from axonal hillock to distal segments of ALM neuron was affected in both syd-2 and unc-10 mutants but SNB-1 travel was affected only in syd-2 mutant background. In another approach, assuming that an additional linker between RAB-3 and UNC-104 exists, deletion of the motor’s PH domain may affect the colocalization between UNC-104 and RAB-3 to a lesser extent as compared to SNB-1 and UNC-104. Indeed, the colocalization between SNB-1 and UNC-104 was largely affected when deleting the PH domain, while RAB-3 and UNC-104 colocalization remained unaffected. Consistently with our model, in syd-2 and unc-10 mutant animals, UNC-104 (with a deleted PH domain) and RAB-3 colocalization was strongly diminished. RAB-3 and SNB-1 also exhibited interactions in BiFC assays and this complex further colocalizes with UNC-104 in somas. From these results, we may indeed conclude that UNC-10/RAB-3 complex is required to additionally strengthen the association of SYD-2 with UNC-104, further enhancing the connectivity of motor to its cargo. Abstract II A plethora of human diseases are based on cilia dysfunctions including polycystic kidney disease, Bardet-Biedl and Meckel-Gruber syndrome. Fortunately, basic mechanisms underlying cilia development and intraflagellar transport (IFT) became more understandable in recent years. Though at the same time a complex ciliary machinery was unraveled leading to new questions, specifically, how IFT cargo assembles at the cilia base, how it localizes to cilia, and how “IFT trains” are regulated. One intriguing recent finding describes the ciliary regulating function of two C. elegans kinases DYF-5 and DYF-18, and we hypothesized that even more kinases and phosphatases may be uncovered. We therefore employed data mining tools to identify kinases and phosphatases specifically expressing in C. elegans ciliated sensory neurons. We then used a broad range of methods such as dye-filling, chemotaxis, IFT component expression pattern etc. to investigate the effects of selected kinases and phosphatases from a candidate screen on ciliogenesis and IFT, and have identified PKG-1 as well as GCK-2 as potential candidates significantly affecting cilia development as well as intraflagellar cargo transport. In pkg-1 mutants, severe accumulation of homodimeric kinesin-2 OSM-3 at the cilia tip was observed in conjunction with an overall reduction in retrograde speeds of ciliary dynein XBX-1, leading to abnormal cilia morphology, likely as a function of reduced tubulin acetylation. While in gck-2 mutants OSM-3 and IFT-particle A (CHE-11) motility was significantly elevated in conjunction with increased tubulin acetylation, KAP-1 motility was decreased, confirming a recent model in which the slow KAP-1 motor restricts the motility of the fast OSM-3 motor. Crucially, all observed effects in mutant animals can be rescued by overexpressing the respective protein PKG-1 or GCK-2 (under the cilia specific Posm-5 promoter). Both, PKG-1 and GCK-2 follow similar expression pattern in cilia and also display 75 movements owing to their colocalization with other investigated IFT machinery components. Because PKG-1 is related to cGMP pathways, we also studied the effect of upstream effectors DAF-11 and ODR-1, respectively, leading to similar observations compared with the pkg-1 mutants. Vice versa, since GCK-2 is related to the mTOR pathway, we used rapamycin to inhibit this pathway leading to similar effects as seen in gck-2 mutants. Importantly, we also manipulated the histone deacetylases HDA-4 and SIR-2.1 to understand their role in regulating cGMP and mTOR pathways. In summary, we identified and characterized the distinct functions of two novel kinases affecting ciliogenesis and IFT in C. elegans. Abstract III Various neurological diseases bearing defects in neurofilaments (NF’s) are known including Parkinson’s disease, Amyotrophic Lateral Sclerosis, Charcot-Marie-Tooth disease and Tauopathies. Though a critical role of NF’s has been ascertained in neuropathological diseases, little is known about how these diseases develop on the molecular level, critical to facilitate future drug design. Model organisms, such as Zebrafish, Drosophila and C.elegans, are employed to study and understand how these diseases develop. However, whether a neurofilament (NF) homolog exist in the nematode still remains unclear. Therefore, the major goal of this study was to identify and characterize a putative NF-like protein in C. elegans. Using bioinformatics tools, we identified TAG-63 with numerous sequence homologies to NEFH (BLAST e-value of 5E-09), three coiled coils as well as various phosphorylation sites. We then employed a broad range of techniques such as Western blotting, transmission electron microscopy (TEM), worm imaging, motility analysis etc. to delineate the role of tag-63 in C.elegans. To identify NF homologs, using KEGG database, we received hits for NEFH orthologs from various animals. Using this bioinformatics tool, we also note a cluster of NEFH orthologs in a rooted phylogenetic tree emerging from a common TAG-63 ancestor. Though we cannot detect KSP repeats in TAG-63, we identified nine potential phosphorylation sites using Scansite tool. Furthermore, the coiled coil prediction tool identified three potential coiled coils in TAG-63. To understand if tag-63 is expressed in neuronal tissues, we generated a transcriptional tag-63 reporter that expresses in a broad range of body, head and tail neurons. Most importantly, TAG-63 also exhibits features of NF-L such as molecular weight of around 70 kDa, lack of KSP 134 repeats and the ability to form filamentous structures when viewed under transmission electron microscopy. Moreover, as it has been reported that NF’s affect axonal transport, we investigated the effect of tag-63 knockout on synaptic vesicle transport. We found that anterograde transport of UNC-104 is significantly reduced in tag-63 mutant animals pointing to a motor-activating role of TAG-63. Though we do not reveal similar effects on UNC-104’s cargo SNB-1, we did measure increased retrograde movements of this cargo. Further, velocity and flux of UNC-104(KIF1A) are largely diminished in tag-63 knockout worms. In addition, fluorescence intensity of SNB-1 increases in the cell body while near the synapse of HSN neuron it decreases in tag-63 mutant background. In summary, we identified and characterized a NF-like protein in C.elegans, and also demonstrate that lack of this protein limits axonal transport efficiencies, suggesting that this model organism may be used for studying neurofilament-based neurological diseases.