利用果蠅基因組缺失的範圍掃描來尋找影響Cadherin回收的基因片段

Genome-Wide Deficiency Screen for the Genomic Region Responsible for Cadherin Internalization in Drosophila Melanogaster

劉佑碩

指導教授 ： 徐瑞洲

國立清華大學/生命科學院/分子醫學研究所/碩士(2014年)

https://doi.org/10.6843/NTHU.2014.00181

摘要

E-cadherin是一種重要的細胞黏著蛋白，它分布在adherens junction，參與許多重要的生理功能，像是生物發育、腫瘤生成、以及維持組織的結構等等。Echinoid (nectin的同系物)也是一種細胞黏著蛋白，與cadherin一樣的分布在adherens junction上，且會間接的與細胞骨架相連接。在我們實驗室之前的研究為了進一步探索echinoid的功能，我們設計了一種嵌合異構物：Ed-cad，它擁有echinoid的胞外區段以及cadherin的胞內區段。藉由在果蠅身上過度表現Ed-cad，我們想觀察這種異構物是否能取代內生性的cadherin或echinoid的功能。實驗意外的發現，過度表現Ed-cad會產生胞吞作用使得內生性cadherin表現量下降。於是，我們想要找尋產生這個胞吞作用的因子為何。目前已知胞吞cadherin的機轉，主要是透過clathrin的途徑。不過我們發現在過度表現Ed-cad的情況下，還有其它未知的途徑來參與胞吞作用。本文就是藉由果蠅基因組缺失的範圍掃描來找尋這個未知的因子。
在果蠅成蟲下過度表現Ed-cad會產生rough eye與翅膀缺陷的表現型。我們即利用這個特性，藉由掃描時觀察這些表型的變化來挑選可能參與影響的基因片段。實驗後發現，7個可能的候選片段被挑選出來，這些片段有些包含了已知參與細胞內運輸的蛋白基因，或是有潛力參與胞內運輸功能的基因，像是reck1、delta、sec10、Centaurin B1、lanB1、fuzzy onion等等。
這項研究目前還停留在一個很初步的階段。未來我們需要進一步針對這些可疑的基因進行單獨的測試，像是knockdown assay。我們也需要更多的生裡上的證據來證實這些基因的參與程度。

關鍵字

基因組缺失 ； 範圍掃描 ； Cadherin回收

並列摘要

E-cadherin is the major cell adhesion molecule (CAM) of adherens junctions (AJs), and it involves in many important cellular physiology. Echinoid (Ed), a nectin homologue, also belongs to CAM and localizes to AJs. To take a deeper look on the function of Ed in our previous study, we generated a chimeric construct which has an extracellular domain of Ed with an intracellular domain of cadherin (Ed-cad), and overexpressed Ed-cad in Drosophila tissue to see if it could replace the function of Ed or cadherin. Surprisingly, overexpression of Ed-cad led to endogenous cadherin endocytosis, so we looked for the factors which participate in cadherin internalization in this situation. Cadherin can be retrieved by different mechanism, mainly via clathrin-mediated endocytosis in most situation. However, there were still other unknown factors which involved in cadherin internalization when overexpressing Ed-cad.
Overexpression of Ed-cad in adult Drosophila caused rough eye and wing defect. Here in this article, we used these features, and obtained Drosophila deficiency kit to generate a genome-wide screening to find out which factors may involve in cadherin internalization. After the screening, 7 genomic regions were identified, while some of them contain candidate genes which were already known or have the potential role involving in vesicular trafficking, including reck1, delta, sec10, Centaurin B1, lanB1, and fuzzy onion.
Nonetheless, the study presented here is only the initial work to find out the unknown factors of cadherin recycling. In the future, we have to examine the candidate genes one by one by such as knock down assay, and need more biological evidence to prove the exactly roles of these specific genes.

參考文獻

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