解析細胞自噬相關構造上的 LC3/GABARAP 蛋白分布

Mapping the LC3/GABARAP Distributions on Autophagic Structures with Photoactivated Localization Microscopy

謝廷松(Ting-Sung Hsieh)

指導教授 ： 楊維元

國立臺灣大學/生命科學院/生化科學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10469

摘要

在真核生物的演化歷程中，細胞自噬（巨型自噬）是一個高度保守的作用機制，它調控著經由溶小體所處理的巨分子或胞器分解。自噬作用在開始時，細胞質內會形成一個由脂質膜與多種蛋白構成的杯狀構造，叫做吞噬胞，它隨後會包圍細胞質中等待被分解的物質，最後封閉開口端產生一個由雙層脂質膜包覆的囊狀構造，稱為自噬體。在自噬體形成的過程中，把Atg8蛋白在哺乳動物細胞中的同源分子LC3和GABARAP接合到磷脂醯乙醇胺，以及隨後募集這些分子到自噬構造上是相當關鍵的步驟。目前在哺乳動物細胞中已經發現了四個LC3和四個GABARAP相關蛋白。近來的研究認為這些LC3和GABARAP蛋白可能分別在自噬體形成的不同階段裡作用，而且在吞噬胞的延拓或者吞噬目標物的選擇這兩個過程中具有獨特的功能。然而，迄今我們仍不清楚這些LC3/GABARAP蛋白究竟如何協同合作來促進自噬體形成，以及它們到底以怎樣的方式分布在延展中的吞噬胞上。在本篇研究中，我們利用光活化定位顯微術解析了細胞中的LC3/GABRAP蛋白分布至分子尺度。我們架構了光活化定位顯微術的儀器設備並開發了一套MATLAB程式組，Palmy PALM 803，來分析資料與繪製光活化定位顯微影像。到目前為止，透過單色光活化定位顯微術，我們已經揭露了細胞中mEos2-LC3B蛋白在可能為自噬構造上的分布狀態。進一步利用雙色或三色光活化定位顯微術，各種LC3/GABARAP分子間的相對位置也可以被決定。此外，細胞內的膜狀結構可以透過穿透式電子顯微技術來拍攝。結合穿透式電子顯微影像與透過光活化定位顯微術定出的LC3/GABARAP蛋白分布，我們將能對自噬體成熟進程中的膜狀結構動態以及各個LC3/GABARAP蛋白在自噬作用中的運作模式有更進一步的了解。

關鍵字

Atg8 ； LC3 ； GABARAP ； 細胞自噬 ； 光活化定位顯微術

並列摘要

Macroautophagy is a highly conserved intracellular process mediating lyso-some-dependent degradation of macromolecules or organelles. This process involves the formation of a cup-shaped membrane structure called phagophore, which subsequently engulfs selected cargos and matures into a double membrane-bound autophagosome. Conjugation of mammalian Atg8 homologues, LC3s and GABARAPs, to phosphatidylethanolamine (PE) and subsequent recruitment of LC3/GABARAP–PE to autophagic structures are crucial for autophagosome biogenesis. Currently in mammals there are four LC3 and four GABARAP variants identified. It has been suggested that LC3s and GABARAPs act at different stages in autophagosome formation and possibly play distinct roles in phagophore expansion or cargo selection control. Exactly how these LC3/GABARAP variants function cooperatively to achieve autophagosome formation and how they distribute on an expanding phagophore remain largely unknown. In this study, we employed photoactivated localization microscopy (PALM) to visualize LC3/GABARAP in vivo at molecular resolution. We set up PALM instrumentation and developed a suite of MATLAB scripts—Palmy PALM 803—for data analysis and image rendering. So far single-color PALM imaging has revealed mEos2-LC3B distributions on possible autophagic structures in the cell. Applying dual- or triple-color PALM, the respective localizations and the relative distributions of individual LC3/GABARAP can be determined. Intracellular membrane structures can also be imaged with transmission electron microscopy (TEM). Combining TEM images with identified LC3/GABARAP distributions by PALM, membrane dynamics through the autophagosome maturation and individual action modes of LC3/GABARAP variants in this autophagic process may be further clarified.

並列關鍵字

Atg8 ； LC3 ； GABARAP ； autophagy ； photoactivated localization microscopy

參考文獻

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