黏著斑是一種靠近細胞膜(15 nm以內)之大型且動態變化的蛋白質複合體。黏著斑可以當作細胞骨架與細胞外間質之物理性連結；同時也可以是細胞生化訊息的樞紐，因為此處聚集並同時指揮與活化許多細胞訊息蛋白質分子於細胞膜上integrin接受器與細胞外間質結合與作用之處。在細胞移行過程中，黏著斑的相互協調與動態調控是相當重要的，包含其新生與瓦解。黏著斑的瓦解或周轉是由鈣離子觸發鈣蛋白酶進行相關蛋白質分子水解導致而成，主要是發生在移行細胞的尾端。鈣蛋白酶藉由水解多種與組成黏著斑相關的蛋白來調控黏著斑的周轉，例如FAK, Paxillin, Talin與Vinculin。黏著斑的構造是由許多不同之黏著斑蛋白分子於特定的空間位置和功能位區組成多層此的立體建構，這些蛋白質分子彼此互相影響與依存，藉以調控黏著斑的形態與功能。許多先前的研究已經證實黏著斑的周轉可以受到鈣離子訊號的調控，但是大部分的研究並無顯示出在活細胞中黏著斑周轉過程之空間與時間的即時變化細節。本研究之目標在於探討活細胞鈣離子訊息所引發黏著斑周轉過程。利用全反射螢光顯微鏡之技術，結果顯示鈣離子可以造成不同黏著斑分子具有不同降解過程，而同時也使用三個具有抵抗鈣蛋白酶降解能力的黏著斑突變蛋白分子來釐清分子之間相互作用與依存的關係。結果顯示Vinculin不容易受到降解外，也不容易受到具有抵抗鈣蛋白酶降解能力的黏著斑突變蛋白分子影響其降解過程。利用3D螢光造影來確認黏著斑複合體之結構與瓦解過程之關係，結果顯示黏著斑複合分子之結構與其瓦解過程有關。傳統上用來研究黏著斑動態變化的方法包括藥理上的刺激或是遺傳上的操作；然而，這些方法在空間上和時間上的精準度並不精確。光遺傳學是一種結合遺傳學與光學去即時與精密的調控一些組織特定細胞內之明確的事件。光敏感通道ChR2已經被發展運用於光遺傳學刺激方法上用來精準的調控某些特定細胞的反應，其是一種可以直接被光切換之具有陽離子選擇性的離子通道，最大吸收光譜為480 nm附近之藍光。因此，當此光敏感通道打開時，會對帶正一價與正二價之陽離子產生一較大的通透度，可以通導包括氫離子、鈉離子、鉀離子與鈣離子等陽離子。由結果顯示光敏感通道ChR2可造成整體或局部的鈣離子進入細胞內。綜合實驗結果，證明黏著斑複合體的結構在鈣離子訊號誘發個別的黏著斑蛋白分子之周轉與動態變化扮演重要的角色。此外，利用光遺傳學技術可以細部地探討鈣離子訊號是如何誘發黏著斑之周轉。

Focal adhesions are large, dynamic protein complexes which are close to the plasma membrane (within 15 nm). Focal adhesions serve as the mechanical linkages between the cytoskeleton, and the extracellular matrix, and as a biochemical signaling hub to concentrate and direct numerous signaling proteins at the sites of integrin binding and clustering. The coordinated and dynamic regulation of focal adhesion is required for cell migration, including assembly and degradation. Degradation, or turnover of focal adhesion, is the major event in the rear end of a migratory cell, which is in a Ca2+/calpain-dependent proteolysis. In addition, calpain has been shown to regulate the turnover of focal adhesion by proteolysis of multiple focal adhesion related proteins, such as FAK, Paxillin, Vinculin, and Talin. The organization of focal adhesion proteins indicates a composite of several laminar architectures with spatial and functional compartments that mediate the interdependent functions of focal adhesion. The effects of Ca2+ signal on focal adhesion turnover have been shown, but most of them have not shown the detailed space-time cascades of focal adhesion turnover in real time. The aim of this study is to investigate how a Ca2+ signal induces cascades of focal adhesion turnover in living cell. The results of TIRFM images showed Ca2+ induces different processes of each focal adhesion molecule. In addition, three calpain-resistant mutant focal adhesion molecules were used to clarify the interactions between focal adhesion molecules. The results showed Vinculin is difficult to be degraded and not be significantly affected by calpain-resistant mutanted focal adhesion molecules. The 3D fluorescence imaging was performed to confirm the relationship between architecture and degradation of focal adhesion. The results showed architecture of focal adhesion is related to the trend of degradation of focal adhesion molecules. Traditional approaches in the study of focal adhesion dynamics have been via pharmacological stimulation or genetic manipulation. However, these methods have poor temporal and spatial precision. An optogenetics is the combination of genetics and optics to control well-defined events within specific cells of living tissue in real time. Optogenetic stimulation approach that uses channelrhodopsin-2 (ChR2) has been developed for providing more precise and targeted stimulation effect on cells. ChR2 is a directly light-switched cation-selective ion channel, which absorbs blue light with an absorption maximum of 480 nm. Then, this channel opens rapidly to generate a large degree of permeability for monovalent and divalent cations, conducting H+, Na+, K+, and Ca2+ ions. The results showed that ChR2 can be used to cause global and local Ca2+ influxes. Taken together, this study suggests that the 3D architecture of focal adhesion plays an important role in Ca2+-mediated focal adhesion turnover. Therefore, optogenetic stimulation of ChR2 can be used to operate detailed focal adhesion turnover.

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