雷射捕陷誘發重水中細胞骨架蛋白之管狀結構形成

Laser Trapping-Induced Fibrous Structure Formation of Cytoskeletal Proteins in Deuterium Oxide

施仰欣(Yang-Hsin Shih)

指導教授 ： 杉山輝樹

國立交通大學/理學院/分子科學研究所/碩士(2019年)

摘要

我們藉由線性偏振之連續式1064 nm雷射聚焦在微管蛋白/重水溶液的固液氣三相介面，成功的利用雷射捕陷誘發重水中細胞骨架蛋白之管狀結構形成。在先前的研究中已成功在H2O溶液中進行了雷射捕陷並證明了微管形成的時空控制。然而，雷射捕陷不僅會增加微管蛋白的局部濃度，也同時會升高局部溶液的溫度。因此，並無法清楚知道雷射照射引起的濃度升高或溫度升高為主要微管形成的原因，與H2O相比，在D2O中通過雷射捕陷增加的局部溫度被抑制了20倍，由此確立雷射捕陷對微管形成的影響。
在不同濃度的微管蛋白下對微管形成進行系統性的研究，範圍為20至80 μM。當雷射光束聚焦在樣品溶液時，微管蛋白在雷射焦點處產生凝聚並且延伸至外部，最終可達直徑約為10 μm。在照射期間，雷射焦點周圍在正交偏光鏡條件下觀察到各向異性區域。
由於微管低溫下能夠溶解，因此為了確認各向異性區域是否為微管結構，我們在4℃的低溫條件下觀察各向異性區域的時間變化，結果顯示，在所有濃度下，各向異性區域會在1小時內消失，此外，高濃度的微管會耗費較久的時間消失。我們還發現可能可以通過激光偏振方向影響微管的定向。基於此處得到的結果，我們提出了雷射捕陷誘導管狀結構形成的動力學和機制，以便在固液氣三相界面上進行有效雷射捕陷。
由微管蛋白二聚體所形成的細胞微管在細胞中扮演重要的角色，並且對維持細胞形狀至關重要。雖然有必要進一步研究以討論形成機制的細節，但我們相信本研究的結果將為未來微管形成的機制提供新的見解。

關鍵字

雷射捕陷 ； 細胞骨架蛋白

並列摘要

We here present laser trapping-formed microtubules consisting of tubulin protein by focusing a continuous wave laser of 1064 nm at air/solution/glass interface in D2O. It has been already preformed the laser trapping experiments in H2O and the spatiotemporal control of microtubule formation was successfully demonstrated. Laser trapping increases not only local concentration of tubulin but also local temperature elevation at laser focus. Therefore, it has not been cleared that either concentration or temperature caused by laser irradiation mainly contribute to microtubule formation because, for example, 20 µmol/l tubulin forms microtubules at high temperature (30 ºC). In D2O, local temperature increased by laser trapping is suppressed twenty times compared to that in H2O, by which laser trapping effects on microtubule formation becomes clear.
A systematic study on microtubule formation was performed under various concentration of tubulin, ranging from 20 to 80 μM. When a focused laser beam was introduced to sample solutions, the condensed area of tubulin was generated at the laser focus and it was extended to the outside, eventually to be approximately 10 μm in diameter. During the irradiation, an anisotropic area, which was confirmed under a cross Nicol condition, was observed around the laser focus.
In order to confirm whether or not microtubules are involved in the formed anisotropic area, the temporal change in anisotropic area was observed under a low temperature of 4 ºC, because the microtubules are dissolved under such a low temperature. As a result, at all concentrations the anisotropic area disappeared within 1 hour, and it took more time at high concentrations. We also found that orientation of microtubules might be aligned by laser polarization direction. Based on all on the results obtained here, we propose dynamics and mechanism of laser trapping-induced fibrous structure formation in view of effective laser trapping at air/solution/glass interface.
Microtubule consisting of cytoskeleton plays a crucial role in the cell from tubulin dimer, and contribute a great part to maintain the cell shape. Although further investigation should be necessary to discuss the details of the formation mechanism, we believe that the findings in this study will give a new insight into the mechanism of microtubule formation in future.

並列關鍵字

Laser Trapping ； Cytoskeletal Proteins ； Tubulin

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