研究在擁擠環境中由多個驅動蛋白拖曳的貨物速度

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黃雅婷(Ya-Ting Huang) 查詢紙本館藏

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物理學系

論文名稱

研究在擁擠環境中由多個驅動蛋白拖曳的貨物速度
(Study of cargo velocity hauled by multiple kinesins in a crowded environment)

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摘要(中)

Kinesin是一種依賴ATP的細胞內馬達，沿著微管長距離運輸貨物。由多個驅動蛋白牽引貨物的運輸距離被發現更遠，但是對於多個驅動蛋白從0.3到3 extmu m/s的貨物速度沒有統一的說法，因為很少有人知道它們如何在團隊合作中工作。為了解決這個問題，我們在擁擠的環境中構建珠子測定法，並使用漲落定理判定驅動蛋白的數量，以研究擁擠效應對多個驅動蛋白拖動的貨物速度的影響。單個驅動蛋白的貨物速度被觀察到被擁擠的背景減慢。隨著驅動蛋白數量的增加，貨物速度增加並達到1 extmu m/s的單個馬達速度，特別是在更擁擠的背景下發生顯著變化。我們的研究結果表明，驅動蛋白協同行走以分擔貨物的負載，因此在擁擠的環境中比單個馬達移動得更快，但貨物速度如何超過1 extmu m/s的問題仍未解決。

摘要(英)

Kinesin, an ATP-dependent intracellular motor, transports cargo along the microtubule for a long distance. It was revealed that cargo hauled by multiple kinesins travels farther, but there is no unified statement about cargo velocity with multiple kinesins from 0.3 to 3 extmu m/s because it is rarely known how they work in teamwork. To answer this, we construct the bead assay in a crowded environment and identify the number of kinesins using the fluctuation theorem to investigate the crowding effect on cargo velocity dragged by multiple kinesins. The cargo velocity of a single kinesin is observed to be slowed by the crowded background. As the number of kinesins increases, the cargo velocity increases and reaches the single motor velocity of 1 extmu m/s, particularly dramatically changing in the more crowded background. Our findings show that kinesins walk in collaboration to share the load on cargo and, thus, move faster than a single motor does in the crowded environment, but the question of how cargo velocity might exceed 1 extmu m/s remains unsolved.

關鍵字(中)

★ 驅動蛋白
★ 擁擠環境
★ 貨物速度
★ 漲落理論
★ 負載共享模型

關鍵字(英)

★ Kinesin
★ Crowded environment
★ Cargo velocity
★ Fluctuation theorem
★ Shared-load model

論文目次

中文摘要 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Biological Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.1 Cell structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.2 Microtubule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
2.3 Kinesin motor proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3. Experimental setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
3.1 Differential interference contrast (DIC) microscopy . . . . . . . . . . . 13
3.2 Optical tweezers (OT) . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.2.1 Basic principle . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3.2.2 Experimental Setup . . . . . . . . . . . . . . . . . . . . . . . . 16
3.2.3 Stiffness calibration . . . . . . . . . . . . . . . . . . . . . . . . 17
Equipartition analysis . . . . . . . . . . . . . . . . . . . . . . . 17
Power spectrum analysis . . . . . . . . . . . . . . . . . . . . . . 17
3.2.4 Determination of a conversion constant . . . . . . . . . . . . . 19
4. Fluctuation theorem 21
4.1 Fluctuation theorem . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4.2 Stochastic model of cargo motion hauled by kinesins . . . . . . . . . . 22
4.3 Application to kinesin motion . . . . . . . . . . . . . . . . . . . . . . . 23
5. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
5.1 Sample flow channel preparation . . . . . . . . . . . . . . . . . . . . . 25
5.2 Viscosity measurement of Xanthan gum . . . . . . . . . . . . . . . . . 26
5.3 Behavior of cargo motion hauled by kinesin-1 motor moving on microtubule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
5.4 Cargo motion hauled by an unknown number of kinesins in complex solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
5.5 Counting the number of kinesins in transportation . . . . . . . . . . . 32
5.6 Load-sharing by cooperation between multiple kinesins in crowded environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
A. Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
A.1 Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
A.1.1 Cleaning cover slip . . . . . . . . . . . . . . . . . . . . . . . . . 41
A.1.2 Tubulin protein aliquot . . . . . . . . . . . . . . . . . . . . . . 41
A.1.3 Microtubule (MT) polymerization . . . . . . . . . . . . . . . . 42
A.1.4 DBMT buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
A.2 Assay buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
A.2.1 Diluted poly-L-lysine solution (PLL) . . . . . . . . . . . . . . . 42
A.2.2 CDB buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
A.2.3 Kinesin motility assay buffer in Xanthan solution (XKAB) . . 43
A.2.4 Kinesin bead assay . . . . . . . . . . . . . . . . . . . . . . . . . 43
A.3 Xanthan gum solution . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

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指導教授

田溶根(Yonggun Jun)

審核日期

2022-6-23

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