研究膜內臨界行為對膜間融合過程的影響;Investigating the Influence of the Intramembrane Critical Behavior on the Progress of the Intermembrane Fusion Process

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Title:	研究膜內臨界行為對膜間融合過程的影響;Investigating the Influence of the Intramembrane Critical Behavior on the Progress of the Intermembrane Fusion Process
Authors:	莊芮綺;Chuang, Jui-Chi
Contributors:	化學工程與材料工程學系
Keywords:	臨界波動;膜融合;螢光共振能量轉移;critical fluctuations;membrane fusion;fluorescence resonance energy transfer, FRET
Date:	2024-08-15
Issue Date:	2024-10-09 15:28:19 (UTC+8)
Publisher:	國立中央大學
Abstract:	膜融合是一種重要的生物現象，指兩個或多個原本獨立的脂雙層膜融合為一體的過程，這種現象廣泛存在於各種生物環境中，如胞吞作用(endocytosis)、胞吐作用(exocytosis)、細胞與細胞間(cell-to-cell)的融合。雖然我們先前的研究揭示了膜內混溶性的臨界波動(critical fluctuations)（一種當系統接近其臨界點時會出現的短暫和局部的組成域(domain)現象）可能會阻礙融合，但在融合過程的哪步驟受到影響仍不清楚。為了解決這個問題，我們必須準確地辨識臨界波動在融合過程中的影響時機。為此，我們採用螢光共振能量轉移（fluorescence resonance energy transfer, FRET）技術來檢查兩個融合膜中脂質分子的混合情形。當兩個螢光分子(供體(donor)與受體(acceptor))靠近時，供體分子的激發能量可以轉移到受體分子，使受體發出螢光，此技術對供體和受體之間的距離非常敏感，因此可以用來測量分子間的距離變化。在本實驗中，我們利用由低熔點脂質1,2-dioleoyi-sn-glycero-3-phosphocholine (DOPC) 、高熔點脂質1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)與膽固醇(cholesterol)所組成的仿生膜進行研究，因為它們的成分與細胞膜相似。透過對不同成分的脂質進行實驗，我們發現在液態單相中，隨著DSPC增加使組成接近液態有序相，脂質混合程度有增加的趨勢。我們認為臨界波動可能是透過水合排斥力來(hydration force)阻礙膜與膜之間相互靠近來影響融合進行，而不是對融合中間體結構(stalk)或融合孔的形成造成影響。而在液-液共存相中，脂質混合程度不隨膽固醇的比例增加而變化。我們推測促進莖結構的形成與水合排斥力呈現相反作用且互相影響，所以推斷兩者會互相抵銷使脂質混合無趨勢變化。;Membrane fusion is a fundamental biological process wherein individual membranes coalesce into a single entity, a pervasive phenomenon in various biological contexts including endocytosis, exocytosis, and cell-to-cell fusion. While our prior research has unveiled that the critical fluctuations in miscibility (a critical phenomenon which manifesters as short-lived and localized compositional domain formation when a system is in the close proximity of its critical point) within membranes might impede fusion, the underlying mechanism for this correlation remain elusive. To resolve this issue, it is imperative to precisely discern when during the fusion process the critical fluctuations exert their influence. To this end, we employ fluorescence resonance energy transfer (FRET), a technique sensitive to the distance between two energy-exchanging fluorescent probes, to scrutinize the fusion step where the lipid molecules in two coalescing membranes start mixing together, in the presence and absence of the critical fluctuations. We utilize biomimetic membranes composed of the low melting temperature lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), high melting temperature lipid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and cholesterol for our study because of their compositional similarity to cell membranes. Through experiments on different lipid compositions, we found that in a single liquid phase, as the DSPC increases and the composition approaches the liquid ordered phase, the extent of lipid mixing tends to increase. We speculate that critical fluctuations may influence membrane fusion by preventing the membranes from approaching each other through hydration repulsive force, rather than affecting the formation of the fusion intermediate structure (stalk) or the fusion pore. In the liquid-liquid coexistence phase, the extent of lipid mixing does not change with an increasing proportion of cholesterol. We speculate that the promotion of stalk formation and hydration forces have opposite effects and influence each other, leading to the cancellation of their effects, resulting in no significant change in lipid mixing.
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