生物分子凝聚體（biomolecular condensates）的形成和消散是細胞用於區隔各種化學反應的一環，參與生物分子的濃度調節、緩衝、感應外界的刺激及信號傳遞等過程。這些生物分子凝聚體的形成原理是生物高分子的液-液相分離（LLPS），使系統產生兩個分離的相（phase），其中一個相是含有高濃度生物高分子的液滴（droplet），另一個相則是生物高分子濃度較低的水溶液。隨著生物分子的濃度增加，凝聚體可能會進一步轉變為膠態或固態，最終形成聚集體（aggregate），而這些聚集體可能與神經退化性疾病有關。研究顯示，在細胞中許多含有低複雜度區域（LCD）的蛋白質，例如FUS和TDP-43，會參與這些生物分子凝聚體的相變。為了研究生物分子凝聚體相變在細胞中的作用，必須控制凝聚體的物理狀態。然而，目前仍缺乏有效的工具來調控這些凝聚體的相變。
為了解決這個問題，我們希望利用光控胜肽作為一個新的工具來控制FUS蛋白的凝聚，並在光的刺激下誘發FUS蛋白的固化。通過分析FUS蛋白的序列，我們首先找出LCD中一段富含酪胺酸，且容易形成聚集體的片段。藉著將該片段與帶正電的多精胺鏈結合後，我們創造了能形成液滴的胜肽（JSF1）。照光後，我們發現JSF1可以釋放多精胺鏈，使LCD片段形成類澱粉纖維（amyloid-like fibril）。通過光學顯微鏡、原子力顯微鏡和電子顯微鏡，我們也鑑定了JSF1在照光前後物理性質的改變。
利用JSF1的相變特性，我們成功將JSF1用於調控FUS蛋白的相變。當FUS蛋白加入JSF1時，FUS蛋白的凝聚會被增強，而照光之後的JSF1成功誘導了FUS蛋白的纖維化。當我們將JSF1加入神經細胞株（N2A）時，發現JSF1能穿透細胞膜並存在於細胞質中。通過Fluorescence recovery after photobleaching （FRAP）的實驗，我們發現JSF1增加了細胞質FUS蛋白凝聚體的流動性。而照光之後，FUS凝聚體的流動性顯著降低。最後，我們分析了神經細胞的存活率，發現FUS凝聚體的流動性與細胞存活率之間存在正相關。
在這個研究中，我們開發了在試管及細胞中調控FUS蛋白相變的新方法。未來，黃老師實驗室可利用JSF1，以及蛋白質體學和RNA定序等方法，繼續探索FUS相變與其蛋白質病理（proteinopathy）之間的關聯。

The biomolecular condensates are important for the subcellular compartmentalization for controlling of cellular reactions such as biomolecule concentration, buffering, stimulus sensing, and signaling. These biomolecular condensates are formed via liquid-liquid phase separation (LLPS), by which the biopolymers condense to form liquid droplet (dense phase) separated from the environment depleting of the biopolymers (dilute phase). However, aberrant LLPS might lead to liquid-to-gel or liquid-to-solid phase transition (maturation) of the condensates, possibly resulting in neurodegenerative disease-related pathological aggregates. It has also been revealed that many proteins with low complexity domain (LCD) such as fused in sarcoma (FUS) and TAR DNA-binding protein 43 (TDP-43) are responsible for these processes. To reveal the roles of phase transition of biomolecular condensates, controlling the phases of condensates is necessary. However, a useful tool to modulate the physical states of the biomolecular condensates is still lacking.
To tackle this issue, we aim to create a peptide-based phase modulator capable of modulating FUS protein condensation and transforming FUS condensates into solid-like aggregates in response to external stimuli. Through the analyzing the sequence of FUS, an aggregation-prone, tyrosine-rich fragment in the LCD of FUS was identified. By conjugating the fragment to a positively-charged tract, we created a peptide, JSF1, which could undergo LLPS and form spherical droplets spontaneously. After photoinitiation, the poly-arginine tract was released, leading to formation of amyloid-like fibrils of the LCD fragment. With the aid of optical microscopy, atomic force microscopy, and transmission electron microscopy, the distinct physical properties of JSF1 before and after photoinitiation was characterized.
Taking advantage of the phase transition properties of JSF1, we successfully applied it in modulating the phases of FUS protein condensates. While JSF1 was incubated with FUS in vitro, the condensation of FUS was effectively enhanced. Upon photoinitiation, JSF1 induced the fibrilization of FUS. When we added JSF1 to live neuronal cell line, we found JSF1 could penetrated cell membrane and mainly localize in the cytosol. Through the fluorescence recovery after photobleaching assay, we found JSF1 increased the fluidity of cytosolic FUS condensates. After photoinitiation, the fluidity of FUS condensates was dramatically decreased. Finally, the cell viability assay demonstrated the positive correlation between the fluidity of FUS condensates and cell viability of neuronal cells.
In this study, we successfully created a novel approach for modulating the phases of FUS protein both in vitro and in cells. By applying proteomics and RNA-seq, Joseph Huang's Lab could further explore the correlation between the FUS phase transition and its proteinopathy in the near future.

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