Tian Ya, Zhang Qiang, Li Jiangtao, Tsutsumi Motosuke, Inose Tomoko, Taemaitree Farsai, Hirai Kenji, Kanekura Kohsuke, Uji-I Hiroshi
Research Institute for Electronic Science (RIES) and Division of Information Science and Technology, Hokkaido University, N20W10, Kita ward, Sapporo, Hokkaido 001-0020, Japan.
School of Medicine, Shihezi University, Shihezi 832003, China.
ACS Omega. 2025 Aug 25;10(35):40056-40065. doi: 10.1021/acsomega.5c04844. eCollection 2025 Sep 9.
Membraneless organelles (MLOs), formed through liquid-liquid phase separation (LLPS), are vital for various cellular functions, including gene expression and RNA metabolism. Destructions of LLPS dynamics, often due to aberrant condensate formation, are implicated in neurodegenerative diseases. Polypeptides composed of repeated dipeptides, namely arginine-rich dipeptide repeat proteins, poly-(proline-arginine) (poly-PR) and poly-(glycine-arginine) (poly-GR), translated from mutated repeats, undergo LLPS and disrupt RNA and protein homeostasis. Although these peptides have similar net charges, they exhibit different diffusion behaviors in liquid droplets, indicating that sequence-specific charge distribution may influence LLPS dynamics. In this study, we combined fluorescence recovery after photobleaching (FRAP) and Raman spectroscopy to examine the density heterogeneity and diffusion behavior of LLPS droplets formed by arginine-rich dipeptides and homopolymeric adenine RNA (poly-A RNA). We analyzed (PR), (PR), (same components but different charge pattern), and (GR) (same net charge as (PR), but different sequence). Our results showed that droplets formed with different dipeptides displayed heterogeneous diffusion behaviors, which strongly correlated to internal density gradients within individual droplets detected by Raman spectroscopy. This suggests that charge patterning, not just net charge, critically influences LLPS dynamics. FRAP results of small dye molecules with different net charges align with the concentration gradient of RNA and dipeptides obtained by Raman spectroscopy. Raman spectroscopy combined with FRAP provides a powerful approach for revealing the biophysical properties of biomolecular condensates.
通过液-液相分离（LLPS）形成的无膜细胞器（MLOs）对包括基因表达和RNA代谢在内的各种细胞功能至关重要。LLPS动态过程的破坏，通常是由于异常凝聚物的形成，与神经退行性疾病有关。由重复二肽组成的多肽，即富含精氨酸的二肽重复蛋白、聚（脯氨酸-精氨酸）（poly-PR）和聚（甘氨酸-精氨酸）（poly-GR），由突变重复序列翻译而来，会经历LLPS并破坏RNA和蛋白质的稳态。尽管这些肽具有相似的净电荷，但它们在液滴中表现出不同的扩散行为，这表明序列特异性电荷分布可能会影响LLPS动态过程。在本研究中，我们结合光漂白后荧光恢复（FRAP）和拉曼光谱来研究由富含精氨酸的二肽和同聚腺嘌呤RNA（poly-A RNA）形成的LLPS液滴的密度异质性和扩散行为。我们分析了（PR）、（PR）（相同成分但不同电荷模式）和（GR）（与（PR）具有相同净电荷，但序列不同）。我们的结果表明，由不同二肽形成的液滴表现出异质扩散行为，这与通过拉曼光谱检测到的单个液滴内的内部密度梯度密切相关。这表明电荷模式，而不仅仅是净电荷，对LLPS动态过程起着关键作用。具有不同净电荷的小染料分子的FRAP结果与通过拉曼光谱获得的RNA和二肽的浓度梯度一致。拉曼光谱与FRAP相结合为揭示生物分子凝聚物的生物物理特性提供了一种强大的方法。
