Shinya Tahara

Biomolecular condensates formed via liquid–liquid phase separation often contain multiple molecular components whose collective interactions determine their physical properties. However, how different molecular species cooperatively regulate condensate mechanics remains poorly understood. In this study, we integrated Raman–Brillouin imaging with fluorescence recovery after photobleaching (FRAP) analysis to quantify the molecular composition, mobility, and high‐frequency viscoelastic responses of multicomponent condensates composed of heterochromatin protein 1α (HP1α), DNA, and lipids. DNA incorporation rendered condensates morphologically distorted yet mechanically soft, while DNA within the condensates exhibited limited mobility, suggesting the formation of partially immobilized DNA‐rich structures that shape the condensate morphology without rigidifying the interior. Subsequent lipid incorporation selectively confined HP1α mobility and increased condensate viscoelasticity without altering the DNA dynamics. These results reveal the opposing mechanical effects of DNA and lipids, arising from distinct DNA–protein coupling states within the condensates. DNA‐rich structures exhibit dynamics decoupled from HP1α within the same condensates, whereas lipid incorporation enhances effective DNA–protein coupling, thereby reinforcing condensate mechanics. These findings establish lipid‐mediated DNA–protein coupling as a key physicochemical mechanism regulating the mechanical properties of multicomponent biomolecular condensates.

Ataxin-3, the causative protein of Machado-Joseph disease, comprises an enzymatic domain, Josephin domain (JD), and a polyglutamine repeat (polyQ). The polyQ expansion promotes the fibrillogenesis of ataxin-3, which is responsible for the disease onset. The fibrillogenesis proceeds through two distinct steps: protofibril formation, in which JD undergoes self-assembly, and subsequent fibrillation. Both polyQ elongation and metal ions accelerate fibrillation; however, the effects of these factors on each stage remain unclear. This study comprehensively analyzed the effects of metal ions on ataxin-3 protofibril formation using spectroscopic analyses. The protofibril formation of JD and that with a 28-residue polyQ tract (Q28) did not complete within 14 d under metal-free conditions. In contrast, the introduction of Cu²⁺ or Zn²⁺ markedly accelerated the process: JD denatured within 1 d, and protofibrils were formed within 4 d. The acceleration was similar between JD and Q28, indicating that metal binding primarily affects JD. Thiol (-SH) assays and mass spectrometry showed that Cu²⁺ binds to thiols and induces intra- and intermolecular disulfide bonds, whereas Zn²⁺ binds to thiols but does not promote disulfide formation. These observations suggest that Cu²⁺ and Zn²⁺ enhance the protofibril formation through interactions with specific JD sites, but the underlying mechanisms differ between the two metal ions.

High-concentration PEG-based liquid–liquid phase separation concentrates biomolecules into droplets, enabling highly sensitive, label-free Raman analysis of enzymatic reactions and protein–small-molecule interactions in dilute, small-volume samples.

Liquid–liquid phase separation (LLPS) is a phenomenon where homogeneous solutions of biomacromolecules separate into two liquid phases and generate liquid droplets enriched in specific biomolecules. LLPS of neurodegeneration-related proteins, including fused in sarcoma (FUS), promotes their aggregation, causing fatal diseases such as amyotrophic lateral sclerosis (ALS). Recent studies showed that RNAs regulate LLPS of these proteins and inhibit their aggregation, which may play an important role in preventing the disease onset; however, the underlying molecular mechanisms remain elusive. It is also unknown whether endogenous RNAs regulate LLPS and subsequent aggregation in cells. In this study, we investigated features of RNAs that enable their entrance into FUS droplets and inhibition of FUS aggregation via droplets and clarified the underlying mechanisms using Raman microscopy. We found that RNA length is one of the primary factors governing both the aggregation-inhibition effect and the localization of RNAs in the droplets in buffer solutions. Short (1000-nt) RNAs were not concentrated and dissolved the droplets. Raman imaging of living cells revealed that intracellular FUS droplets are enriched with endogenous RNAs at levels comparable to in vitro droplets and exhibit high fluidity, confirming that endogenous RNAs play a crucial role in suppressing droplet-to-aggregate transition of FUS in cells. These findings indicate that short RNAs stabilize FUS droplets through heterotypic RNA–FUS interactions that compete with homotypic FUS–FUS direct contacts responsible for aggregation, whereas binding of long RNAs enhances FUS solubility and promotes droplet dissolution. Our study highlights the protective role of RNAs against pathogenic aggregation of neurodegeneration-related proteins via droplets.

We present the results of Raman imaging of molecular crowding environments in a living cell and a liquid droplet formed by liquid-liquid phase separation. Using the Raman band of water as an internal intensity standard, we propose an in situ quantification method for evaluating biomolecular concentrations. Based on these concentration measurements, intracellular crowding environments can be quantitatively assessed. A single liquid droplet in a buffer solution is found to have a very high biomolecular concentration, exceeding a few millimolar concentrations, and the concentration within a droplet varies depending on the surrounding environment. The biomolecular concentration in a droplet increases as conditions facilitate droplet formation, and we propose a model in which changes in the surrounding environment lead to the formation of dense droplets, which subsequently transform into aggregates. Concentration quantification in a single droplet was also performed in a living cell, demonstrating that the biomolecular concentration in a droplet is not much different from that in the surrounding intracellular environment. This result indicates that within a droplet in a cell, certain molecules are highly concentrated, while other molecules are excluded, and the overall concentration is comparable to that of the surrounding intracellular environment. The droplet formation within a cell can be regarded as a redistribution of biomolecules constituting a molecular crowding environment.

Rhodopsins, an important group of photoreceptor proteins, possess a retinal chromophore. It has long been believed that the photoisomerization of the chromophore is the initial event triggering its photocycle. However, we recently reported that protein structural changes around the chromophore precede photoisomerization in bacteriorhodopsin (BR). In this study, we performed deep-ultraviolet femtosecond stimulated Raman measurements for H⁺-pump rhodopsin (RxR) and photosensor rhodopsin (NpSRII) to investigate whether these ultrafast protein dynamics are common among rhodopsins. We observed that the protein structural changes occur within 0.2 ps after photoexcitation and precede the chromophore photoisomerization, similar to the case for BR. This strongly indicates that the protein structural change precedes the photoisomerization in rhodopsins, regardless of the difference in their origins and functions. Furthermore, we found that only limited protein changes occur on the time scale of the photoisomerization, suggesting that the protein environment is already optimized for the structural change of the chromophore. These observations raise the possibility that the ultrafast protein change arranges the environment around the chromophore to facilitate the photoisomerization.

A highly sensitive method is proposed for obtaining the Raman spectra of low-concentration proteins and nucleic acids in an aqueous solution using liquid–liquid phase separation.