Liquid-liquid Phase Separation Research to Start Now.

Liquid-liquid phase separation (LLPS) is a phenomenon in which certain molecules assemble locally within a cell to form aggregates (droplets) of biomolecules with liquid-like properties. In recent years, LLPS has attracted much attention as it has been shown to affect many biological processes in the cell. Although the study of droplets formed by phase separation is still in its infancy, elucidating how these biological phenomena affect cellular functions and the pathogenesis of disease is considered key to the development of new therapeutic strategies.

Reference: E. Dolgin, Nature, 2018, DOI: 10.1038/d41586-018-03070-2.

◯Autophagy
Autophagy, the degradation process for recycling or metabolising unwanted proteins and organelles in cells, is attracting attention because it has been linked to neurodegenerative diseases such as Parkinson's disease and ageing^*1. Zheng and colleagues have shown that LLPS is involved in the regulation of autophagy activity. Induction of autophagy triggers LLPS of FIP200, an essential factor for autophagosome formation, which triggers a Ca2+ transient at the ER surface regulated by the protein EPG-4/EI24. Furthermore, the droplet-like structure of FIP200 is dependent on the ER proteins VAPs and ATLs for its association, suggesting that it functions as an autophagosome initiation site.




*1 Qiaoxia Zheng et al., Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites., Cell, 2022, 185(22), 4082-4098.

◯Ferroptosis
Iron ion-dependent accumulation of lipid peroxides has been shown to be one of the cell death pathways, ferroptosis, distinct from apoptosis as a form of programmed cell death, and is attracting attention as a new target for cancer therapy*2. A compound called icFSP1 has been identified as a potent inhibitor of FSP1 and has been shown to inhibit FSP1 from the plasma membrane to the cell surface. to intracellular relocalisation of FSP1 to form droplet-like structures, thereby promoting ferroptosis.


*2 Toshitaka Nakamura et al., Phase separation of FSP1 promotes ferroptosis., Nature, 2023, 619, 371-377.

Generally, when starting LLPS research, it is often necessary to confirm that the target protein forms droplets in a cell-free system.
However, the optimal conditions for this step also differ depending on the protein, and there are various considerations and precautions such as pH, salt type, and the presence or absence of crowding agents*.
We have prepared two types of kits that are ideal for those who are just starting out in LLPS research.

*Crowding agent (molecular adulterant reproducer): A polymer such as PEG or Ficoll that is added to reproduce a crowded intracellular environment.

LL01 LLPS Starter Kit: Ready-to-use kit for the first time LLPS research
LL02 LLPS Forming Condition Screening Kit: Ideal for optimization of conditions for target protein droplets

Various interactions are involved in the LLPS-induced droplet formation of proteins, with typical examples being electrostatic interactions using the charge of the protein and hydrophobic interactions with highly hydrophobic proteins. Alpha-synuclein (αSyn), one of the causative proteins of Parkinson's disease, is known to cause droplet formation by LLPS. S129E, a phosphorylation-mimetic mutant of αSyn, promotes droplet formation and it has been reported that this enhancement of droplet formation is due to altered interaction^*3. Phosphorylation-mimetic mutations of αSyn are known to promote aggregation involved in lesion formation^*4, and the identification of changes in protein-protein interactions in droplet formation is expected to be a new therapeutic strategy.

Here, the LLPS Starter Kit and LLPS Forming Condition Screening Kit were used to identify differences in protein-protein interactions in droplet formation between Bovine serum albumin (BSA) and Lactoferrin (LT). When the electrostatic interaction was inhibited by increasing the salt concentration (NaCl), LT droplet formation was inhibited, confirming that LT droplets are mainly formed and maintained by electrostatic interaction, as reported. On the other hand, droplet formation of BSA was enhanced in a NaCl concentration-dependent manner, presumably because the increase in salt concentration brought the proteins closer together and strengthened hydrophobic interactions.

*3 Manisha Poudyal et al., Intermolecular interactions underlie protein/peptide phase separation irrespective of sequence and structure at crowded milieu., Nature, 2023, 14, 6199.
*4 Soumik Ray et al., α-Synuclein aggregation nucleates through liquid–liquid phase separation., Nature, 2020, 12, 705-716.