The phosphatidylinositol (4,5)-bisphosphate-Rab35 axis regulates migrasome formation.

Migrasomes are recently discovered organelles, which are formed on the ends or branch points of retraction fibers at the trailing edge of migrating cells. Previously, we showed that recruitment of integrins to the site of migrasome formation is essential for migrasome biogenesis. In this study, we found that prior to migrasome formation, PIP5K1A, a PI4P kinase which converts PI4P into PI(4,5)P2, is recruited to migrasome formation sites. The recruitment of PIP5K1A results in generation of PI(4,5)P2 at the migrasome formation site. Once accumulated, PI(4,5)P2 recruits Rab35 to the migrasome formation site by interacting with the C-terminal polybasic cluster of Rab35. We further demonstrated that active Rab35 promotes migrasome formation by recruiting and concentrating integrin α5 at migrasome formation sites, which is likely mediated by the interaction between integrin α5 and Rab35. Our study identifies the upstream signaling events orchestrating migrasome biogenesis.

Single-molecule techniques to visualize and to characterize liquid-liquid phase separation and phase transition.

Biomolecules forming membraneless structures via liquid-liquid phase separation (LLPS) is a common event in living cells. Some liquid-like condensates can convert into solid-like aggregations, and such a phase transition process is related to some neurodegenerative diseases. Liquid-like condensates and solid-like aggregations usually exhibit distinctive fluidity and are commonly distinguished via their morphology and dynamic properties identified through ensemble methods. Emerging single-molecule techniques are a group of highly sensitive techniques, which can offer further mechanistic insights into LLPS and phase transition at the molecular level. Here, we summarize the working principles of several commonly used single-molecule techniques and demonstrate their unique power in manipulating LLPS, examining mechanical properties at the nanoscale, and monitoring dynamic and thermodynamic properties at the molecular level. Thus, single-molecule techniques are unique tools to characterize LLPS and liquid-to-solid phase transition under close-to-physiological conditions.