Quantitative real-time in-cell imaging reveals heterogeneous clusters of proteins prior to condensation.

Our current understanding of biomolecular condensate formation is largely based on observing the final near-equilibrium condensate state. Despite expectations from classical nucleation theory, pre-critical protein clusters were recently shown to form under subsaturation conditions in vitro; if similar long-lived clusters comprising more than a few molecules are also present in cells, our understanding of the physical basis of biological phase separation may fundamentally change. Here, we combine fluorescence microscopy with photobleaching analysis to quantify the formation of clusters of NELF proteins in living, stressed cells. We categorise small and large clusters based on their dynamics and their response to p38 kinase inhibition. We find a broad distribution of pre-condensate cluster sizes and show that NELF protein cluster formation can be explained as non-classical nucleation with a surprisingly flat free-energy landscape for a wide range of sizes and an inhibition of condensation in unstressed cells.

A bioinspired glycopolymer for capturing membrane proteins in native-like lipid-bilayer nanodiscs.

Amphiphilic copolymers that directly extract membrane proteins and lipids from cellular membranes to form nanodiscs combine the advantages of harsher membrane mimics with those of a native-like membrane environment. Among the few commercial polymers that are capable of forming nanodiscs, alternating diisobutylene/maleic acid (DIBMA) copolymers have gained considerable popularity as gentle and UV-transparent alternatives to aromatic polymers. However, their moderate hydrophobicities and high electric charge densities render all existing aliphatic copolymers rather inefficient under near-physiological conditions. Here, we introduce Glyco-DIBMA, a bioinspired glycopolymer that possesses increased hydrophobicity and reduced charge density but nevertheless retains excellent solubility in aqueous solutions. Glyco-DIBMA outperforms established aliphatic copolymers in that it solubilizes lipid vesicles of various compositions much more efficiently, thereby furnishing smaller, more narrowly distributed nanodiscs that preserve a bilayer architecture and exhibit rapid lipid exchange. We demonstrate the superior performance of Glyco-DIBMA in preparative and analytical applications by extracting a broad range of integral membrane proteins from cellular membranes and further by purifying a membrane-embedded voltage-gated K+ channel, which was fluorescently labeled and analyzed with the aid of microfluidic diffusional sizing (MDS) directly within native-like lipid-bilayer nanodiscs.