The formation of biomolecular condensates underpins many cellular processes; however, our current understanding of condensate formation within cells is largely based on observing the final near-equilibrium condensate state. It is less clear how proteins behave before condensates form or at concentrations at which condensation does not occur in cells. Here, we use a combination of fluorescence microscopy and photobleaching analysis to quantify phase separation of negative elongation factor (NELF) in living and stressed cells. We use the recently reported system of stress-induced condensation of NELF in human nuclei as a model to study the behaviour of proteins before condensation. We find that pre-condensate heterogeneous clusters both grow and shrink and are not freely diffusing. Unexpectedly, we also find such small dynamic clusters in unstressed cells in which condensates do not form. We provide a categorisation of small and large clusters based on their dynamics and their response to p38 kinase inhibition. Overall, our data are best explained as non-classical nucleation with a flat free-energy landscape for clusters of a range of sizes and an inhibition of condensation.

No abstract

The effect of the melting temperature on the cerium oxidation state and crystallization of cerium phosphate glasses with a molar composition 30CeO 2 70P 2 O 5 was investigated. Ce 3+ and Ce 4+ ion concentration changes in the glass attributable to the melting temperature were investigated by X-ray photoelectron spectroscopy analysis. The crystallization kinetics of the glasses and the activation energy for crystallization were evaluated under non-isothermal conditions using differential thermal analysis (DTA) performed at different heating rates. Each DTA curve exhibited one exothermic peak associated with the crystallization of the glass. The crystalline phase was identified as CePO 4 via X-ray diffractometry analysis. The Kissinger and Marotta methods were used to calculate the local activation energies for the glass samples. The amount of precipitated CePO 4 with heating at 890 K increased as the melting temperature (or Ce 3+ ) increased. The catalytic properties were studied by thermogravimetric analysis, which showed that a greater amount of precipitated CePO 4 led to poorer catalytic properties of the glass.

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.

No abstract