Quantification of the Onset of Condensation in Negative Elongation Factors

Abstract

Several biomolecules condensate in cells to form membraneless organelles via liquid-liquid phase separation, usually in response to changes in their environment. Recent studies suggest that biological phase separation for example regulates cell survival and pathology. The process of biological phase separation itself is not well understood. It is for example unclear how many proteins of a kind are necessary to induce a dense phase in cells – or if the local concentration is the limiting factor. Here we employ highly inclined and laminated optical sheet microscopy and single-molecule fluorescence methods to quantify early steps of phase separation of the negative elongation factor (NELF). NELF downregulates transcription and condensates upon heat and toxin stress. We treat the NELF as a model system to quantify the threshold concentration and rapidness of NELF condensation at low expression levels in fixed and live cells. by counting the number of NELF proteins in the dense phase in a time dependent manner we will contribute to understand the molecular mechanisms underlying liquid-liquid phase separation in cells.