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CPP 11.3: Vortrag
Montag, 16. März 2020, 10:00–10:15, SCH A251
Measuring protein concentrations in biomolecular condensates via quantitative phase microscopy — •Patrick M McCall1,2, K Kim3, J Wang1, AW Fritsch1, A Poznyakovskiy1, B Diederich4, M Kreysing1, R Heintzmann4, J Guck3, S Alberti3, J Brugués1,2, and AA Hyman1 — 1MPI-CBG, Dresden — 2MPI-PKS, Dresden — 3TU Dresden — 4Leibniz IPhT, Jena
Many compartments in eukaryotic cells are protein-rich biomolecular condensates formed via phase separation from the cyto- or nucleoplasm. Although knowledge of condensate composition is essential for a full description of condensate properties and potential functions, measurements of composition pose a number of technical challenges. To address these, we use quantitative phase microscopy and optical diffraction tomography to measure the refractive index of model condensates, from which the protein concentration may be inferred. Here, model condensates are formed by phase separation of purified protein constructs derived from the primarily disordered RNA-binding domain (RBD) of TAF15. Surprisingly, we find that phase separation of TAF15(RBD) is attenuated only weakly by salt (0.05-3 M KCl) or temperature (10-50 ∘C), suggesting that Coulombic and entropic interactions, respectively, play only minor roles in controlling the phase equilibria. Interestingly, we also find that partition coefficients determined by fluorescence microscopy dramatically underestimate protein concentrations in condensates. A simple model including inner filter and excited-state saturation effects suggests that the discrepancy stems primarily from reduced fluorescence quantum yields in condensates.