Quantitative phase microscopy enables precise and efficient determination of biomolecular condensate composition.

First Authors Patrick M McCall
Authors Patrick M McCall, Kyoohyun Kim, Anatol W Fritsch, Juan M Iglesias-Artola, Louise Jawerth, Jie Wang, Martine Ruer, Jan Peychl, Andrey Poznyakovskiy, Jochen Guck, Simon Alberti, Anthony A. Hyman, Jan Brugues
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Last Authors Jan Brugues
Journal Name bioRxiv (bioRxiv)
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Article Number https://doi.org/10.1101/2020.10.25.352823
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Abstract Many compartments in eukaryotic cells are protein-rich biomolecular condensates demixed from the cyto- or nucleoplasm. Although much has been learned in recent years about the integral roles condensates play in many cellular processes as well as the biophysical properties of reconstituted condensates, an understanding of their most basic feature, their composition, remains elusive. Here we combined quantitative phase microscopy (QPM) and the physics of sessile droplets to develop a precise method to measure the shape and composition of individual model condensates. This technique does not rely on fluorescent dyes or tags, which we show can significantly alter protein phase behavior, and requires 1000-fold less material than traditional label-free technologies. We further show that this QPM method measures the protein concentration in condensates to a 3-fold higher precision than the next best label-free approach, and that commonly employed strategies based on fluorescence intensity dramatically underestimate these concentrations by as much as 50-fold. Interestingly, we find that condensed-phase protein concentrations can span a broad range, with PGL3, TAF15(RBD) and FUS condensates falling between 80 and 500 mg/ml under typical in vitro conditions. This points to a natural diversity in condensate composition specified by protein sequence. We were also able to measure temperature-dependent phase equilibria with QPM, an essential step towards relating phase behavior to the underlying physics and chemistry. Finally, time-resolved QPM reveals that PGL3 condensates undergo a contraction-like process during aging which leads to doubling of the internal protein concentration coupled to condensate shrinkage. We anticipate that this new approach will enable understanding the physical properties of biomolecular condensates and their function.
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DOI 10.1101/2020.10.25.352823
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Created By herbst
Added Date 2020-10-28
Last Edited By thuem
Last Edited Date 2020-10-30 11:13:16.202
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