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      Surface tension and viscosity of protein condensates quantified by micropipette aspiration

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          Abstract

          The material properties of biomolecular condensates have been suggested to play important biological and pathological roles. Despite the rapid increase in the number of biomolecules identified that undergo liquid-liquid phase separation, quantitative studies and direct measurements of the material properties of the resulting condensates have been severely lagging behind. Here, we develop a micropipette-based technique that uniquely, to our knowledge, allows quantifications of both the surface tension and viscosity of biomolecular condensates, independent of labeling and surface-wetting effects. We demonstrate the accuracy and versatility of this technique by measuring condensates of LAF-1 RGG domains and a polymer-based aqueous two-phase system. We further confirm our measurements using established condensate fusion and fluorescence recovery after photobleaching assays. We anticipate the micropipette-based technique will be widely applicable to biomolecular condensates and will resolve several limitations regarding current approaches.

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          Biomolecular condensates: organizers of cellular biochemistry

          In addition to membrane-bound organelles, eukaryotic cells feature various membraneless compartments, including the centrosome, the nucleolus and various granules. Many of these compartments form through liquid–liquid phase separation, and the principles, mechanisms and regulation of their assembly as well as their cellular functions are now beginning to emerge.
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            Liquid phase condensation in cell physiology and disease.

            Phase transitions are ubiquitous in nonliving matter, and recent discoveries have shown that they also play a key role within living cells. Intracellular liquid-liquid phase separation is thought to drive the formation of condensed liquid-like droplets of protein, RNA, and other biomolecules, which form in the absence of a delimiting membrane. Recent studies have elucidated many aspects of the molecular interactions underlying the formation of these remarkable and ubiquitous droplets and the way in which such interactions dictate their material properties, composition, and phase behavior. Here, we review these exciting developments and highlight key remaining challenges, particularly the ability of liquid condensates to both facilitate and respond to biological function and how their metastability may underlie devastating protein aggregation diseases.
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              Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates

              Evidence is now mounting that liquid-liquid phase separation (LLPS) underlies the formation of membraneless compartments in cells. This realization has motivated major efforts to delineate the function of such biomolecular condensates in normal cells and their roles in contexts ranging from development to age-related disease. There is great interest in understanding the underlying biophysical principles and the specific properties of biological condensates with the goal of bringing insights into a wide range of biological processes and systems. The explosion of physiological and pathological contexts involving LLPS requires clear standards for their study. Here, we propose guidelines for rigorous experimental characterization of LLPS processes in vitro and in cells, discuss the caveats of common experimental approaches, and point out experimental and theoretical gaps in the field.
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                Author and article information

                Journal
                9918266001106676
                50936
                Biophys Rep (N Y)
                Biophys Rep (N Y)
                Biophysical reports
                2667-0747
                7 May 2022
                08 September 2021
                11 August 2021
                14 October 2022
                : 1
                : 1
                : 100011
                Affiliations
                [1 ]Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, New Jersey
                [2 ]Department of Chemical and Biochemical Engineering, Rutgers University, Piscataway, New Jersey
                [3 ]Laboratory of Molecular Neuroscience, German Center for Neurodegenerative Diseases, Berlin, Germany
                Author notes

                AUTHOR CONTRIBUTIONS

                Z.S., B.S.S., and D.M. conceived the project. H.W. and Z.S. carried out the experiments and analyzed the data. F.M.K. purified the proteins. Z.S., H.W., B.S.S., and D.M. wrote the manuscript.

                [* ]Correspondence: zheng.shi@ 123456rutgers.edu
                Article
                NIHMS1802015
                10.1016/j.bpr.2021.100011
                9563586
                36247368
                c5d961c7-ef16-4fe8-8f51-942ecc539bd9

                This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/).

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