Characterization of liquid–liquid phase separation using super-resolution and single-molecule imaging

Hongchen Zhang; Shipeng Shao; Yujie Sun

doi:10.52601/bpr.2022.210043

Volume 8 Issue 1

Feb. 2022

Turn off MathJax

Article Contents

Article Navigation > Biophysics Reports > 2022 > 8(1): 2-13

Hongchen Zhang, Shipeng Shao, Yujie Sun. Characterization of liquid–liquid phase separation using super-resolution and single-molecule imaging[J]. Biophysics Reports, 2022, 8(1): 2-13. doi: 10.52601/bpr.2022.210043

Citation:

Hongchen Zhang, Shipeng Shao, Yujie Sun. Characterization of liquid–liquid phase separation using super-resolution and single-molecule imaging[J]. Biophysics Reports, 2022, 8(1): 2-13. doi: 10.52601/bpr.2022.210043

Citation:

PDF (7453 KB)

Characterization of liquid–liquid phase separation using super-resolution and single-molecule imaging

doi: 10.52601/bpr.2022.210043

Hongchen Zhang^{1, 2},
Shipeng Shao^{3
,
,},
Yujie Sun^{1
,
,}

1.
State Key Laboratory of Membrane Biology & Biomedical Pioneering Innovation Center (BIOPIC) & School of Future Technology, Peking University, Beijing 100871, China
2.
School of Life Sciences, Peking University, Beijing 100871, China
3.
Beijing Institute of Heart Lung and Blood Vessel Disease, Beijing Anzhen Hospital, Capital Medical University, Beijing 100029, China

More Information

Corresponding author: shaoshipengdq@163.com (S. Shao); sun_yujie@pku.edu.cn (Y. Sun)
Received Date: 23 August 2021
Accepted Date: 08 February 2022

Available Online: 17 March 2022

Publish Date: 28 February 2022

Abstract

Abstract

Liquid – liquid phase separation (LLPS) is an emerging phenomenon involved in various biological processes. The formation of phase-separated condensates is crucial for many intrinsically disordered proteins to fulfill their biological functions. Using the recombinant protein to reconstitute the formation of condensates in vitro has become the standard method to investigate the behavior and function of LLPS. Meanwhile, there is an urgent need to characterize the LLPS in living cells. Importantly, condensates formed through LLPS at physical relevant concentrations are often smaller than the optical diffraction limit, which makes precise characterization and quantification inaccurate due to the scatter of light. The booming development of super-resolution optical microscopy enables the visualization of multiple obscured subcellular components and processes, which is also suitable for the LLPS research. In this protocol, we provide step-by-step instructions to help users take advantage of super-resolution imaging to depict the morphology and quantify the molecule number of endogenous condensates in living cells using RNA Pol II as an example. This streamlined workflow offers exceptional robustness, sensitivity, and precision, which could be easily implemented in any laboratory with an inverted total internal reflection microscope. We expect that super-resolution microscopy will contribute to the investigation of both large and tiny condensates under physiological and pathological conditions and lead our understanding of the mechanism of LLPS to a higher and deeper layer.
- Liquid–liquid phase separation,
- Super-resolution imaging,
- Single-molecule imaging

FullText(HTML)

References(20)

References

[1]	Azaldegui CA, Vecchiarelli AG, Biteen JS (2021) The emergence of phase separation as an organizing principle in bacteria. Biophys J 120(7): 1123−1138 doi: 10.1016/j.bpj.2020.09.023
[2]	Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313(5793): 1642−1645 doi: 10.1126/science.1127344
[3]	Boija A, Klein IA, Sabari BR, Dall'Agnese A, Coffey EL, Zamudio AV, Li CH, Shrinivas K, Manteiga JC, Hannett NM, Abraham BJ, Afeyan LK, Guo YE, Rimel JK, Fant CB, Schuijers J, Lee TI, Taatjes DJ, Young RA (2018) Transcription factors activate genes through the phase-separation capacity of their activation domains. Cell 175(7): 1842−1855 doi: 10.1016/j.cell.2018.10.042
[4]	Cai D, Feliciano D, Dong P, Flores E, Gruebele M, Porat-Shliom N, Sukenik S, Liu Z, Lippincott-Schwartz J (2019) Phase separation of YAP reorganizes genome topology for long-term YAP target gene expression. Nat Cell Biol 21(12): 1578−1589
[5]	Fasciani A, D'Annunzio S, Poli V, Fagnocchi L, Beyes S, Michelatti D, Corazza F, Antonelli L, Gregoretti F, Oliva G, Belli R, Peroni D, Domenici E, Zambrano S, Intartaglia D, Settembre C, Conte I, Testi C, Vergyris P, Ruocco G, Zippo A (2020) MLL4-associated condensates counterbalance Polycomb-mediated nuclear mechanical stress in Kabuki syndrome. Nat Genet 52(12): 1397−1411 doi: 10.1038/s41588-020-00724-8
[6]	Feric M, Vaidya N, Harmon TS, Mitrea DM, Zhu L, Richardson TM, Kriwacki RW, Pappu RV, Brangwynne CP (2016) Coexisting liquid phases underlie nucleolar subcompartments. Cell 165(7): 1686−1697 doi: 10.1016/j.cell.2016.04.047
[7]	Guillen-Boixet J, Kopach A, Holehouse AS, Wittmann S, Jahnel M, Schlussler R, Kim K, Trussina I, Wang J, Mateju D, Poser I, Maharana S, Ruer-Gruss M, Richter D, Zhang X, Chang YT, Guck J, Honigmann A, Mahamid J, Hyman AA, Pappu RV, Alberti S, Franzmann TM (2020) RNA-induced conformational switching and clustering of G3BP drive stress granule assembly by condensation. Cell 181(2): 346−361 doi: 10.1016/j.cell.2020.03.049
[8]	Guo YE, Manteiga JC, Henninger JE, Sabari BR, Dall'Agnese A, Hannett NM, Spille JH, Afeyan LK, Zamudio AV, Shrinivas K, Abraham BJ, Boija A, Decker TM, Rimel JK, Fant CB, Lee TI, Cisse, II, Sharp PA, Taatjes DJ, Young RA (2019) Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 572(7770): 543−548 doi: 10.1038/s41586-019-1464-0
[9]	Hess ST, Girirajan TPK, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91(11): 4258−4272 doi: 10.1529/biophysj.106.091116
[10]	Levet F, Hosy E, Kechkar A, Butler C, Beghin A, Choquet D, Sibarita JB (2015) SR-Tesseler: a method to segment and quantify localization-based super-resolution microscopy data. Nat Methods 12(11): 1065−1071 doi: 10.1038/nmeth.3579
[11]	Lu H, Yu D, Hansen AS, Ganguly S, Liu R, Heckert A, Darzacq X, Zhou Q (2018) Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II. Nature 558(7709): 318−323 doi: 10.1038/s41586-018-0174-3
[12]	Lu Y, Wu T, Gutman O, Lu H, Zhou Q, Henis YI, Luo K (2020) Phase separation of TAZ compartmentalizes the transcription machinery to promote gene expression. Nat Cell Biol 22(4): 453−464 doi: 10.1038/s41556-020-0485-0
[13]	Ovesny M, Krizek P, Borkovec J, Svindrych Z, Hagen GM (2014) ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 30(16): 2389−2390 doi: 10.1093/bioinformatics/btu202
[14]	Protter DSW, Rao BS, Van Treeck B, Lin Y, Mizoue L, Rosen MK, Parker R (2018) Intrinsically disordered regions can contribute promiscuous interactions to RNP granule assembly. Cell Rep 22(6): 1401−1412 doi: 10.1016/j.celrep.2018.01.036
[15]	Razin SV, Gavrilov AA (2020) The role of liquid-liquid phase separation in the compartmentalization of cell nucleus and spatial genome organization. Biochemistry (Mosc) 85(6): 643−650 doi: 10.1134/S0006297920060012
[16]	Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3(10): 793−796 doi: 10.1038/nmeth929
[17]	Shin Y, Berry J, Pannucci N, Haataja MP, Toettcher JE, Brangwynne CP (2017) Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets. Cell 168(1-2): 159−171 doi: 10.1016/j.cell.2016.11.054
[18]	Shin Y, Brangwynne CP (2017) Liquid phase condensation in cell physiology and disease. Science 357(6357): eaaf4382. https://doi.org/10.1126/science.aaf4382
[19]	Tsang B, Pritisanac I, Scherer SW, Moses AM, Forman-Kay JD (2020) Phase separation as a missing mechanism for interpretation of disease mutations. Cell 183(7): 1742−1756 doi: 10.1016/j.cell.2020.11.050
[20]	Zhang H, Shao S, Zeng Y, Wang X, Qin Y, Ren Q, Xiang S, Wang Y, Xiao J, Sun Y (2022) Reversible phase separation of HSF1 is required for an acute transcriptional response during heat shock. Nat Cell Biol. https://doi.org/10.1038/s41556-022-00846-7