基于激光扫描共聚焦显微镜的蛋白质相分离研究方法

基于激光扫描共聚焦显微镜的蛋白质相分离研究方法

赵  明， 韩秋影，李卓欣，赵  冰，申业壮，王  凯，徐  欣，於  宇，夏  天^*

（1.国家生物医学分析中心，北京100850；2.吉林大学第一医院，肿瘤中心，吉林 长春 130021 ）

摘  要 激光扫描共聚焦显微镜（LSCM）具有高分辨率、高对比度和可定量测量等优势，其在细胞及组织成像等方面的广泛应用极大地推动了生命科学领域的发展。相分离的研究极度依赖显微成像技术，本文着重总结了基于激光扫描共聚焦显微镜的相分离研究方法，并探讨了相分离研究的难点，为相关研究人员了解蛋白质相分离及显微镜在相分离领域的应用提供了参考，也为开展相分离研究提供了技术依据。

关键词  相分离；共聚焦显微镜；蛋白质

中图分类号：Q334；Q331；Q233   文献标识码：A      doi:10.3969/j.issn.1000-6281.2023.01.016

The research methods of protein phase separation based on laser scanning confocal microscope

ZHAO Ming , HAN Qiu-ying, LI Zhuo-xin , ZHAO Bing , SHEN Ye-zhuang , WANG Kai , XU Xin ,YU Yu , XIA Tian^*

（1. National Center of Biomedical Analysis, Beijing  100850；2. Cancer Center, The First Hospital of Jilin University, Changchun Jilin130021，China）

Abstract    Laser scanning confocal microscope (LSCM) has played awidespread role in acquiring the images of cells and tissues with the advantages of high resolution, high contrastand quantitative measurements, which has greatly contributed to the development of life sciences. The study of phase separation heavily relies on microscopic imaging. Here, we summarized and discussed the application of laser scanning confocal microscopy in phase separation, which may  provide references to understand protein LLPS and also provides technical basis for the study of phase separation.

Keywords    phase separation；laser scanning confocal microscope；protein 

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[1]  PADDOCK S W, ELICEIRI K W. Laser scanning confocal microscopy: history, applications, and related optical sectioning techniques [J]. Methods Mol Biol, 2014, 1075: 9-47.

[2]  MINSKY M.  Memoir on inventing the confocal scanning microscope [J]. Scanning, 1988, 10(4): 128-138.

[3]  CANETTE A, BRIANDET R. Microscopy | Confocal laser scanning microscopy [M]. France: BATT C A, TORTORELLO M L. Encyclopedia of Food Microbiology (Second Edition). Oxford：Academic Press, 2014: 676-683.

[4]  WILSON E B.  The structure of protoplasm [J]. Science, 1899, 10(237): 33-45.

[5]  BRANGWYNNE C P, ECKMANN C R, COURSON D S, et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation [J]. Science, 2009, 324(5935): 1729-1732.

[6]  BOULAY G, SANDOVAL G J, RIGGI N, et al. Cancer-specific retargeting of BAF complexes by a prion-like domain [J]. Cell, 2017, 171(1): 163-178 e19.

[7]  ZHU G, XIE J, KONG W, et al. Phase separation of disease-associated SHP2 mutants underlies MAPK hyperactivation [J]. Cell, 2020, 183(2): 490-502 e18.

[8]  PATEL A, LEE H O, JAWERTH L, et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation [J]. Cell, 2015, 162(5): 1066-1077.

[9]  SHIN Y, BRANGWYNNE C P. Liquid phase condensation in cell physiology and disease [J]. Science, 2017, 357(6357).

[10]  TSANG B, PRITISANAC I, SCHERER S W, et al. Phase separation as a missing mechanism for interpretation of disease mutations [J]. Cell, 2020, 183(7): 1742-1756.

[11]  SU X, DITLEV J A, HUI E, et al. Phase separation of signaling molecules promotes T cell receptor signal transduction [J]. Science, 2016, 352(6285): 595-599.

[12]  SHEU-GRUTTADAURIA J, MACRAE I J. Phase transitions in the assembly and function of human miRISC [J]. Cell, 2018, 173(4): 946-957 e16.

[13]  JIANG H, WANG S, HUANG Y, et al. Phase transition of spindle-associated protein regulate spindle apparatus assembly [J]. Cell, 2015, 163(1): 108-122.

[14]  ZHANG G, WANG Z, DU Z, et al. mTOR regulates phase separation of PGL granules to modulate their autophagic degradation [J]. Cell, 2018, 174(6): 1492-1506 e22.

[15]  ZHENG Q, CHEN Y, CHEN D, et al. Calcium transients on the ER surface trigger liquid-liquid phase separation of FIP200 to specify autophagosome initiation sites [J]. Cell, 2022, 185(22): 4082-4098 e22.

[16]  KANG J Y, WEN Z, PAN D, et al.  LLPS of FXR1 drives spermiogenesis by activating translation of stored mRNAs [J]. Science, 2022, 377(6607): eabj6647.

[17]  ROMERO P, OBRADOVIC Z, LI X, et al. Sequence complexity of disordered protein [J]. Proteins, 2001, 42(1): 38-48.

[18]  QUIROZ F G, CHILKOTI A. Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers [J]. Nat Mater, 2015, 14(11): 1164-1171.

[19]  BRANGWYNNE CLIFFORD P, TOMPA P, PAPPU ROHIT V. Polymer physics of intracellular phase transitions [J]. Nature Physics, 2015, 11(11): 899-904.

[20]  XIAO Q, MCATEE C K, SU X. Phase separation in immune signalling [J]. Nat Rev Immunol, 2022, 22(3): 188-199.

[21]  ZHAO M, YU Y, SUN L M, et al. GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein [J]. Nat Commun, 2021, 12(1): 2114.

[22]  KATO M, HAN T W, XIE S, et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels [J]. Cell, 2012, 149(4): 753-767.

[23]  LI P, BANJADE S, CHENG H C, et al. Phase transitions in the assembly of multivalent signalling proteins [J]. Nature, 2012, 483(7389): 336-340.

[24]  ZHAO M, XIA T, XING J Q, et al. The stress granule protein G3BP1 promotes pre-condensation of cGAS to allow rapid responses to DNA [J]. EMBO Rep, 2022, 23(1): e53166.

[25]  SCHNEIDER M W G, GIBSON B A, OTSUKA S, et al. A mitotic chromatin phase transition prevents perforation by microtubules [J]. Nature, 2022, 609(7925): 183-190.

[26]  ZHANG M, ZHU C, DUAN Y, et al. The intrinsically disordered region from PP2C phosphatases functions as a conserved CO₂ sensor [J]. Nat Cell Biol, 2022, 24(7): 1029-1037.

[27]  DU M, EA C K, FANG Y, et al. Liquid phase separation of NEMO induced by polyubiquitin chains activates NF-kappaB [J]. Mol Cell, 2022, 82(13): 2415-2426 e5.

[28]  BOYD-SHIWARSKI C R, SHIWARSKI D J, GRIFFITHS S E, et al. WNK kinases sense molecular crowding and rescue cell volume via phase separation [J]. Cell, 2022, 185(24): 4488-4506 e20.

[29]  SHIN Y, BERRY J, PANNUCCI N, et al. Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets [J]. Cell, 2017, 168(1/2): 159-171 e14.

[30]  ZHANG H, SHAO S, ZENG Y, et al. Reversible phase separation of HSF1 is required for an acute transcriptional response during heat shock [J]. Nat Cell Biol, 2022, 24(3): 340-352.

[31]  SCHNEIDER N, WIELAND F G, KONG D, et al. Liquid-liquid phase separation of light-inducible transcription factors increases transcription activation in mammalian cells and mice [J]. Sci Adv, 2021, 7(1).

[32]  YU X, ZHANG L, SHEN J, et al. The STING phase-separator suppresses innate immune signalling [J]. Nat Cell Biol, 2021, 23(4): 330-340.

[33]  ALBERTI S, SAHA S, WOODRUFF J B, et al. A user's guide for phase separation assays with purified proteins [J]. J Mol Biol, 2018, 430(23): 4806-4820.