富含精氨酸的肽可以主动介导液-液相分离

Steven Boeynaems; Mathias De Decker; Peter Tompa; Ludo Van Den Bosch

doi:10.21769/BioProtoc.2525

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Peer-reviewed

Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation

富含精氨酸的肽可以主动介导液-液相分离

Steven Boeynaems

MD Mathias De Decker

PT Peter Tompa

LB Ludo Van Den Bosch email

发布: 2017年09月05日第7卷第17期 DOI: 10.21769/BioProtoc.2525 浏览次数: 13540

评审: Nicoletta CordaniWeiyan JiaMirko Messa

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The authors used this protocol in:

Cover of Molecular Cell, featuring study using the protocol.

Mar 2017

实验方案合集

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Abstract

Studying liquid-liquid phase separation (LLPS) of proteins provides key insights into the biogenesis of membraneless organelles and pathological protein aggregation in disease. We have established a protocol for inducing the phase separation of arginine-rich peptides, which allows for studying their molecular determinants and dynamics (Boeynaems et al., 2017).

Keywords: LLPS (LLPS)

Amyotrophic lateral sclerosis (肌萎缩性侧索硬化)

Peptides (肽)

Arginine (精氨酸)

Phase separation (相分离)

Mass spec (质谱仪)

C9orf72 (C9orf72)

Stress granule (应激颗粒)

Background

Arginine-rich disordered domains are often found in RNA binding proteins, including the ones associated with neurodegenerative diseases (e.g., FUS, FMRP, hnRNPA1) (Varadi et al., 2015; Boeynaems et al., 2017). Also toxic arginine-rich repeat peptides (i.e., polyGR and polyPR) are produced in amyotrophic lateral sclerosis patients carrying the C9orf72 repeat expansion (Kwon et al., 2014; Mizielinska et al., 2014; Varadi et al., 2015; Boeynaems et al., 2016). While phase separation of uncharged low complexity domains had been studied before (Kato et al., 2012; Burke et al., 2015; Lin et al., 2015; Molliex et al., 2015; Patel et al., 2015), we developed this protocol to test whether arginine-rich domains could also contribute to phase separation. A schematic representation of the protocol can be seen in Figure 1.

Figure 1. Workflow of protein droplet formation and analysis. A diffuse arginine-rich peptide solution can be induced to undergo liquid-liquid phase separation by addition of RNA or molecular crowder (PEG). Resulting droplets can be further analyzed by fluorescence microscopy and FRAP.

Materials and Reagents

Pipette tips
Amicon^® Ultra–0.5 ml centrifugal filters 3K (Merck, catalog number: UFC500396 )
Cell counting slides (Bio-Rad Laboratories, catalog number: 1450015 )
Clear adhesive tape
Custom 20 to 60 amino acid peptides with free carboxy and amino-termini (Pepscan)
Custom Alexa labeled RNA 30 ribonucleotide oligomers (IDT)
MilliQ water
Alexa Fluor^® Protein Labeling Kit (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A10235 )
PEG300 (Sigma-Aldrich, catalog number: 81160 )
Note: This product has been discontinued.
Potassium phosphate monobasic (KH₂PO₄)
Potassium phosphate dibasic (K₂HPO₄)
Polyuridylic acid potassium salt (Sigma-Aldrich, catalog number: P9528 )
Clear nail varnish
10x potassium buffer, pH 7 (see Recipes)

Equipment

Pipette
Bench-top centrifuge (Eppendorf, models: 5430 and 5810 R )
trUView cuvettes (Bio-Rad Laboratories, catalog number: 1702510 )
Note: This product has been discontinued.
SmartSpec Plus spectrophotometer (Bio-Rad Laboratories, model: SmartSpec Plus )
LSM 780 Meta NLO confocal microscope (ZEISS, model: LSM 780 NLO ) with 20x long range objective (ZEISS, model: LD Plan-Neofluor 20x/0.4 Corr Ph2 M27, catalog number: 421351-9970-000 )

Software

Zen software (ZEISS)
GraphPad Prism (GraphPad)
Excel (Microsoft)

Procedure

English

中文翻译

文章信息

版权信息

如何引用

Boeynaems, S., De Decker, M., Tompa, P. and Van Den Bosch, L. (2017). Arginine-rich Peptides Can Actively Mediate Liquid-liquid Phase Separation. Bio-protocol 7(17): e2525. DOI: 10.21769/BioProtoc.2525.