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Title: Controllable protein phase separation and modular recruitment to form responsive membraneless organelles

Abstract

Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles. Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. Here we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. First, we demonstrate enzymatically triggered droplet assembly and disassembly, whereby miscibility and RGG domain valency are tuned by protease activity. Second, we control droplet composition by selectively recruiting cargo molecules via protein interaction motifs. We then demonstrate protease-triggered controlled release of cargo. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells. This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells.

Authors:
ORCiD logo; ; ; ; ; ORCiD logo; ; ; ORCiD logo
Publication Date:
Research Org.:
Univ. of Pennsylvania, Philadelphia, PA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1461590
Alternate Identifier(s):
OSTI ID: 1511484
Grant/Contract Number:  
SC0007063
Resource Type:
Published Article
Journal Name:
Nature Communications
Additional Journal Information:
Journal Name: Nature Communications Journal Volume: 9 Journal Issue: 1; Journal ID: ISSN 2041-1723
Publisher:
Nature Publishing Group
Country of Publication:
United Kingdom
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Schuster, Benjamin S., Reed, Ellen H., Parthasarathy, Ranganath, Jahnke, Craig N., Caldwell, Reese M., Bermudez, Jessica G., Ramage, Holly, Good, Matthew C., and Hammer, Daniel A. Controllable protein phase separation and modular recruitment to form responsive membraneless organelles. United Kingdom: N. p., 2018. Web. doi:10.1038/s41467-018-05403-1.
Schuster, Benjamin S., Reed, Ellen H., Parthasarathy, Ranganath, Jahnke, Craig N., Caldwell, Reese M., Bermudez, Jessica G., Ramage, Holly, Good, Matthew C., & Hammer, Daniel A. Controllable protein phase separation and modular recruitment to form responsive membraneless organelles. United Kingdom. doi:10.1038/s41467-018-05403-1.
Schuster, Benjamin S., Reed, Ellen H., Parthasarathy, Ranganath, Jahnke, Craig N., Caldwell, Reese M., Bermudez, Jessica G., Ramage, Holly, Good, Matthew C., and Hammer, Daniel A. Mon . "Controllable protein phase separation and modular recruitment to form responsive membraneless organelles". United Kingdom. doi:10.1038/s41467-018-05403-1.
@article{osti_1461590,
title = {Controllable protein phase separation and modular recruitment to form responsive membraneless organelles},
author = {Schuster, Benjamin S. and Reed, Ellen H. and Parthasarathy, Ranganath and Jahnke, Craig N. and Caldwell, Reese M. and Bermudez, Jessica G. and Ramage, Holly and Good, Matthew C. and Hammer, Daniel A.},
abstractNote = {Many intrinsically disordered proteins self-assemble into liquid droplets that function as membraneless organelles. Because of their biological importance and ability to colocalize molecules at high concentrations, these protein compartments represent a compelling target for bio-inspired materials engineering. Here we manipulated the intrinsically disordered, arginine/glycine-rich RGG domain from the P granule protein LAF-1 to generate synthetic membraneless organelles with controllable phase separation and cargo recruitment. First, we demonstrate enzymatically triggered droplet assembly and disassembly, whereby miscibility and RGG domain valency are tuned by protease activity. Second, we control droplet composition by selectively recruiting cargo molecules via protein interaction motifs. We then demonstrate protease-triggered controlled release of cargo. Droplet assembly and cargo recruitment are robust, occurring in cytoplasmic extracts and in living mammalian cells. This versatile system, which generates dynamic membraneless organelles with programmable phase behavior and composition, has important applications for compartmentalizing collections of proteins in engineered cells and protocells.},
doi = {10.1038/s41467-018-05403-1},
journal = {Nature Communications},
number = 1,
volume = 9,
place = {United Kingdom},
year = {2018},
month = {7}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1038/s41467-018-05403-1

Citation Metrics:
Cited by: 15 works
Citation information provided by
Web of Science

Figures / Tables:

Fig. 1 Fig. 1: Phase separation of RGG-based IDP constructs. a Illustration of four-cell C. elegans embryo. LAF-1 is present in P granules, which contribute to germline specification. b Domain organization of LAF-1, RGG, RGG-RGG, and RGG-RGG-RGG. c Representative turbidity measurements show temperaturedependent phase behavior of RGG, RGG-RGG, and RGG-RGG-RGG at proteinmore » concentrations of 0.2 mg/mL (approximately 12 μM RGG domain concentration) in 150 mM NaCl buffer, pH 7.5. d Phase diagram of RGG-RGG as a function of salt and protein concentrations. Green markers indicateconditions at which optically resolvable droplets were observed. Inset: microscopy image of phase-separated RGG-RGG protein droplets. Scale bar: 10μm. e Schematic illustrating design goals (i) inducible phase separation and (ii) cargo recruitment and triggered rel« less

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