Designer membraneless organelles sequester native factors for control of cell behavior

Garabedian, Mikael V.; Wang, Wentao; Dabdoub, Jorge B.; Tong, Michelle; Caldwell, Reese M.; Benman, William; Schuster, Benjamin S.; Deiters, Alexander; Good, Matthew C.

doi:10.1038/s41589-021-00840-4

Title: Designer membraneless organelles sequester native factors for control of cell behavior

Journal Article · Mon Aug 02 00:00:00 EDT 2021 · Nature Chemical Biology

DOI:https://doi.org/10.1038/s41589-021-00840-4· OSTI ID:1991581

^[1]; Wang, Wentao ^[1]; Dabdoub, Jorge B. ^[1]; Tong, Michelle ^[1]; Caldwell, Reese M. ^[1]; Benman, William ^[1];

^[2];

^[3];

^[1]

Univ. of Pennsylvania, Philadelphia, PA (United States)
Rutgers Univ., Piscataway, NJ (United States)
Univ. of Pittsburgh, PA (United States)

Subcellular compartmentalization of macromolecules increases flux and prevents inhibitory interactions to control biochemical reactions. Inspired by this functionality, we sought to build designer compartments that function as hubs to regulate the flow of information through cellular control systems. Here we report a synthetic membraneless organelle platform to control endogenous cellular activities through sequestration and insulation of native proteins. We engineer and express a disordered protein scaffold to assemble micron size condensates and recruit endogenous clients via genomic tagging with high-affinity dimerization motifs. By relocalizing up to ninety percent of a targeted enzymes to synthetic condensates, we efficiently control cellular behaviors, including proliferation, division, and cytoskeletal organization. Further, we demonstrate multiple strategies for controlled cargo release from condensates to switch cells between functional states. These synthetic organelles offer a powerful and generalizable approach to modularly control cell decision-making in a variety of model systems with broad applications for cellular engineering.

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Research Organization:: Univ. of Pennsylvania, Philadelphia, PA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Institute of Biomedical Imaging and Bioengineering; National Science Foundation (NSF)

Grant/Contract Number:: SC0007063; R01 EB028320; DMR1720530; CHE-1404836

OSTI ID:: 1991581

Journal Information:: Nature Chemical Biology, Vol. 17, Issue 9; ISSN 1552-4450

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

References (57)

Modular Thermal Control of Protein Dimerization Piraner, Dan I.; Wu, Yan; Shapiro, Mikhail G. ACS Synthetic Biology, Vol. 8, Issue 10 https://doi.org/10.1021/acssynbio.9b00275	journal	September 2019
Protein Phase Separation Provides Long-Term Memory of Transient Spatial Stimuli Dine, Elliot; Gil, Agnieszka A.; Uribe, Giselle Cell Systems, Vol. 6, Issue 6 https://doi.org/10.1016/j.cels.2018.05.002	journal	June 2018
Polarity establishment requires localized activation of Cdc42 Woods, Benjamin; Kuo, Chun-Chen; Wu, Chi-Fang Journal of Cell Biology, Vol. 211, Issue 1 https://doi.org/10.1083/jcb.201506108	journal	October 2015
Optochemical Control of Protein Localization and Activity within Cell-like Compartments Caldwell, Reese M.; Bermudez, Jessica G.; Thai, David Biochemistry, Vol. 57, Issue 18 https://doi.org/10.1021/acs.biochem.8b00131	journal	April 2018
Remote control of therapeutic T cells through a small molecule–gated chimeric receptor Wu, Chia-Yung; Roybal, Kole T.; Puchner, Elias M. Science, Vol. 350, Issue 6258 https://doi.org/10.1126/science.aab4077	journal	October 2015
RGG/RG Motif Regions in RNA Binding and Phase Separation Chong, P. Andrew; Vernon, Robert M.; Forman-Kay, Julie D. Journal of Molecular Biology, Vol. 430, Issue 23 https://doi.org/10.1016/j.jmb.2018.06.014	journal	November 2018
Role of the Yeast Gin4p Protein Kinase in Septin Assembly and the Relationship between Septin Assembly and Septin Function Longtine, Mark S.; Fares, Hanna; Pringle, John R. Journal of Cell Biology, Vol. 143, Issue 3 https://doi.org/10.1083/jcb.143.3.719	journal	November 1998
Displacement of Formins from Growing Barbed Ends by Bud14 Is Critical for Actin Cable Architecture and Function Chesarone, Melissa; Gould, Christopher J.; Moseley, James B. Developmental Cell, Vol. 16, Issue 2 https://doi.org/10.1016/j.devcel.2008.12.001	journal	February 2009
Large protein organelles form a new iron sequestration system with high storage capacity Giessen, Tobias W.; Orlando, Benjamin J.; Verdegaal, Andrew A. eLife, Vol. 8 https://doi.org/10.7554/eLife.46070	journal	July 2019
SPLIT: Stable Protein Coacervation Using a Light Induced Transition Reed, Ellen H.; Schuster, Benjamin S.; Good, Matthew C. ACS Synthetic Biology, Vol. 9, Issue 3 https://doi.org/10.1021/acssynbio.9b00503	journal	February 2020
Programming self-organizing multicellular structures with synthetic cell-cell signaling Toda, Satoshi; Blauch, Lucas R.; Tang, Sindy K. Y. Science, Vol. 361, Issue 6398 https://doi.org/10.1126/science.aat0271	journal	July 2018
Compositional Control of Phase-Separated Cellular Bodies Banani, Salman F.; Rice, Allyson M.; Peeples, William B. Cell, Vol. 166, Issue 3 https://doi.org/10.1016/j.cell.2016.06.010	journal	July 2016
The nucleus is insulated from large cytosolic calcium ion changes Al-Mohanna, Futwan A.; Caddy, Keith W. T.; Bolsover, Stephen R. Nature, Vol. 367, Issue 6465 https://doi.org/10.1038/367745a0	journal	February 1994
Analysis of cell-cycle specific localization of the Rdi1p RhoGDI and the structural determinants required for Cdc42p membrane localization and clustering at sites of polarized growth Toenjes, Kurt A.; Cole, Karen C.; Wasserman, Ben T. Current Genetics, Vol. 45, Issue 6 https://doi.org/10.1007/s00294-004-0505-9	journal	June 2004
Controllable protein phase separation and modular recruitment to form responsive membraneless organelles Schuster, Benjamin S.; Reed, Ellen H.; Parthasarathy, Ranganath Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-05403-1	journal	July 2018
The involvement of stress granules in aging and aging‐associated diseases Cao, Xiuling; Jin, Xuejiao; Liu, Beidong Aging Cell, Vol. 19, Issue 4 https://doi.org/10.1111/acel.13136	journal	March 2020
Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior Schuster, Benjamin S.; Dignon, Gregory L.; Tang, Wai Shing Proceedings of the National Academy of Sciences, Vol. 117, Issue 21 https://doi.org/10.1073/pnas.2000223117	journal	May 2020
Synthetic protein scaffolds provide modular control over metabolic flux Dueber, John E.; Wu, Gabriel C.; Malmirchegini, G. Reza Nature Biotechnology, Vol. 27, Issue 8, p. 753-759 https://doi.org/10.1038/nbt.1557	journal	August 2009
Optogenetic control with a photocleavable protein, PhoCl Zhang, Wei; Lohman, Alexander W.; Zhuravlova, Yevgeniya Nature Methods, Vol. 14, Issue 4 https://doi.org/10.1038/nmeth.4222	journal	March 2017
Scaffold Proteins: Hubs for Controlling the Flow of Cellular Information Good, M. C.; Zalatan, J. G.; Lim, W. A. Science, Vol. 332, Issue 6030 https://doi.org/10.1126/science.1198701	journal	May 2011
Modular construction of mammalian gene circuits using TALE transcriptional repressors Li, Yinqing; Jiang, Yun; Chen, He Nature Chemical Biology, Vol. 11, Issue 3 https://doi.org/10.1038/nchembio.1736	journal	February 2015
Yeast formins regulate cell polarity by controlling the assembly of actin cables Sagot, Isabelle; Klee, Saskia K.; Pellman, David Nature Cell Biology, Vol. 4, Issue 1 https://doi.org/10.1038/ncb719	journal	December 2001
Client proximity enhancement inside cellular membrane-less compartments governed by client-compartment interactions Song, Daesun; Jo, Yongsang; Choi, Jeong-Mo Nature Communications, Vol. 11, Issue 1 https://doi.org/10.1038/s41467-020-19476-4	journal	November 2020
ERK signalling as a regulator of cell motility Tanimura, Susumu; Takeda, Kohsuke The Journal of Biochemistry, Vol. 162, Issue 3 https://doi.org/10.1093/jb/mvx048	journal	July 2017
Engineering dynamical control of cell fate switching using synthetic phospho-regulons Gordley, Russell M.; Williams, Reid E.; Bashor, Caleb J. Proceedings of the National Academy of Sciences, Vol. 113, Issue 47 https://doi.org/10.1073/pnas.1610973113	journal	November 2016
Modular engineering of cellular signaling proteins and networks Gordley, Russell M.; Bugaj, Lukasz J.; Lim, Wendell A. Current Opinion in Structural Biology, Vol. 39 https://doi.org/10.1016/j.sbi.2016.06.012	journal	August 2016
Phase transitions in the assembly of multivalent signalling proteins Li, Pilong; Banjade, Sudeep; Cheng, Hui-Chun Nature, Vol. 483, Issue 7389 https://doi.org/10.1038/nature10879	journal	March 2012
The budding yeast Polo-like kinase Cdc5 is released from the nucleus during anaphase for timely mitotic exit Botchkarev, Vladimir V.; Rossio, Valentina; Yoshida, Satoshi Cell Cycle, Vol. 13, Issue 20 https://doi.org/10.4161/15384101.2014.953882	journal	October 2014
Mapping Local and Global Liquid Phase Behavior in Living Cells Using Photo-Oligomerizable Seeds Bracha, Dan; Walls, Mackenzie T.; Wei, Ming-Tzo Cell, Vol. 175, Issue 6 https://doi.org/10.1016/j.cell.2018.10.048	journal	November 2018
Scaffolds, adaptors and linkers of TCR signaling: theory and practice Burack, W. Richard; Cheng, Alec M.; Shaw, Andrey S. Current Opinion in Immunology, Vol. 14, Issue 3 https://doi.org/10.1016/S0952-7915(02)00347-3	journal	June 2002
The Yeast Actin Cytoskeleton: from Cellular Function to Biochemical Mechanism Moseley, James B.; Goode, Bruce L. Microbiology and Molecular Biology Reviews, Vol. 70, Issue 3 https://doi.org/10.1128/MMBR.00013-06	journal	September 2006
Specific early-G1 blocks accompanied with stringent response in Saccharomyces cerevisiae lead to growth arrest in resting state similar to the G0 of higher eucaryotes. Iida, H.; Yahara, I. Journal of Cell Biology, Vol. 98, Issue 4 https://doi.org/10.1083/jcb.98.4.1185	journal	April 1984
De novo engineering of intracellular condensates using artificial disordered proteins Dzuricky, Michael; Rogers, Bradley A.; Shahid, Abdulla Nature Chemistry, Vol. 12, Issue 9 https://doi.org/10.1038/s41557-020-0511-7	journal	August 2020
Polo-Like Kinase Cdc5 Controls the Local Activation of Rho1 to Promote Cytokinesis Yoshida, Satoshi; Kono, Keiko; Lowery, Drew M. Science, Vol. 313, Issue 5783 https://doi.org/10.1126/science.1126747	journal	July 2006
An auxin-based degron system for the rapid depletion of proteins in nonplant cells Nishimura, Kohei; Fukagawa, Tatsuo; Takisawa, Haruhiko Nature Methods, Vol. 6, Issue 12 https://doi.org/10.1038/nmeth.1401	journal	November 2009
Formation and functionalization of membraneless compartments in Escherichia coli Wei, Shao-Peng; Qian, Zhi-Gang; Hu, Chun-Fei Nature Chemical Biology, Vol. 16, Issue 10 https://doi.org/10.1038/s41589-020-0579-9	journal	June 2020
SYNZIP Protein Interaction Toolbox: in Vitro and in Vivo Specifications of Heterospecific Coiled-Coil Interaction Domains Thompson, Kenneth Evan; Bashor, Caleb J.; Lim, Wendell A. ACS Synthetic Biology, Vol. 1, Issue 4 https://doi.org/10.1021/sb200015u	journal	February 2012
Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets Shin, Yongdae; Berry, Joel; Pannucci, Nicole Cell, Vol. 168, Issue 1-2 https://doi.org/10.1016/j.cell.2016.11.054	journal	January 2017
Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes Najem, Joseph S.; Taylor, Graham J.; Armendarez, Nick Journal of Visualized Experiments, Issue 145 https://doi.org/10.3791/58998	journal	January 2019
Photocleavable proteins that undergo fast and efficient dissociation Lu, Xiaocen; Wen, Yurong; Zhang, Shuce Chemical Science, Vol. 12, Issue 28 https://doi.org/10.1039/D1SC01059J	journal	January 2021
Engineered Protein Nano-Compartments for Targeted Enzyme Localization Choudhary, Swati; Quin, Maureen B.; Sanders, Mark A. PLoS ONE, Vol. 7, Issue 3 https://doi.org/10.1371/journal.pone.0033342	journal	March 2012
Cas9-mediated gene editing in Saccharomyces cerevisiae Anand, Ranjith; Memisoglu, Gonen; Haber, James Protocol Exchange https://doi.org/10.1038/protex.2017.021a	journal	April 2017
Designer membraneless organelles enable codon reassignment of selected mRNAs in eukaryotes Reinkemeier, Christopher D.; Girona, Gemma Estrada; Lemke, Edward A. Science, Vol. 363, Issue 6434 https://doi.org/10.1126/science.aaw2644	journal	March 2019
Coexisting Liquid Phases Underlie Nucleolar Subcompartments Feric, Marina; Vaidya, Nilesh; Harmon, Tyler S. Cell, Vol. 165, Issue 7 https://doi.org/10.1016/j.cell.2016.04.047	journal	June 2016
Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions Nakamura, Hideki; Lee, Albert A.; Afshar, Ali Sobhi Nature Materials, Vol. 17, Issue 1 https://doi.org/10.1038/nmat5006	journal	November 2017
CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. Adams, A. E.; Johnson, D. I.; Longnecker, R. M. Journal of Cell Biology, Vol. 111, Issue 1 https://doi.org/10.1083/jcb.111.1.131	journal	July 1990
The Anchor-Away Technique: Rapid, Conditional Establishment of Yeast Mutant Phenotypes Haruki, Hirohito; Nishikawa, Junichi; Laemmli, Ulrich K. Molecular Cell, Vol. 31, Issue 6 https://doi.org/10.1016/j.molcel.2008.07.020	journal	September 2008
Domains, Motifs, and Scaffolds: The Role of Modular Interactions in the Evolution and Wiring of Cell Signaling Circuits Bhattacharyya, Roby P.; Reményi, Attila; Yeh, Brian J. Annual Review of Biochemistry, Vol. 75, Issue 1 https://doi.org/10.1146/annurev.biochem.75.103004.142710	journal	June 2006
The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics Elbaum-Garfinkle, Shana; Kim, Younghoon; Szczepaniak, Krzysztof Proceedings of the National Academy of Sciences, Vol. 112, Issue 23 https://doi.org/10.1073/pnas.1504822112	journal	May 2015
Cell Signaling in Space and Time: Where Proteins Come Together and When They’re Apart Scott, John D.; Pawson, Tony Science, Vol. 326, Issue 5957 https://doi.org/10.1126/science.1175668	journal	November 2009
RNA-Based Coacervates as a Model for Membraneless Organelles: Formation, Properties, and Interfacial Liposome Assembly Aumiller, William M.; Pir Cakmak, Fatma; Davis, Bradley W. Langmuir, Vol. 32, Issue 39 https://doi.org/10.1021/acs.langmuir.6b02499	journal	September 2016
Biomolecular condensates: organizers of cellular biochemistry Banani, Salman F.; Lee, Hyun O.; Hyman, Anthony A. Nature Reviews Molecular Cell Biology, Vol. 18, Issue 5 https://doi.org/10.1038/nrm.2017.7	journal	February 2017
Prokaryotic nanocompartments form synthetic organelles in a eukaryote Lau, Yu Heng; Giessen, Tobias W.; Altenburg, Wiggert J. Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-03768-x	journal	April 2018
Light-based control of metabolic flux through assembly of synthetic organelles Zhao, Evan M.; Suek, Nathan; Wilson, Maxwell Z. Nature Chemical Biology, Vol. 15, Issue 6 https://doi.org/10.1038/s41589-019-0284-8	journal	May 2019
Using Engineered Scaffold Interactions to Reshape MAP Kinase Pathway Signaling Dynamics Bashor, C. J.; Helman, N. C.; Yan, S. Science, Vol. 319, Issue 5869 https://doi.org/10.1126/science.1151153	journal	March 2008
Bacterial encapsulins as orthogonal compartments for mammalian cell engineering Sigmund, Felix; Massner, Christoph; Erdmann, Philipp Nature Communications, Vol. 9, Issue 1 https://doi.org/10.1038/s41467-018-04227-3	journal	May 2018
Signal transduction: hanging on a scaffold Burack, W. Richard; Shaw, Andrey S. Current Opinion in Cell Biology, Vol. 12, Issue 2 https://doi.org/10.1016/S0955-0674(99)00078-2	journal	April 2000

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59 BASIC BIOLOGICAL SCIENCES
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synthetic condense
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Title: Designer membraneless organelles sequester native factors for control of cell behavior

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References (57)

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