Synergistic Phase Separation of Two Pathways Promotes Integrin Clustering and Integrin Adhesion Complex Formation

Integrin adhesion complexes (IACs) are integrin-based plasma membrane-associated comp iartments where cells sense environmental cues. The physical mechanisms and molecular interactions that mediate nascent IAC formation are unclear. We found that both p130Cas ("Cas") and Focal adhesion kinase ("FAK") undergo liquid-liquid phase separation in vitro under physiologic conditions. Cas- and FAK- driven phase separation is sufficient to reconstitute kindlindependent integrin clustering in vitro. In vitro condensates and cellular IACs exhibit similar sensitivities to environmental perturbations including changes in temperature and pH. Furthermore, mutations that inhibit or enhance phase separation in vitro reduce or increase the number of IACs in cells, respectively. Finally, we find that the Cas and FAK pathways act synergistically to promote phase separation, integrin clustering and IAC formation in vitro and in cells. We propose that Cas- and FAK- driven phase separation provides an intracellular trigger for integrin clustering and nascent IAC formation.

Liquid–Liquid Phase Separation in Crowded Environments

International Journal of Molecular Sciences ◽

10.3390/ijms21165908 ◽

2020 ◽

Vol 21 (16) ◽

pp. 5908 ◽

Cited By ~ 4

Author(s):

Alain A. M. André ◽

Evan Spruijt

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Macromolecular Crowding ◽

Liquid Phase Separation ◽

The Impact ◽

Crowded Environments ◽

Biomolecular condensates play a key role in organizing cellular fluids such as the cytoplasm and nucleoplasm. Most of these non-membranous organelles show liquid-like properties both in cells and when studied in vitro through liquid–liquid phase separation (LLPS) of purified proteins. In general, LLPS of proteins is known to be sensitive to variations in pH, temperature and ionic strength, but the role of crowding remains underappreciated. Several decades of research have shown that macromolecular crowding can have profound effects on protein interactions, folding and aggregation, and it must, by extension, also impact LLPS. However, the precise role of crowding in LLPS is far from trivial, as most condensate components have a disordered nature and exhibit multiple weak attractive interactions. Here, we discuss which factors determine the scope of LLPS in crowded environments, and we review the evidence for the impact of macromolecular crowding on phase boundaries, partitioning behavior and condensate properties. Based on a comparison of both in vivo and in vitro LLPS studies, we propose that phase separation in cells does not solely rely on attractive interactions, but shows important similarities to segregative phase separation.

Concentration and dosage sensitivity of proteins driving liquid-liquid phase separation

10.1101/2021.02.19.430946 ◽

2021 ◽

Author(s):

Nazanin Farahi ◽

Tamas Lazar ◽

Shoshana J. Wodak ◽

Peter Tompa ◽

Rita Pancsa

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Liquid Phase Separation ◽

Condensate Formation ◽

Associated Proteins ◽

In Cells ◽

Dosage Sensitivity

AbstractLiquid-liquid phase separation (LLPS) is a molecular process that leads to the formation of membraneless organelles (MLOs), i.e. functionally specialized liquid-like cellular condensates formed by proteins and nucleic acids. Integration of data on LLPS-associated proteins from dedicated databases revealed only modest overlap between them and resulted in a confident set of 89 human LLPS driver proteins. Since LLPS is highly concentration-sensitive, the underlying experiments are often criticized for applying higher-than-physiological protein concentrations. To clarify this issue, we performed a naive comparison of in vitro applied and quantitative proteomics-derived protein concentrations and discuss a number of considerations that rationalize the choice of apparently high in vitro concentrations in most LLPS studies. The validity of in vitro LLPS experiments is further supported by in vivo phase-separation experiments and by the observation that the corresponding genes show a strong propensity for dosage sensitivity. This observation implies that the availability of the respective proteins is tightly regulated in cells to avoid erroneous condensate formation. In all, we propose that although local protein concentrations are practically impossible to determine in cells, proteomics-derived cellular concentrations should rather be considered as lower limits of protein concentrations, than strict upper bounds, to be respected by in vitro experiments.

Targeting NSD2-mediated SRC-3 liquid–liquid phase separation sensitizes bortezomib treatment in multiple myeloma

Nature Communications ◽

10.1038/s41467-021-21386-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jing Liu ◽

Ying Xie ◽

Jing Guo ◽

Xin Li ◽

Jingjing Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Phase Separation ◽

Liquid Phase ◽

Liquid Phase Separation ◽

Acquired Drug Resistance ◽

Bortezomib Treatment ◽

AbstractDevelopment of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

G-quadruplex DNA inhibits unwinding activity but promotes liquid–liquid phase separation by the DEAD-box helicase Ded1p

Chemical Communications ◽

10.1039/d1cc01479j ◽

2021 ◽

Author(s):

Jun Gao ◽

Zhaofeng Gao ◽

Andrea A. Putnam ◽

Alicia K. Byrd ◽

Sarah L. Venus ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Liquid Phase Separation ◽

Intrinsically Disordered ◽

Dead Box ◽

G4 Dna ◽

G Quadruplex ◽

The Dead ◽

G-quadruplex (G4) DNA inhibits RNA unwinding activity but promotes liquid–liquid phase separation of the DEAD-box helicase Ded1p in vitro and in cells. This highlights multifaceted effects of G4DNA on an enzyme with intrinsically disordered domains.

Taurine suppresses liquid-liquid phase separation of lysozyme protein

10.1101/2021.01.26.428332 ◽

2021 ◽

Author(s):

Kanae Tsubotani ◽

Sayuri Maeyama ◽

Shigeru Murakami ◽

Stephen W Schaffer ◽

Takashi Ito

Keyword(s):

Phase Separation ◽

Polyethylene Glycol ◽

Critical Temperature ◽

Liquid Phase ◽

Liquid Phase Separation ◽

Compatible Osmolyte ◽

Hen Egg ◽

AbstractTaurine is a compatible osmolyte that infers stability to proteins. Recent studies have revealed that liquid-liquid phase separation (LLPS) of proteins underlie the formation of membraneless organelles in cells. In the present study, we evaluated the role of taurine on LLPS of hen egg lysozyme. We demonstrated that taurine decreases the turbidity of the polyethylene glycol-induced crowding solution of lysozyme. We also demonstrated that taurine attenuates LLPS-dependent cloudiness of lysozyme solution with 0.5 or 1M NaCl at a critical temperature. Moreover, we observed that taurine inhibits LLPS formation of a heteroprotein mix solution of lysozyme and ovalbumin. These data indicate that taurine can modulate the formation of LLPS of proteins.

Small molecules for modulating protein driven liquid-liquid phase separation in treating neurodegenerative disease

10.1101/721001 ◽

2019 ◽

Cited By ~ 8

Author(s):

Richard J. Wheeler ◽

Hyun O. Lee ◽

Ina Poser ◽

Arun Pal ◽

Thom Doeleman ◽

...

Keyword(s):

Phase Separation ◽

Neurodegenerative Disease ◽

Stress Granules ◽

Life Time ◽

Stress Granule ◽

Phase Behaviour ◽

Granule Proteins ◽

Lateral Sclerosis ◽

AbstractAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with few avenues for treatment. Many proteins implicated in ALS associate with stress granules, which are examples of liquid-like compartments formed by phase separation. Aberrant phase transition of stress granules has been implicated in disease, suggesting that modulation of phase transitions could be a possible therapeutic route. Here, we combine cell-based and protein-based screens to show that lipoamide, and its related compound lipoic acid, reduce the propensity of stress granule proteins to aggregate in vitro. More significantly, they also prevented aggregation of proteins over the life time of Caenorhabditis elegans. Observations that they prevent dieback of ALS patient-derived (FUS mutant) motor neuron axons in culture and recover motor defects in Drosophila melanogaster expressing FUS mutants suggest plausibility as effective therapeutics. Our results suggest that altering phase behaviour of stress granule proteins in the cytoplasm could be a novel route to treat ALS.

Liquid-liquid phase separation of the chromosomal passenger complex drives parallel bundling of midzone microtubules

10.1101/2022.01.07.475460 ◽

2022 ◽

Author(s):

Ewa Niedzialkowska ◽

Tan M Truong ◽

Luke A Eldredge ◽

Stefanie Redemann ◽

Denis Chretien ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Chromosome Segregation ◽

Dynamic Structure ◽

Liquid Phase Separation ◽

Chromosomal Passenger Complex ◽

Chromosomal Passenger ◽

Spindle Midzone ◽

The spindle midzone is a dynamic structure that forms during anaphase, mediates chromosome segregation, and provides a signaling platform to position the cleavage furrow. The spindle midzone comprises two antiparallel bundles of microtubules (MTs) but the process of their formation is poorly understood. Here, we show that the Chromosomal Passenger Complex (CPC) undergoes liquid-liquid phase separation (LLPS) to generate parallel MT bundles in vitro when incubated with free tubulin and GTP. MT bundles emerge from CPC droplets with protruding minus-ends that then grow into long, tapered MT structures. During this growth, the CPC in condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for LLPS or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data uncovers a kinase-independent function of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle.

Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

10.1101/2020.12.18.423429 ◽

2020 ◽

Author(s):

Florian Geiger ◽

Guido Papa ◽

William E. Arter ◽

Julia Acker ◽

Kadi L. Saar ◽

...

Keyword(s):

Phase Separation ◽

Unique Feature ◽

Rna Viruses ◽

Therapeutic Approaches ◽

Subcellular Organelles ◽

Selective Enrichment ◽

Novel Therapeutic ◽

AbstractRNA viruses induce formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation. We demonstrate that rotavirus proteins NSP5 and NSP2 undergo phase separation in vitro and form RNA-rich condensates in vivo that can be reversibly dissolved by aliphatic diols. During infection, these RNA-protein condensates became less dynamic and impervious to aliphatic diols, indicating a transition from a liquid to solid state. Some aspects of assembly of rotavirus replication factories mirror the formation of cytoplasmic ribonucleoprotein granules, while the selective enrichment of viral transcripts appears to be a unique feature of these condensates. Such complex RNA-protein condensates that underlie replication of RNA viruses represent an attractive target for developing novel therapeutic approaches.

Phase separation and toxicity of C9orf72 poly(PR) depends on alternate distribution of arginine

The Journal of Cell Biology ◽

10.1083/jcb.202103160 ◽

2021 ◽

Vol 220 (11) ◽

Author(s):

Chen Chen ◽

Yoshiaki Yamanaka ◽

Koji Ueda ◽

Peiying Li ◽

Tamami Miyagi ◽

...

Keyword(s):

Phase Separation ◽

Charge Distribution ◽

Protein Interactions ◽

Protein Protein Interactions ◽

C9orf72 Gene ◽

And Diffusion ◽

Lateral Sclerosis ◽

Dipeptide Repeat ◽

Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid–liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein–protein interactions.

Intrinsic Disorder-Based Emergence in Cellular Biology: Physiological and Pathological Liquid-Liquid Phase Transitions in Cells

Polymers ◽

10.3390/polym11060990 ◽

2019 ◽

Vol 11 (6) ◽

pp. 990 ◽

Cited By ~ 17

Author(s):

April L. Darling ◽

Boris Y. Zaslavsky ◽

Vladimir N. Uversky

Keyword(s):

Phase Transitions ◽

Phase Separation ◽

Liquid Phase ◽

Intrinsic Disorder ◽

Living Cells ◽

Cellular Biology ◽

Physiological Functions ◽

Pathological Conditions ◽

The visible outcome of liquid-liquid phase transitions (LLPTs) in cells is the formation and disintegration of various proteinaceous membrane-less organelles (PMLOs). Although LLPTs and related PMLOs have been observed in living cells for over 200 years, the physiological functions of these transitions (also known as liquid-liquid phase separation, LLPS) are just starting to be understood. While unveiling the functionality of these transitions is important, they have come into light more recently due to the association of abnormal LLPTs with various pathological conditions. In fact, several maladies, such as various cancers, different neurodegenerative diseases, and cardiovascular diseases, are known to be associated with either aberrant LLPTs or some pathological transformations within the resultant PMLOs. Here, we will highlight both the physiological functions of cellular liquid-liquid phase transitions as well as the pathological consequences produced through both dysregulated biogenesis of PMLOs and the loss of their dynamics. We will also discuss the potential downstream toxic effects of proteins that are involved in pathological formations.