Polarisome scaffolder Spa2-mediated macromolecular condensation of Aip5 for actin polymerization

Abstract A multiprotein complex polarisome nucleates actin cables for polarized cell growth in budding yeast and filamentous fungi. However, the dynamic regulations of polarisome proteins in polymerizing actin under physiological and stress conditions remains unknown. We identify a previously functionally unknown polarisome member, actin-interacting-protein 5 (Aip5), which promotes actin assembly synergistically with formin Bni1. Aip5-C terminus is responsible for its activities by interacting with G-actin and Bni1. Through N-terminal intrinsically disordered region, Aip5 forms high-order oligomers and generate cytoplasmic condensates under the stresses conditions. The molecular dynamics and reversibility of Aip5 condensates are regulated by scaffolding protein Spa2 via liquid-liquid phase separation both in vitro and in vivo. In the absence of Spa2, Aip5 condensates hamper cell growth and actin cable structures under stress treatment. The present study reveals the mechanisms of actin assembly for polarity establishment and the adaptation in stress conditions to protect actin assembly by protein phase separation.

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Nuclear condensates of the Polycomb protein chromobox 2 (CBX2) assemble through phase separation

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.006620 ◽

2018 ◽

Vol 294 (5) ◽

pp. 1451-1463 ◽

Cited By ~ 92

Author(s):

Roubina Tatavosian ◽

Samantha Kent ◽

Kyle Brown ◽

Tingting Yao ◽

Huy Nguyen Duc ◽

...

Keyword(s):

Phase Separation ◽

Polycomb Group ◽

Site Directed Mutagenesis ◽

Developmental Defects ◽

Heterochromatin Formation ◽

Intrinsically Disordered ◽

Polycomb Protein ◽

Condensate Formation

Polycomb group (PcG) proteins repress master regulators of development and differentiation through organization of chromatin structure. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the cell nucleus, and these condensates are the physical sites of PcG-targeted gene silencing via formation of facultative heterochromatin. However, the physiochemical principles underlying the formation of PcG condensates remain unknown, and their determination could shed light on how these condensates compact chromatin. Using fluorescence live-cell imaging, we observed that the Polycomb repressive complex 1 (PRC1) protein chromobox 2 (CBX2), a member of the CBX protein family, undergoes phase separation to form condensates and that the CBX2 condensates exhibit liquid-like properties. Using site-directed mutagenesis, we demonstrated that the conserved residues of CBX2 within the intrinsically disordered region (IDR), which is the region for compaction of chromatin in vitro, promote the condensate formation both in vitro and in vivo. We showed that the CBX2 condensates concentrate DNA and nucleosomes. Using genetic engineering, we report that trimethylation of Lys-27 at histone H3 (H3K27me3), a marker of heterochromatin formation produced by PRC2, had minimal effects on the CBX2 condensate formation. We further demonstrated that the CBX2 condensate formation does not require CBX2–PRC1 subunits; however, the condensate formation of CBX2–PRC1 subunits depends on CBX2, suggesting a mechanism underlying the assembly of CBX2–PRC1 condensates. In summary, our results reveal that PcG condensates assemble through liquid–liquid phase separation (LLPS) and suggest that phase-separated condensates can organize PcG-bound chromatin.

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Identifying sequence perturbations to an intrinsically disordered protein that determine its phase-separation behavior

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2000223117 ◽

2020 ◽

Vol 117 (21) ◽

pp. 11421-11431 ◽

Cited By ~ 21

Author(s):

Benjamin S. Schuster ◽

Gregory L. Dignon ◽

Wai Shing Tang ◽

Fleurie M. Kelley ◽

Aishwarya Kanchi Ranganath ◽

...

Keyword(s):

Phase Separation ◽

Intrinsically Disordered Proteins ◽

Lipid Membrane ◽

Coarse Grained ◽

Disordered Proteins ◽

Sequence Features ◽

Intrinsically Disordered ◽

Arginine Residues

Phase separation of intrinsically disordered proteins (IDPs) commonly underlies the formation of membraneless organelles, which compartmentalize molecules intracellularly in the absence of a lipid membrane. Identifying the protein sequence features responsible for IDP phase separation is critical for understanding physiological roles and pathological consequences of biomolecular condensation, as well as for harnessing phase separation for applications in bioinspired materials design. To expand our knowledge of sequence determinants of IDP phase separation, we characterized variants of the intrinsically disordered RGG domain from LAF-1, a model protein involved in phase separation and a key component of P granules. Based on a predictive coarse-grained IDP model, we identified a region of the RGG domain that has high contact probability and is highly conserved between species; deletion of this region significantly disrupts phase separation in vitro and in vivo. We determined the effects of charge patterning on phase behavior through sequence shuffling. We designed sequences with significantly increased phase separation propensity by shuffling the wild-type sequence, which contains well-mixed charged residues, to increase charge segregation. This result indicates the natural sequence is under negative selection to moderate this mode of interaction. We measured the contributions of tyrosine and arginine residues to phase separation experimentally through mutagenesis studies and computationally through direct interrogation of different modes of interaction using all-atom simulations. Finally, we show that despite these sequence perturbations, the RGG-derived condensates remain liquid-like. Together, these studies advance our fundamental understanding of key biophysical principles and sequence features important to phase separation.

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Mechanism and cellular function of Bud6 as an actin nucleation–promoting factor

Molecular Biology of the Cell ◽

10.1091/mbc.e11-05-0404 ◽

2011 ◽

Vol 22 (21) ◽

pp. 4016-4028 ◽

Cited By ~ 45

Author(s):

Brian R. Graziano ◽

Amy Grace DuPage ◽

Alphee Michelot ◽

Dennis Breitsprecher ◽

James B. Moseley ◽

...

Keyword(s):

Actin Assembly ◽

Actin Cable ◽

Polarized Cell Growth ◽

Cable Assembly ◽

Nucleation Promoting Factor ◽

Actin Cables ◽

Elongation Phase

Formins are a conserved family of actin assembly–promoting factors with diverse biological roles, but how their activities are regulated in vivo is not well understood. In Saccharomyces cerevisiae, the formins Bni1 and Bnr1 are required for the assembly of actin cables and polarized cell growth. Proper cable assembly further requires Bud6. Previously it was shown that Bud6 enhances Bni1-mediated actin assembly in vitro, but the biochemical mechanism and in vivo role of this activity were left unclear. Here we demonstrate that Bud6 specifically stimulates the nucleation rather than the elongation phase of Bni1-mediated actin assembly, defining Bud6 as a nucleation-promoting factor (NPF) and distinguishing its effects from those of profilin. We generated alleles of Bud6 that uncouple its interactions with Bni1 and G-actin and found that both interactions are critical for NPF activity. Our data indicate that Bud6 promotes filament nucleation by recruiting actin monomers to Bni1. Genetic analysis of the same alleles showed that Bud6 regulation of formin activity is critical for normal levels of actin cable assembly in vivo. Our results raise important mechanistic parallels between Bud6 and WASP, as well as between Bud6 and other NPFs that interact with formins such as Spire.

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Polycomb Cbx2 Condensates Assemble through Phase Separation

10.1101/468926 ◽

2018 ◽

Cited By ~ 1

Author(s):

Roubina Tatavosian ◽

Samantha Kent ◽

Kyle Brown ◽

Tingting Yao ◽

Huy Nguyen Duc ◽

...

Keyword(s):

Phase Separation ◽

Polycomb Group ◽

Developmental Defects ◽

Intrinsically Disordered ◽

Condensate Formation ◽

Development And Differentiation ◽

Polycomb Repressive Complex 1 ◽

Liquid Liquid Phase Separation

AbstractPolycomb group (PcG) proteins are master regulators of development and differentiation. Mutation and dysregulation of PcG genes cause developmental defects and cancer. PcG proteins form condensates in the nucleus of cells and these condensates are the physical sites of PcG-targeted gene silencing. However, the physiochemical principles underlying the PcG condensate formation remain unknown. Here we show that Polycomb repressive complex 1 (PRC1) protein Cbx2, one member of the Cbx family proteins, contains a long stretch of intrinsically disordered region (IDR). Cbx2 undergoes phase separation to form condensates. Cbx2 condensates exhibit liquid-like properties and can concentrate DNA and nucleosomes. We demonstrate that the conserved residues within the IDR promote the condensate formation in vitro and in vivo. We further indicate that H3K27me3 has minimal effects on the Cbx2 condensate formation while depletion of core PRC1 subunits facilitates the condensate formation. Thus, our results reveal that PcG condensates assemble through liquid-liquid phase separation (LLPS) and suggest that PcG-bound chromatin is in part organized through phase-separated condensates.

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Distinct phospho-forms of cortactin differentially regulate actin polymerization and focal adhesions

AJP Cell Physiology ◽

10.1152/ajpcell.00238.2008 ◽

2008 ◽

Vol 295 (5) ◽

pp. C1113-C1122 ◽

Cited By ~ 37

Author(s):

Anne E. Kruchten ◽

Eugene W. Krueger ◽

Yu Wang ◽

Mark A. McNiven

Keyword(s):

Cell Migration ◽

Focal Adhesion ◽

Actin Polymerization ◽

Control Cell ◽

Focal Adhesions ◽

Serine Phosphorylation ◽

Actin Binding ◽

Actin Assembly

Cortactin is an actin-binding protein that is overexpressed in many cancers and is a substrate for both tyrosine and serine/threonine kinases. Tyrosine phosphorylation of cortactin has been observed to increase cell motility and invasion in vivo, although it has been reported to have both positive and negative effects on actin polymerization in vitro. In contrast, serine phosphorylation of cortactin has been shown to stimulate actin assembly in vitro. Currently, the effects of cortactin serine phosphorylation on cell migration are unclear, and furthermore, how the distinct phospho-forms of cortactin may differentially contribute to cell migration has not been directly compared. Therefore, we tested the effects of different tyrosine and serine phospho-mutants of cortactin on lamellipodial protrusion, actin assembly within cells, and focal adhesion dynamics. Interestingly, while expression of either tyrosine or serine phospho-mimetic cortactin mutants resulted in increased lamellipodial protrusion and cell migration, these effects appeared to be via distinct processes. Cortactin mutants mimicking serine phosphorylation appeared to predominantly affect actin polymerization, whereas mutation of cortactin tyrosine residues resulted in alterations in focal adhesion turnover. Thus these findings provide novel insights into how distinct phospho-forms of cortactin may differentially contribute to actin and focal adhesion dynamics to control cell migration.

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The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1504822112 ◽

2015 ◽

Vol 112 (23) ◽

pp. 7189-7194 ◽

Cited By ~ 484

Author(s):

Shana Elbaum-Garfinkle ◽

Younghoon Kim ◽

Krzysztof Szczepaniak ◽

Carlos Chih-Hsiung Chen ◽

Christian R. Eckmann ◽

...

Keyword(s):

Phase Separation ◽

Phase Boundary ◽

Single Molecule ◽

Protein Interactions ◽

Driving Forces ◽

Early Embryo ◽

P Granules ◽

Intrinsically Disordered

P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA–protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA–protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.

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WASP-interacting Protein Is Important for Actin Filament Elongation and Prompt Pseudopod Formation in Response to a Dynamic Chemoattractant Gradient

Molecular Biology of the Cell ◽

10.1091/mbc.e05-10-0994 ◽

2006 ◽

Vol 17 (10) ◽

pp. 4564-4575 ◽

Cited By ~ 8

Author(s):

Scott A. Myers ◽

Laura R. Leeper ◽

Chang Y. Chung

Keyword(s):

Actin Polymerization ◽

Time Course ◽

Leading Edge ◽

Delayed Response ◽

Dependent Manner ◽

Actin Assembly ◽

Interacting Protein

The role of WASP-interacting protein (WIP) in the process of F-actin assembly during chemotaxis of Dictyostelium was examined. Mutations of the WH1 domain of WASP led to a reduction in binding to WIPa, a newly identified homolog of mammalian WIP, a reduction of F-actin polymerization at the leading edge, and a reduction in chemotactic efficiency. WIPa localizes to sites of new pseudopod protrusion and colocalizes with WASP at the leading edge. WIPa increases F-actin elongation in vivo and in vitro in a WASP-dependent manner. WIPa translocates to the cortical membrane upon uniform cAMP stimulation in a time course that parallels F-actin polymerization. WIPa-overexpressing cells exhibit multiple microspike formation and defects in chemotactic efficiency due to frequent changes of direction. Reduced expression of WIPa by expressing a hairpin WIPa (hp WIPa) construct resulted in more polarized cells that exhibit a delayed response to a new chemoattractant source due to delayed extension of pseudopod toward the new gradient. These results suggest that WIPa is required for new pseudopod protrusion and prompt reorientation of cells toward a new gradient by initiating localized bursts of actin polymerization and/or elongation.

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Molecular determinants of phase separation forDrosophilaDNA replication licensing factors

10.1101/2021.04.12.439485 ◽

2021 ◽

Author(s):

Matthew W. Parker ◽

Jonchee Kao ◽

Alvin Huang ◽

James M. Berger ◽

Michael R. Botchan

Keyword(s):

Phase Separation ◽

Self Assembly ◽

A Priori ◽

Initiation Factor ◽

Sequence Composition ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Partner Proteins

ABSTRACTLiquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. We have shown that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separationin vitroand chromosome bindingin vivo, and that initiator condensates selectively recruit specific partner proteins. How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. UsingD. melanogaster (Dm)Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6- hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive self-assembly and condensate specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and specificitya priori.

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Molecular determinants of phase separation for Drosophila DNA replication licensing factors

eLife ◽

10.7554/elife.70535 ◽

2021 ◽

Vol 10 ◽

Author(s):

Matthew W Parker ◽

Jonchee A Kao ◽

Alvin Huang ◽

James M Berger ◽

Michael R Botchan

Keyword(s):

Phase Separation ◽

Dna Replication ◽

A Priori ◽

Initiation Factor ◽

Sequence Composition ◽

Intrinsically Disordered ◽

Intrinsically Disordered Regions ◽

Partner Proteins

Liquid-liquid phase separation (LLPS) of intrinsically disordered regions (IDRs) in proteins can drive the formation of membraneless compartments in cells. Phase-separated structures enrich for specific partner proteins and exclude others. Previously, we showed that the IDRs of metazoan DNA replication initiators drive DNA-dependent phase separation in vitro and chromosome binding in vivo, and that initiator condensates selectively recruit replication-specific partner proteins (Parker et al., 2019). How initiator IDRs facilitate LLPS and maintain compositional specificity is unknown. Here, using D. melanogaster (Dm) Cdt1 as a model initiation factor, we show that phase separation results from a synergy between electrostatic DNA-bridging interactions and hydrophobic inter-IDR contacts. Both sets of interactions depend on sequence composition (but not sequence order), are resistant to 1,6-hexanediol, and do not depend on aromaticity. These findings demonstrate that distinct sets of interactions drive condensate formation and specificity across different phase-separating systems and advance efforts to predict IDR LLPS propensity and partner selection a priori.

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Thermodynamic and sequential characteristics of phase separation and droplet formation for an intrinsically disordered region/protein ensemble

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008672 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1008672

Author(s):

Wen-Ting Chu ◽

Jin Wang

Keyword(s):

Phase Separation ◽

High Temperatures ◽

Electrostatic Interactions ◽

Sequence Length ◽

Intrinsically Disordered ◽

Dynamics Simulations ◽

Conventional Behavior ◽

Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) of some IDPs/IDRs can lead to the formation of the membraneless organelles in vitro and in vivo, which are essential for many biological processes in the cell. Here we select three different IDR segments of chaperon Swc5 and develop a polymeric slab model at the residue-level. By performing the molecular dynamics simulations, LLPS can be observed at low temperatures even without charge interactions and disappear at high temperatures. Both the sequence length and the charge pattern of the Swc5 segments can influence the critical temperature of LLPS. The results suggest that the effects of the electrostatic interactions on the LLPS behaviors can change significantly with the ratios and distributions of the charged residues, especially the sequence charge decoration (SCD) values. In addition, three different forms of swc conformation can be distinguished on the phase diagram, which is different from the conventional behavior of the free IDP/IDR. Both the packed form (the condensed-phase) and the dispersed form (the dilute-phase) of swc chains are found to be coexisted when LLPS occurs. They change to the fully-spread form at high temperatures. These findings will be helpful for the investigation of the IDP/IDR ensemble behaviors as well as the fundamental mechanism of the LLPS process in bio-systems.

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