Tie-lines reveal interactions driving heteromolecular condensate formation

Qian, Daoyuan; Welsh, Timothy J.; Erkamp, Nadia A.; Qamar, Seema; Nixon‐Abell, Jonathon; George-Hyslop, Peter St; Michaels, Thomas C. T.; Knowles, Tuomas P. J.

doi:10.1101/2022.02.22.481401

Cited by 13 publications

(37 citation statements)

References 38 publications

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“…S3). The same may be the case for a significant number of other homo- and heterotypic IDP-based condensates that have been reported in the presence of PEG (4), for example a recent preprint by Knowles and co-workers indicated PEG interacts with FUS-protein condensates (51).…”

Section: Resultsmentioning

confidence: 63%

Crowding-induced phase separation and solidification by co-condensation of PEG in NPM1-rRNA condensates

André¹,

Yewdall²,

Spruijt³

2022

Preprint

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The crowdedness of the cell calls for adequate intracellular organization. Biomolecular condensates, formed by liquid-liquid phase separation of intrinsically disordered proteins and nucleic acids, are important organizers of cellular fluids. To underpin the molecular mechanisms of protein condensation, cell-free studies are often used where the role of crowding is not investigated in detail. Here, we investigate the effects of macromolecular crowding on the formation and material properties of a model heterotypic biomolecular condensate, consisting of nucleophosmin (NPM1) and ribosomal RNA (rRNA). We studied the effect of the macromolecular crowding agent PEG, which is often considered an inert crowding agent. We observed that PEG could induce both homotypic and heterotypic phase separation of NPM1 and NPM1-rRNA, respectively. Crowding increases the condensed concentration of NPM1 and decreases its equilibrium dilute phase concentration, while no significant change in the concentration of rRNA in the dilute phase was observed. Interestingly, the crowder itself is concentrated in the condensates, suggesting that co-condensation rather than excluded volume interactions underlie the enhanced phase separation by PEG. Fluorescence recovery after photobleaching (FRAP) measurements indicated that both NPM1 and rRNA become immobile at high PEG concentrations, indicative of a liquid-to-gel transition. Together, these results shed new light onto the role of synthetic crowding agents in phase separation, and demonstrate that condensate properties determined in vitro depend strongly on the addition of crowding agents.

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Section: Resultsmentioning

confidence: 63%

Crowding-induced phase separation and solidification by co-condensation of PEG in NPM1-rRNA condensates

André¹,

Yewdall²,

Spruijt³

2022

Preprint

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“…To our knowledge, this work is the first experimental investigation of dilute-phase organization in a biphasic system at equilibrium and our findings pave the way to further experimental and theoretical investigations of how complexes in the dilute phase could regulate condensate stability and dynamics. For example, recent advances in the field have demonstrated many elegant ways to experimentally measure tie-lines 32,34,35 . These measurements should also be possible for the EPYC1-Rubisco system studied here and would enable more detailed and quantitative testing of theoretical models and simulations 13,36,37 .…”

Section: Discussionmentioning

confidence: 99%

Phase-separating pyrenoid proteins form complexes in the dilute phase

GrandPre

Wilson

et al. 2022

Preprint

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While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play a significant role in the stability, dynamics, and regulation of condensates.

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“…Protein structure [17,18] Parameter Electrophoretic mobility (μ) [19] Molecular weight (Mw) [20] Half maximal inhibitory concentration (IC50) [21][22][23][24] Folding energy (ΔΔG) [25] Hydrodynamic radius (Rₕ) [19][20][21][22] Solubility constant (Kₛₚ) [30,31] Binding constant (Kd) [32][33][34][35] Partition coefficient (K) [36][37][38] Saturation concentration (Csat) [39][40][41][42][43] Colloidal stability [44,45] Diffusion coefficient (D) [37][38][39] Critical size (Rc) [49] Contact angle (θ) [50] Tie-lines [51] Condensate structure [52,53] Viscoelastic moduli (E, G, γ) [54,55] X-ray scattering (III) To measure viscosity, protein-rich phase and protein-lean phase were co-flowed and the velocity of probe beads were used to measure the viscosity of the condensate phase [99] (C) (I) Microfluidic device for measuring the relationship between shear stress and gelation of condensates (II) By varying the flow rate within the channel, protein condensates were sheared to form fibres and a controlled pressure. The solid fibres recoiled when the flow was turned off, compared to the condensate only which did not.…”

Section: Confocal Microfluidicsmentioning

confidence: 99%