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Phase transitions in the assembly of multivalent signalling proteins

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Abstract

Cells are organized on length scales ranging from ångström to micrometres. However, the mechanisms by which ångström-scale molecular properties are translated to micrometre-scale macroscopic properties are not well understood. Here we show that interactions between diverse synthetic, multivalent macromolecules (including multi-domain proteins and RNA) produce sharp liquid–liquid-demixing phase separations, generating micrometre-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to the valency of the interacting species. In the case of the actin-regulatory protein called neural Wiskott–Aldrich syndrome protein (N-WASP) interacting with its established biological partners NCK and phosphorylated nephrin1, the phase transition corresponds to a sharp increase in activity towards an actin nucleation factor, the Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions may be used to spatially organize and biochemically regulate information throughout biology.

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Figure 1: Macroscopic and microscopic phase transitions in multivalent SH3–PRM systems.
Figure 2: Multivalency drives phase separation and probably drives a sol–gel transition in the droplet phase.
Figure 3: Coexpression of SH3 5 and PRM 5 in cells produces dynamic puncta.
Figure 4: Phase transition correlates with biochemical activity transition in the nephrin–NCK–N-WASP system.

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Acknowledgements

We thank J. Onuchic and S. Padrick for discussion of the theoretical aspects of this study, L. Rice for sharing his fluorescence microscope, M. Socolich for a gift of purified eGFP, K. Luby-Phelps and A. Bugde for advice on FRAP experiments, S. Padrick and L. Doolittle for help in purifying actin and the Arp2/3 complex and for sharing reagents, N. Grishin and S. Shi for help with database searches, K. Lynch for providing the PTB expression construct, D. Billadeau and T. Gomez for providing antibodies, A. Ramesh, W. Winkler and P.-L. Tsai for advice on RNA experiments, K. Roybal and C. Wülfing for sharing unpublished data, and J. Liu for help with cryo-electron tomography. This work was supported by the following: the Howard Hughes Medical Institute and grants from the National Institutes of Health (NIH) (R01-GM56322) and Welch Foundation (I–1544) to M.K.R., a Chilton Foundation Fellowship to H.-C.C., an NIH EUREKA award (R01-GM088745) to Q.-X.J., an NIH Cancer Biology T32 Training Grant to M.L., a National Science Foundation award (DMR-1005707) to P.S.R. and a Gates Millennium Fund award to J.V.H. Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract number W-31-109-ENG-38. BioCAT is NIH-supported Research Center RR-08630.

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M.K.R. oversaw the project, helped analyse all of the data and wrote the paper with assistance from all of the authors. P.L., H.-.C.C. and M.K.R. conceived of the project. P.L. developed and interpreted the theoretical and computational models, which promoted much of the experimentation. S.B. performed and analysed experiments on the nephrin–NCK–N-WASP system and performed monovalent competition studies. H.-.C.C. mapped and analysed the phase diagrams, and collected FRAP data, on the engineered model systems. S.K. performed and analysed the cellular experiments. S.B., B.C., L.G. and B.T.N. collected and/or analysed the SAXS data. S.B., M.L. and Q.-.X. J. collected and/or analysed the electron microscopy data. S.B., J.V.H. and P.S.R. collected and/or analysed the multi-angle DLS data. H.-.C.C. and S.B. collected and analysed the single-angle DLS data. D.S.K. synthesized the octameric PRM dendrimer. S.F.B. analysed the cyclization in the sol–gel transition.

Corresponding author

Correspondence to Michael K. Rosen.

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The authors declare no competing financial interests.

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Supplementary Information

This file contains Supplementary Text and Data, Supplementary Table 1, full legend for Supplementary Movie 1, Supplementary References and Supplementary Figures 1-23. (PDF 24828 kb)

Supplementary Movie 1

This zipped file contains a movie showing large polymer formation – see Supplementary Information file page 23 for full legend. (ZIP 218 kb)

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Li, P., Banjade, S., Cheng, HC. et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012). https://doi.org/10.1038/nature10879

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