Abstract

Many intracellular membraneless organelles form via phase separation of intrinsically disordered proteins (IDPs) or regions (IDRs). These include the Caenorhabditis elegans protein LAF-1, which forms P granule-like droplets in vitro. However, the role of protein disorder in phase separation and the macromolecular organization within droplets remain elusive. Here, we utilize a novel technique, ultrafast-scanning fluorescence correlation spectroscopy, to measure the molecular interactions and full coexistence curves (binodals), which quantify the protein concentration within LAF-1 droplets. The binodals of LAF-1 and its IDR display a number of unusual features, including ‘high concentration’ binodal arms that correspond to remarkably dilute droplets. We find that LAF-1 and other in vitro and intracellular droplets are characterized by an effective mesh size of 3–8 nm, which determines the size scale at which droplet properties impact molecular diffusion and permeability. These findings reveal how specific IDPs can phase separate to form permeable, low-density (semi-dilute) liquids, whose structural features are likely to strongly impact biological function.

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Acknowledgements

We thank H. Zhang and D. M. Mitrea for purifying WHI3 and NPM1, respectively. We acknowledge funding from the Princeton Center for Complex Materials, an MRSEC supported by NSF grant DMR 1420541, and the Eric and Wendy Schmidt Transformative Technology Fund. This work was also supported by an NIH Director's New Innovator Award (1DP2GM105437-01 to C.P.B.), an NSF CAREER award (1253035 to C.P.B.), NIH grants (1K99NS096217-01 to S.E.G. and 5RO1NS056114 to R.V.P.), and an HFSP Program grant (RGP0007/2012 to C.P.B.). A.S.H. is a Bonnie and Kent Lattig graduate fellow in the Center for Biological Systems Engineering at Washington University in Saint Louis. We thank C. Theriault from TAG Optics for providing the TAG lens used in this study.

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Author notes

    • Ming-Tzo Wei
    • , Shana Elbaum-Garfinkle
    •  & Alex S. Holehouse

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey 08544, USA

    • Ming-Tzo Wei
    • , Shana Elbaum-Garfinkle
    • , Carlos Chih-Hsiung Chen
    • , Marina Feric
    • , Rodney D. Priestley
    •  & Clifford P. Brangwynne
  2. Department of Biomedical Engineering and Center for Biological Systems Engineering, Washington University in Saint Louis, Saint Louis, Missouri 63130, USA

    • Alex S. Holehouse
    •  & Rohit V. Pappu
  3. Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, New Jersey 08544, USA

    • Craig B. Arnold

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Contributions

M.W. acquired and analysed data in usFCS development and nano-scale rheology. S.E.G. acquired and analysed data in particle tracking microrheology, FCS and dextran permeability. A.S.H. performed computational simulations and theoretical analysis. C.C.C. created in vivo LAF-1 constructs and injected dextran molecules into C. elegans. M.F. created in vivo NPM1/FIB-1 constructs and injected dextran molecules into X. laevis. M.W., C.B.A., C.P.B. and R.D.P. designed the usFCS measurements. A.S.H. and R.V.P. designed the theoretical analysis with input from M.W., S.E.G. and C.P.B.; M.W., S.E.G. A.S.H., R.V.P. and C.P.B. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Rohit V. Pappu or Clifford P. Brangwynne.

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DOI

https://doi.org/10.1038/nchem.2803

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