Article

Intracellular production of hydrogels and synthetic RNA granules by multivalent molecular interactions

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Abstract

Some protein components of intracellular non-membrane-bound entities, such as RNA granules, are known to form hydrogels in vitro. The physico-chemical properties and functional role of these intracellular hydrogels are difficult to study, primarily due to technical challenges in probing these materials in situ. Here, we present iPOLYMER, a strategy for a rapid induction of protein-based hydrogels inside living cells that explores the chemically inducible dimerization paradigm. Biochemical and biophysical characterizations aided by computational modelling show that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that acts as a size-dependent molecular sieve. We functionalize these polymers with RNA-binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. These results show that iPOLYMER can be used to synthetically reconstitute the nucleation of biologically functional entities, including RNA granules in intact cells.

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Acknowledgements

We are grateful to N. Kedersha and P. Anderson who provided helpful discussions and reagents related to stress granules, to J. L. Pfaltz who collaborated with A.S.A. to develop a modified C++ code for identifying chordless cycles in graphs, and to R. Reed, A. Ewald, H. Sesaki, M. Iijima and S. Regot for sharing their resources for our experiments. We also extend our appreciation to J. P. Gong, I. Hamachi, R. Yoshida for valuable comments on our work. This work was mainly supported by the Johns Hopkins University Catalyst Fund to T.I., and in part by the National Institutes of Health (NIH) (GM092930, DK102910, CA103175 and DK089502 to T.I., and T32GM007445 to A.S.), and the National Science Foundation (NSF) (CCF-1217213 to J.G.).

Author information

Author notes

    • Hideki Nakamura
    •  & Albert A. Lee

    These authors contributed equally to this work.

    • Albert A. Lee

    Present address: Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California 94702, USA.

Affiliations

  1. Department of Cell Biology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA

    • Hideki Nakamura
    • , Albert A. Lee
    • , Shigeki Watanabe
    • , Shiva Razavi
    • , Allister Suarez
    • , Yu-Chun Lin
    • , Makoto Tanigawa
    • , Robert DeRose
    • , Diana Bobb
    •  & Takanari Inoue
  2. Center for Cell Dynamics, Institute for Basic Biomedical Sciences, Johns Hopkins University, Baltimore, Maryland 21205, USA

    • Hideki Nakamura
    • , Albert A. Lee
    • , Elmer Rho
    • , Allister Suarez
    • , Yu-Chun Lin
    • , Brian Huang
    • , Robert DeRose
    • , Diana Bobb
    •  & Takanari Inoue
  3. Center for Imaging Science, Whitaker Biomedical Engineering Institute, Johns Hopkins University, Baltimore, Maryland 21218, USA

    • Ali Sobhi Afshar
    •  & John Goutsias
  4. Department of Biomedical Engineering, Whitaker Biomedical Engineering Institute, Johns Hopkins University, Baltimore, Maryland 21218, USA

    • Shiva Razavi
    • , Makoto Tanigawa
    •  & Takanari Inoue
  5. Department of Biophysics and Biophysical Chemistry, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA

    • William Hong
    •  & Sandra B. Gabelli
  6. Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21287, USA

    • Sandra B. Gabelli
  7. Department of Oncology, School of Medicine, Johns Hopkins University, Baltimore, Maryland 21287, USA

    • Sandra B. Gabelli

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Contributions

H.N., A.A.L. and T.I. conceived the project. H.N., A.A.L., A.S., Y.-C.L., M.T., R.D. and D.B., performed molecular biology as well as cell biology experiments. H.N., A.A.L., S.R. and A.S. purified proteins under the guidance of W.H. and S.B.G. The biochemical and biophysical experiments were mostly performed by H.N. and A.A.L., and partially by S.R. and Y.-C.L. H.N., A.A.L. and T.I. wrote the manuscript with the help of J.G. A.S.A. and J.G. developed the computational model, analysed the computational results, and wrote the computational parts of the paper. A.S.A. wrote appropriate code and conducted the computational experiments. S.W. performed correlated EM measurement and analysis. E.R. and B.H. performed development and demonstration of light-inducible iPOLYMER with H.N.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Ali Sobhi Afshar or Takanari Inoue.

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