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Eph-ephrin signaling modulated by polymerization and condensation of receptors

  1. Scott E. Fraserb,2,3
  1. aBiology Division, California Institute of Technology, Pasadena, CA 91125;
  2. bCenter of Regenerative Medicine in Barcelona, Hospital Duran i Reynals, Hospitalet de Llobregat, 08908 Barcelona, Spain;
  3. cTranslational Imaging Center, University of Southern California, Los Angeles, CA 90089;
  4. dDepartment of Biological Sciences, Molecular and Computational Biology Section, University of Southern California, Los Angeles, CA 90089;
  5. eLaboratory of Theoretical & Applied Mechanics, Department of Mechanical Engineering, Universidade Federal Fluminense, Niterói, RJ 24210-240, Brazil;
  6. fICFO-The Institute of Photonic Sciences, The Barcelona Institute of Science and Technology, 08860 Castelldefels (Barcelona), Spain;
  7. gCenter for Applied Molecular Medicine, University of Southern California, Los Angeles, CA 90033;
  8. hBiomimetic Systems for Cell Engineering Group, Institute for Bioengineering of Catalonia, 08028 Barcelona, Spain;
  9. iBiomedical Research Networking Center in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), 28029 Madrid, Spain;
  10. jEuropean Molecular Biology Laboratory Australia, Australian Regenerative Medicine Institute, Monash University, Clayton, VIC 3800, Australia;
  11. kElectronics Department, University of Barcelona, 08028 Barcelona, Spain;
  12. lInstitució Catalana de Recerca i Estudis Avan?ats, 08010 Barcelona, Spain
  1. Edited by Harry B. Gray, California Institute of Technology, Pasadena, CA, and approved October 31, 2017 (received for review August 1, 2017)


Cell communication is a precisely orchestrated mechanism in which cell receptors translate extracellular cues into intracellular signals. The Eph receptors act as a model guidance system steering cells to defined positions by their ligand ephrin. However, we still lack a mechanistic understanding of how membrane receptors can read a wide range of ligand concentrations and gradients and integrate them into coherent cellular responses. Here we reveal the evolution of Eph aggregation upon ephrin stimulation with unprecedented resolution by extending current imaging methods. The results fit biophysical models of protein aggregation. In these models, two protein oligomerization modes, polymerization and condensation, correlate with the “on/off” switching of the receptor activation, providing a precise, proportional, and dynamic response to variable ephrin inputs.


Eph receptor signaling plays key roles in vertebrate tissue boundary formation, axonal pathfinding, and stem cell regeneration by steering cells to positions defined by its ligand ephrin. Some of the key events in Eph-ephrin signaling are understood: ephrin binding triggers the clustering of the Eph receptor, fostering transphosphorylation and signal transduction into the cell. However, a quantitative and mechanistic understanding of how the signal is processed by the recipient cell into precise and proportional responses is largely lacking. Studying Eph activation kinetics requires spatiotemporal data on the number and distribution of receptor oligomers, which is beyond the quantitative power offered by prevalent imaging methods. Here we describe an enhanced fluorescence fluctuation imaging analysis, which employs statistical resampling to measure the Eph receptor aggregation distribution within each pixel of an image. By performing this analysis over time courses extending tens of minutes, the information-rich 4D space (x, y, oligomerization, time) results were coupled to straightforward biophysical models of protein aggregation. This analysis reveals that Eph clustering can be explained by the combined contribution of polymerization of receptors into clusters, followed by their condensation into far larger aggregates. The modeling reveals that these two competing oligomerization mechanisms play distinct roles: polymerization mediates the activation of the receptor by assembling monomers into 6- to 8-mer oligomers; condensation of the preassembled oligomers into large clusters containing hundreds of monomers dampens the signaling. We propose that the polymerization–condensation dynamics creates mechanistic explanation for how cells properly respond to variable ligand concentrations and gradients.


  • ?1S.O. and F.C. contributed equally to this work.

  • ?2A.R. and S.E.F. contributed equally to this work.

  • ?3To whom correspondence may be addressed. Email: samuelojosnegros{at}gmail.com or sfraser{at}provost.usc.edu.
  • Author contributions: S.O., F.C., D.R., C.L.C., V.H., E.M., M.L., A.R., and S.E.F. designed research; S.O., F.C., D.R., J.J.O., C.L.C., V.H., C.T., A.S., S.M., and S.E.F. performed research; E.M. and S.E.F. contributed new reagents/analytic tools; S.O., F.C., D.R., J.J.O., C.L.C., V.H., S.M., and E.M. analyzed data; and S.O., F.C., D.R., J.J.O., C.L.C., V.H., E.M., M.L., A.R., and S.E.F. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.danielhellerman.com/lookup/suppl/doi:10.1073/pnas.1713564114/-/DCSupplemental.

Published under the PNAS license.

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