Dimeric endophilin A2 stimulates assembly and GTPase activity of dynamin 2

Biophys J. 2011 Feb 2;100(3):729-737. doi: 10.1016/j.bpj.2010.12.3717.


Endophilin, which participates in membrane vesiculation during receptor-mediated endocytosis, is a ∼40 kDa SH3 domain-containing protein that binds to the proline/arginine-rich domain of dynamin, a ∼100 kDa GTPase that is essential for endocytic membrane scission. It has been suggested that endophilin is monomeric in the cytoplasm and dimerizes only after it binds to membranes (or perhaps to dimers or tetramers of dynamin). To clarify this issue, we studied the oligomeric state of endophilin both in vitro using analytical ultracentrifugation and fluorescence anisotropy, and in living cells using two-photon fluorescence fluctuation spectroscopy. We analyzed the fluctuation data using the Q-analysis method, which allowed us to determine the intrinsic brightness of the labeled protein complexes and hence its aggregation state in the cytoplasmic regions of the cell. Although a relatively high K(d) (∼5-15 μM) was observed in vitro, the cell measurements indicate that endophilin is dimeric in the cytoplasm, even at submicromolar concentrations. We also demonstrate that endophilin significantly enhances the assembly of dynamin, and that this enhancement is proportional to the fraction of dimeric endophilin that is present. Moreover, there is correlation between the concentrations of endophilin that promote dynamin self-assembly and those that stimulate dynamin GTPase activity. These findings support the view that endophilin-dynamin interactions play an important role in endocytosis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Anisotropy
  • Cell Survival
  • Dynamin II / chemistry
  • Dynamin II / metabolism*
  • Dynamin II / ultrastructure
  • Green Fluorescent Proteins / metabolism
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Models, Molecular
  • Nephelometry and Turbidimetry
  • Protein Binding
  • Protein Multimerization*
  • Protein Structure, Quaternary
  • Rats
  • Scattering, Small Angle
  • Solutions
  • Spectrometry, Fluorescence
  • X-Ray Diffraction
  • src Homology Domains


  • Intracellular Signaling Peptides and Proteins
  • Solutions
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins
  • Dynamin II