Crosslinking-induced endocytosis of acetylcholine receptors by quantum dots

PLoS One. 2014 Feb 25;9(2):e90187. doi: 10.1371/journal.pone.0090187. eCollection 2014.


In a majority of patients with myasthenia gravis (MG), anti-acetylcholine receptor (AChR) antibodies target postsynaptic AChR clusters and thus compromise the membrane integrity of neuromuscular junctions (NMJs) and lead to muscle weakness. Antibody-induced endocytosis of AChRs in the postsynaptic membrane represents the initial step in the pathogenesis of MG; however, the molecular mechanisms underlying AChR endocytosis remain largely unknown. Here, we developed an approach to mimic the pathogenic antibodies for inducing the crosslinking and internalization of AChRs from the postsynaptic membrane. Using biotin-α-bungarotoxin and quantum dot (QD)-streptavidin, cell-surface and internalized AChRs could be readily distinguished by comparing the size, fluorescence intensity, trajectory, and subcellular localization of the QD signals. QD-induced AChR endocytosis was mediated by clathrin-dependent and caveolin-independent mechanisms, and the trafficking of internalized AChRs in the early endosomes required the integrity of microtubule structures. Furthermore, activation of the agrin/MuSK (muscle-specific kinase) signaling pathway strongly suppressed QD-induced internalization of AChRs. Lastly, QD-induced AChR crosslinking potentiated the dispersal of aneural AChR clusters upon synaptic induction. Taken together, our results identify a novel approach to study the mechanisms of AChR trafficking upon receptor crosslinking and endocytosis, and demonstrate that agrin-MuSK signaling pathways protect against crosslinking-induced endocytosis of AChRs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Agrin / metabolism
  • Animals
  • Clathrin / metabolism
  • Cross-Linking Reagents / chemistry
  • Cross-Linking Reagents / pharmacology*
  • Endocytosis / drug effects*
  • Mice
  • Microtubules / drug effects
  • Microtubules / metabolism
  • Particle Size
  • Quantum Dots* / chemistry
  • Receptor Protein-Tyrosine Kinases / metabolism
  • Receptors, Cholinergic / chemistry*
  • Receptors, Cholinergic / metabolism*
  • Signal Transduction / drug effects
  • Synapses / drug effects
  • Synapses / metabolism


  • Agrin
  • Clathrin
  • Cross-Linking Reagents
  • Receptors, Cholinergic
  • MuSK protein, mouse
  • Receptor Protein-Tyrosine Kinases

Grants and funding

This study was partly supported by a research development grant (186316) from Muscular Dystrophy Association and a start-up grant (2012-067-SU-01) from National University of Singapore to C.W.L., and a General Research Fund grant (662312) from Hong Kong Research Grants Council to H.B.P. and C.W.L. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.