Crosslinking-Induced Endocytosis of Acetylcholine Receptors by Quantum Dots

Abstract

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.

Receptor Crosslinking: A General Method to Trigger Internalization and Lysosomal Targeting of Therapeutic Receptor:Ligand Complexes

“…In order to evaluate delivery of exogenous proteins to lysosomes within the context of ADCs, we sought to exploit the enhanced trafficking to lysosomes that many receptors perform when clustered or crosslinked into “supramultivalent” interactions. This enhanced and sometimes aberrant lysosomal delivery has been observed for many receptors, 12 including rabies G protein, 13 ErbB family receptors such as epidermal growth factor receptor, 14 , 15 acetylcholine receptors, 16 , 17 and FcRn receptors. 18 These findings were demonstrated in a range of cell types, including hamster kidney, 12 mouse neuroblastoma, 13 human kidney, 14 human epidermal, 15 rat muscle, 16 Xenopus muscle, 17 and human endothelial cells.…”

“…This enhanced and sometimes aberrant lysosomal delivery has been observed for many receptors, 12 including rabies G protein, 13 ErbB family receptors such as epidermal growth factor receptor, 14 , 15 acetylcholine receptors, 16 , 17 and FcRn receptors. 18 These findings were demonstrated in a range of cell types, including hamster kidney, 12 mouse neuroblastoma, 13 human kidney, 14 human epidermal, 15 rat muscle, 16 Xenopus muscle, 17 and human endothelial cells. 18 Furthermore, crosslinking was induced in these reports by a range of methods, including streptavidin (SA), 12 , 17 bivalent antibodies, 13 , 16 , 18 natural ligands, 14 , 18 and multivalent designed ankyrin repeat proteins (DARPins).…”

Quantum effects in the brain: A review

“…Halpern as an alternative means to implement a quantum computer 209 . They have also been used to model the mechanism by which anti-acetylcholine receptor antibodies contribute to the neuromuscular disorder myasthenia gravis 210 . Even more recently, graphene quantum dots have been shown to prevent and even undo the protein clumping of neurons in the brain that leads to Parkinson's disease 211 .…”

EGF receptor lysosomal degradation is delayed in the cells stimulated with EGF-Quantum dot bioconjugate but earlier key events of endocytic degradative pathway are similar to that of native EGF

“…Quantum dots (QDs) are fluorescent semiconductor nanoparticles with a high quantum yield and exceptional photostability [ 1 – 4 ]. Their complexing to ligands recognizing surface receptors that undergo internalization makes them an attractive tool for live cell imaging of ligand-receptor complexes behavior and for specific delivery of fluorescent probes into the cells of interest [ 5 – 10 ]. However, functionalized QDs are much larger than native ligands and due to their large surface become quasi-multivalent.…”