Endosomal trafficking of the receptor tyrosine kinase MuSK proceeds via clathrin-dependent pathways, Arf6 and actin

Abstract

Muscle-specific kinase (MuSK), a receptor tyrosine kinase, is the key player during the formation of the neuromuscular junction. Signal transduction events downstream of MuSK activation induce both pre- and postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors at synaptic sites. More recently, regulated MuSK endocytosis and degradation have been implicated as crucial events for MuSK signalling activity, implicating a cross-talk between signalling and endocytosis. In the present study, we use a live imaging approach to study MuSK endocytosis. We find that MuSK is internalized via a clathrin-, dynamin-dependent pathway. MuSK is transported to Rab7-positive endosomes for degradation and recycled via Rab4- and Rab11-positive vesicles. MuSK activation by Dok7 mildly affects the localization of MuSK on the cell surface but has no effect on the rate of MuSK internalization. Interestingly, MuSK colocalizes with actin and Arf6 at the cell surface and during endosomal trafficking. Disruption of the actin cytoskeleton or the proper function of Arf6 concentrates MuSK in cell protrusions. Moreover, inhibition of Arf6 or cytoskeletal rearrangements impairs acetylcholine receptor clustering and phosphorylation. These results suggest that MuSK uses both classical and nonclassical endosomal pathways that involve a variety of different components of the endosomal machinery. Structured digital abstract MuSK and Arf6 colocalize by fluorescence microscopy (View Interaction: 1, 2) MuSK and Rab4 colocalize by fluorescence microscopy (View interaction) MuSK and Rab11 colocalize by fluorescence microscopy (View interaction) MuSK and Rab7 colocalize by fluorescence microscopy (View interaction) Signaling via the receptor tyrosine kinase MuSK is required for the formation of the neuromuscular synapse. Here, we investigate MuSK endocytosis using an in vivo labeling approach. Evidence is provided that ligand-independent MuSK internalization preferentially occurs via a clathrin-dependent pathway. Further, we find the small GTPase Arf6 implicated in MuSK endocytosis and function. © 2013 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of FEBS.