Endosomal trafficking of the receptor tyrosine kinase MuSK proceeds via clathrin‐dependent pathways, A rf6 and actin

Muscle‐specific kinase ( M u SK ), a receptor tyrosine kinase, is the key player during the formation of the neuromuscular junction. Signal transduction events downstream of M u SK activation induce both pre‐ and postsynaptic differentiation, which, most prominently, includes the clustering of acetylcholine receptors at synaptic sites. More recently, regulated M u SK endocytosis and degradation have been implicated as crucial events for M u SK signalling activity, implicating a cross‐talk between signalling and endocytosis. In the present study, we use a live imaging approach to study M u SK endocytosis. We find that M u SK is internalized via a clathrin‐, dynamin‐dependent pathway. Mu SK is transported to Rab7‐positive endosomes for degradation and recycled via Rab4‐ and Rab11‐positive vesicles. Mu SK activation by D ok7 mildly affects the localization of M u SK on the cell surface but has no effect on the rate of M u SK internalization. Interestingly, M u SK colocalizes with actin and Arf6 at the cell surface and during endosomal trafficking. Disruption of the actin cytoskeleton or the proper function of A rf6 concentrates M u SK in cell protrusions. Moreover, inhibition of A rf6 or cytoskeletal rearrangements impairs acetylcholine receptor clustering and phosphorylation. These results suggest that M u SK uses both classical and nonclassical endosomal pathways that involve a variety of different components of the endosomal machinery. Structured digital abstractMuSK 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 )