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 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)

MuSK Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop

“…Constructs carrying a C-terminal HA-Tag were ligated into retroviral vector pBabe/puro 28 51 . S751D and S751A mutations were introduced into a previously described CMV/MuSK construct carrying a N-terminal HA-Tag 52 . Expression plasmids CMV/MuSK-WT, kinase active CMV/MuSK-KA (LS746/747MT) and kinase-dead CMV/MuSK-KD (K608A) have previously been described 16 17 52 .…”

Fluorescence colocalization microscopy analysis can be improved by combining object‐recognition with pixel‐intensity‐correlation

“…In a previous project 23, we had noticed that molecules, which exhibited clearly different localizations in the cell as assessed by fluorescence microscopy, still gave high numerical colocalization values with intensity‐based coefficients such as Pearson's or Manders' correlation parameters. Evaluating all the relevant pixel‐intensity based parameters with the ImageJ plugin JACoP revealed that they were prone to false positive values or rather high standard deviations, which prompted us to develop an object‐based colocalization analysis technique that scores the area fraction of colocalization.…”

“…Since we found that statistical methods of pixel‐intensity correlation do not provide a clear evaluation of colocalization and based on our previous observations that object‐recognition based strategies seem to be superior 23, we thought of combining both approaches. To that end, we designed a macro for the Fiji software package, which automates a series of image processing steps that can be executed in batch mode for a whole series of images in a folder.…”

Centrosome and ciliary abnormalities in fetal akinesia deformation sequence human fibroblasts

“…MuSK and rapsyn are scaffold proteins that play key roles in AChR clustering and NMJ formation. MuSK, a muscle-specific receptor tyrosine kinase, is activated by the 03/09/19 extracellular matrix protein agrin upon agrin-binding to Lpr4, a member of the lowdensity lipoprotein receptor family Luiskandl et al, 2013;Mazhar and Herbst, 2012;Zhang et al, 2008;Zhang et al, 2011). Activated MuSK induces co-clustering of rapsyn and AChRs (Ghazanfari et al, 2011;Marchand et al, 2000;Marchand et al, 2002;Moransard et al, 2003) and downstream signaling of MuSK requires binding of DOK7 to the phosphotyrosinebinding site in MuSK (Okada et al, 2006).…”

“…Signaling downstream of MuSK is only poorly understood, but it involves interactions of both MuSK and rapsyn to all three cytoskeleton networks, i.e. microtubules, the actin cytoskeleton and intermediate filaments (Aittaleb et al, 2015;Antolik et al, 2007;Bartoli et al, 2001;Dobbins et al, 2008;Ghasemizadeh et al, 2019;Li et al, 2016;Luiskandl et al, 2013;Mihailovska et al, 2014;Moransard et al, 2003;Oury et al, 2019). Rapsyn has, moreover, been found in non-muscle cell types Musil et al, 1989) and plays a role in lysosome clustering (Aittaleb et al, 2015).…”