title: Acetylcholine receptor (AChR) clustering is regulated both by glycogen synthase kinase 3β (GSK3β)-dependent phosphorylation and the level of CLIP-associated protein 2 (CLASP2) mediating the capture of microtubule plus-ends.
abstract: The postsynaptic apparatus of the neuromuscular junction (NMJ) traps and anchors acetylcholine receptors (AChRs) at high density at the synapse. We have previously shown that microtubule (MT) capture by CLASP2, a MT plus-end-tracking protein (+TIP), increases the size and receptor density of AChR clusters at the NMJ through the delivery of AChRs and that this is regulated by a pathway involving neuronal agrin and several postsynaptic kinases, including GSK3. Phosphorylation by GSK3 has been shown to cause CLASP2 dissociation from MT ends, and nine potential phosphorylation sites for GSK3 have been mapped on CLASP2. How CLASP2 phosphorylation regulates MT capture at the NMJ and how this controls the size of AChR clusters are not yet understood. To examine this, we used myotubes cultured on agrin patches that induce AChR clustering in a two-dimensional manner. We show that expression of a CLASP2 mutant, in which the nine GSK3 target serines are mutated to alanine (CLASP2-9XS/9XA) and are resistant to GSK3β-dependent phosphorylation, promotes MT capture at clusters and increases AChR cluster size, compared with myotubes that express similar levels of wild type CLASP2 or that are noninfected. Conversely, myotubes expressing a phosphomimetic form of CLASP2 (CLASP2-8XS/D) show enrichment of immobile mutant CLASP2 in clusters, but MT capture and AChR cluster size are reduced. Taken together, our data suggest that both GSK3β-dependent phosphorylation and the level of CLASP2 play a role in the maintenance of AChR cluster size through the regulated capture and release of MT plus-ends.
authors.affiliation: Institute of Physiology, Department of Biomedicine, University of Basel, CH-4056 Basel, Switzerland,; Department of Cell Biology, Erasmus Medical Center, 3015 GE, Rotterdam, Netherlands.
authors.forename: Sreya
authors.initials: S
authors.lastname: Basu
authors.affiliation: Institute of Physiology, Department of Biomedicine, University of Basel, CH-4056 Basel, Switzerland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Sladecek
authors.affiliation: Department of Cell and Tissue Biology, University of California at San Francisco, San Francisco, California 94143, and.
authors.forename: Hayley
authors.initials: H
authors.lastname: Pemble
authors.affiliation: Department of Cell and Tissue Biology, University of California at San Francisco, San Francisco, California 94143, and.
authors.forename: Torsten
authors.initials: T
authors.lastname: Wittmann
authors.affiliation: Optical Imaging Center, Erasmus Medical Center, 3015 GE Rotterdam, Netherlands.
authors.forename: Johan A
authors.initials: JA
authors.lastname: Slotman
authors.affiliation: Optical Imaging Center, Erasmus Medical Center, 3015 GE Rotterdam, Netherlands.
authors.forename: Wiggert
authors.initials: W
authors.lastname: van Cappellen
authors.affiliation: Institute of Physiology, Department of Biomedicine, University of Basel, CH-4056 Basel, Switzerland,. Electronic address: Hans-Rudolf.Brenner@unibas.ch.
authors.forename: Hans-Rudolf
authors.initials: HR
authors.lastname: Brenner
authors.affiliation: Department of Cell Biology, Erasmus Medical Center, 3015 GE, Rotterdam, Netherlands,. Electronic address: n.galjart@erasmusmc.nl.
authors.forename: Niels
authors.initials: N
authors.lastname: Galjart
mesh_terms: D018171:Agrin / Q000502:physiology; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D057809:HEK293 Cells; D006801:Humans; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D010766:Phosphorylation; D061251:Primary Cell Culture; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011950:Receptors, Cholinergic / Q000378:metabolism*
chemical_list: D018171:Agrin; C497851:CLASP2 protein, human; D008869:Microtubule-Associated Proteins; D011950:Receptors, Cholinergic; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; C000605507:Gsk3b protein, mouse; D038362:Glycogen Synthase Kinase 3
keywords: Glycogen Synthase Kinase 3 (GSK-3); Microtubule; Microtubule-associated Protein (MAP); Nicotinic Acetylcholine Receptors (nAChR); Synapse