title: CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles.
abstract: A hallmark of the neuromuscular junction (NMJ) is the high density of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane. The postsynaptic apparatus of the NMJ is organized by agrin secreted from motor neurons. The mechanisms that underlie the focal delivery of AChRs to the adult NMJ are not yet understood in detail. We previously showed that microtubule (MT) capture by the plus end-tracking protein CLASP2 regulates AChR density at agrin-induced AChR clusters in cultured myotubes via PI3 kinase acting through GSK3β. Here we show that knockdown of the CLASP2-interaction partner LL5β by RNAi and forced expression of a CLASP2 fragment blocking the CLASP2/LL5β interaction inhibit microtubule capture. The same treatments impair focal vesicle delivery to the clusters. Consistent with these findings, knockdown of LL5β at the NMJ in vivo reduces the density and insertion of AChRs into the postsynaptic membrane. MT capture and focal vesicle delivery to agrin-induced AChR clusters are also inhibited by microtubule- and actin-depolymerizing drugs, invoking both cytoskeletal systems in MT capture and in the fusion of AChR vesicles with the cluster membrane. Combined our data identify a transport system, organized by agrin through PI3 kinase, GSK3β, CLASP2, and LL5β, for precise delivery of AChR vesicles from the subsynaptic nuclei to the overlying synaptic membrane.
authors.affiliation: 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: Department of Biomedicine, University of Basel, CH-4056 Basel, Switzerland.
authors.forename: Stefan
authors.initials: S
authors.lastname: Sladecek
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology and Program in Neuroscience, University of Michigan, Ann Arbor, MI 48109.
authors.forename: Isabel
authors.initials: I
authors.lastname: Martinez de la Peña y Valenzuela
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology and Program in Neuroscience, University of Michigan, Ann Arbor, MI 48109.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Akaaboune
authors.affiliation: Biomedical Imaging Group, Erasmus Medical Center, 3015 GE Rotterdam, Netherlands.
authors.forename: Ihor
authors.initials: I
authors.lastname: Smal
authors.affiliation: Department of Biomedicine, University of Basel, CH-4056 Basel, Switzerland.
authors.forename: Katrin
authors.initials: K
authors.lastname: Martin
authors.affiliation: Department of Cell Biology, Erasmus Medical Center, 3015 GE Rotterdam, Netherlands.
authors.forename: Niels
authors.initials: N
authors.lastname: Galjart
authors.affiliation: Department of Biomedicine, University of Basel, CH-4056 Basel, Switzerland Hans-Rudolf.Brenner@unibas.ch n.galjart@erasmusmc.nl.
authors.forename: Hans Rudolf
authors.initials: HR
authors.lastname: Brenner
mesh_terms: D000199:Actins / Q000378:metabolism*; D018171:Agrin / Q000378:metabolism; D000818:Animals; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009469:Neuromuscular Junction / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism*; D013570:Synaptic Membranes / Q000378:metabolism*; D022161:Transport Vesicles / Q000378:metabolism*
chemical_list: D000199:Actins; D018171:Agrin; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D008565:Membrane Proteins; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D019869:Phosphatidylinositol 3-Kinases; D011950:Receptors, Cholinergic; C506523:CLASP2 protein, mouse; C000591917:Phldb2 protein, mouse; C000605507:Gsk3b protein, mouse