title: Disruption of adaptor protein 2μ (AP-2μ) in cochlear hair cells impairs vesicle reloading of synaptic release sites and hearing.
abstract: Active zones (AZs) of inner hair cells (IHCs) indefatigably release hundreds of vesicles per second, requiring each release site to reload vesicles at tens per second. Here, we report that the endocytic adaptor protein 2μ (AP-2μ) is required for release site replenishment and hearing. We show that hair cell-specific disruption of AP-2μ slows IHC exocytosis immediately after fusion of the readily releasable pool of vesicles, despite normal abundance of membrane-proximal vesicles and intact endocytic membrane retrieval. Sound-driven postsynaptic spiking was reduced in a use-dependent manner, and the altered interspike interval statistics suggested a slowed reloading of release sites. Sustained strong stimulation led to accumulation of endosome-like vacuoles, fewer clathrin-coated endocytic intermediates, and vesicle depletion of the membrane-distal synaptic ribbon in AP-2μ-deficient IHCs, indicating a further role of AP-2μ in clathrin-dependent vesicle reformation on a timescale of many seconds. Finally, we show that AP-2 sorts its IHC-cargo otoferlin. We propose that binding of AP-2 to otoferlin facilitates replenishment of release sites, for example, via speeding AZ clearance of exocytosed material, in addition to a role of AP-2 in synaptic vesicle reformation.
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: SangYong
authors.initials: S
authors.lastname: Jung
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie (FMP), Berlin, Germany NeuroCure Cluster of Excellence & Collaborative Research Center 958, Freie Universität Berlin, Berlin, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Maritzen
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Wichmann
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Auditory Systems Physiology Group, InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Zhizi
authors.initials: Z
authors.lastname: Jing
authors.affiliation: Bernstein Group Biophysics of Neural Computation, Max Planck Institute for Dynamics and Self-Organization, Göttingen, Germany andreas@nld.ds.mpg.de haucke@fmp-berlin.de tmoser@gwdg.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Neef
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Natalia H
authors.initials: NH
authors.lastname: Revelo
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Hanan
authors.initials: H
authors.lastname: Al-Moyed
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Department of Molecular Structural Biology, Institute for Microbiology and Genetics, University of Göttingen, Göttingen, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Meese
authors.affiliation: Department of Molecular Neurobiology, Max Planck Institute of Experimental Medicine, Göttingen, Germany.
authors.forename: Sonja M
authors.initials: SM
authors.lastname: Wojcik
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany.
authors.forename: Iliana
authors.initials: I
authors.lastname: Panou
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie (FMP), Berlin, Germany.
authors.forename: Haydar
authors.initials: H
authors.lastname: Bulut
authors.affiliation: Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Schu
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Department of Molecular Structural Biology, Institute for Microbiology and Genetics, University of Göttingen, Göttingen, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Ficner
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Molecular Biology of Cochlear Neurotransmission Group, InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Ellen
authors.initials: E
authors.lastname: Reisinger
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Department of Neuro- and Sensory Physiology, University Medical Center Göttingen, Göttingen, Germany Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, Göttingen, Germany.
authors.forename: Silvio O
authors.initials: SO
authors.lastname: Rizzoli
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Jakob
authors.initials: J
authors.lastname: Neef
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Auditory Systems Physiology Group, InnerEarLab, Department of Otolaryngology, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Nicola
authors.initials: N
authors.lastname: Strenzke
authors.affiliation: Leibniz Institut für Molekulare Pharmakologie (FMP), Berlin, Germany NeuroCure Cluster of Excellence & Collaborative Research Center 958, Freie Universität Berlin, Berlin, Germany andreas@nld.ds.mpg.de haucke@fmp-berlin.de tmoser@gwdg.de.
authors.forename: Volker
authors.initials: V
authors.lastname: Haucke
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany Collaborative Research Center 889, University of Göttingen, Göttingen, Germany Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University of Göttingen, Göttingen, Germany andreas@nld.ds.mpg.de haucke@fmp-berlin.de tmoser@gwdg.de.
authors.forename: Tobias
authors.initials: T
authors.lastname: Moser
mesh_terms: D000200:Action Potentials; D033942:Adaptor Proteins, Vesicular Transport / Q000502:physiology*; D000818:Animals; D016057:Evoked Potentials, Auditory, Brain Stem; D006198:Hair Cells, Auditory / Q000502:physiology*; D006309:Hearing; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D013569:Synapses / Q000502:physiology; D009435:Synaptic Transmission; D013572:Synaptic Vesicles / Q000378:metabolism*
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; C000605051:endocytic adaptor protein 2mu, mouse
keywords: active zone; endocytosis; release site clearance; synaptic ribbon; vesicle reformation