title: Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction.
abstract: BACKGROUND
  Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ). We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD)1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1-/- NMJs.
  
  
  METHODS
  Localization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1-/- mouse skeletal muscle.
  
  
  RESULTS
  Endogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1-/- mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1-4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1-/- mouse skeletal muscle than in wild-type skeletal muscle.
  
  
  CONCLUSIONS
  EHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1-/- muscle may be due to functional compensation by other EHD paralogs.
authors.affiliation: Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC, USA. ehoffman@cnmcresearch.org.
authors.forename: Suzanne E
authors.initials: SE
authors.lastname: Mate
authors.forename: Jack H
authors.initials: JH
authors.lastname: Van Der Meulen
authors.forename: Priyanka
authors.initials: P
authors.lastname: Arya
authors.forename: Sohinee
authors.initials: S
authors.lastname: Bhattacharyya
authors.forename: Hamid
authors.initials: H
authors.lastname: Band
authors.forename: Eric P
authors.initials: EP
authors.lastname: Hoffman