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Open Access Research

Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction

Suzanne E Mate12, Jack H Van Der Meulen1, Priyanka Arya34, Sohinee Bhattacharyya35, Hamid Band345 and Eric P Hoffman12*

Author Affiliations

1 Research Center for Genetic Medicine, Children’s National Medical Center, Washington, DC, USA

2 Department of Integrative Systems Biology, George Washington School of Medicine, Washington, DC, USA

3 Eppley Institute for Research in Cancer and Allied Diseases, UNMC-Eppley Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA

4 Department of Genetics, Cell Biology and Anatomy, College of Medicine, University of Nebraska Medical Center, Omaha, NE, USA

5 Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, NE, USA

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Skeletal Muscle 2012, 2:19  doi:10.1186/2044-5040-2-19

Published: 13 September 2012

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.

Conclusion

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.

Keywords:
Neuromuscular junction; Eps homology domain containing protein; Endosomal transport; Endosomal recycling; Bungarotoxin; Endplate; Synapse; Skeletal muscle