Drosophila Nedd4-long reduces Amphiphysin levels in muscles and leads to impaired T-tubule formation

Molecular Biology of the Cell
Frozan SafiDaniela Rotin

Abstract

Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4-short (dNedd4S) and -long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. We previously showed that whereas dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, we searched for binding partners to the N-terminal unique region of dNedd4Lo in larval lysates using mass spectrometry and identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains and regulates muscle transverse tubule (T-tubule) formation in flies. We validated the interaction by coimmunoprecipitation and showed direct binding between dAmph-SH3 domain and dNedd4Lo N-terminus. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and impaired T-tubule formation, phenocopying amph-null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C→A). We propose that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and mu...Continue Reading

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Citations

Dec 7, 2018·The Journal of Biological Chemistry·Samantha S WassermanDaniela Rotin
Sep 10, 2020·International Journal of Molecular Sciences·Sara BachillerRocío Ruiz

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Methods Mentioned

BETA
ubiquitination
coimmunoprecipitation
transgenic
transfection
confocal microscopy
X-Ray

Software Mentioned

Image Studio
Proteome
! Tandem
ToupView
GraphPad Prism
Ethovioson XT
Scaffold
Cyclone
Statistica
Volocity

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