Recently our lab showed that whereas dNedd4S is essential for proper NM synaptogenesis ( Ing et al., 2007 ), dNedd4Lo inhibits it ( Zhong et al., 2011 ). Previously we showed that dNedd4S promotes NM synaptogenesis in flies ( Ing et al., 2007 ) by interacting and ubiquitinating Commissureless (Comm), which leads to endocytosis of Comm from the muscle surface, a step required for NM synaptogenesis ( Wolf et al., 1998 ). Differences between the two isoforms of dNedd4 include an alternate start codon site, resulting in a longer N-terminal region in dNedd4Lo, and an extra exon inserted between those encoding WW1 and WW2 domains ( Zhong et al., 2011 ). Drosophila contains a single dNedd4 gene, which undergoes alternative splicing to produce several splice isoforms, including two prominent ones: dNedd4-short (dNedd4S) and dNedd4-long (dNedd4Lo). Neuronal precursor cell expressed developmentally down-regulated 4 (Nedd4) family members belong to the HECT family of E3 ligases and contain a common C2-WW(n)-HECT domain architecture.
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Studies have shown that the RING-family ubiquitin ligase complex Highwire ( Wan et al., 2000 ) inhibits synapse formation and function by inhibiting the kinase Wallenda/DLK1, the activator of JNK ( Tian and Wu, 2013 ), whereas the deubiquitinating enzyme Fat Facet (Faf DiAntonio et al., 2001 ) targets Liquid Facet/Epsin and promotes synaptic growth ( Bao et al., 2008 Tian and Wu, 2013 ). The ubiquitination cascade involves three enzymes-E1, E2, and E3-with the last responsible for substrate recognition and ubiquitin transfer, either indirectly (e.g., RING E3 ligases) or directly (e.g., HECT E3 ligases Rotin and Kumar, 2009 ).Īlthough many proteins are involved in the regulation of NM synaptogenesis in flies, the role of the ubiquitin system in this process is less well characterized.
Ubiquitination is the process of conjugating ubiquitin onto proteins, and it plays an important role in controlling protein degradation/stability, as well as in trafficking, sorting, and endocytosis of transmembrane proteins ( Glickman and Ciechanover, 2002 ). Throughout development, internal and external cues guide muscles to adopt specific properties that allow them to perform particular functions. Each muscle fiber is distinguishable by size, shape, orientation, number of nuclei, innervation, and tendon attachment sites ( Baylies et al., 1998 ). In Drosophila larva, a repeated pattern of 30 unique muscle fibers is present in each abdominal hemisegment ( Bate, 1990 ), which are innervated by 36 motor neurons ( Landgraf et al., 2003 ). During larval stages, the essential muscle pattern created in the embryo does not change, except that the muscles continue to expand along with the growth of the larva ( Dobi et al., 2015 ). The contractile apparatus is then assembled, and muscles begin to contract. After myoblast fusion, nuclei are positioned correctly throughout the myotube ( Folker and Baylies, 2013 ) and form connections to surrounding tendon cells to establish the myotendinous junction, which is innervated by motorneurons in a process called neuromuscular (NM) synaptogenesis ( Landgraf et al., 1999 Volk, 1999 Collins and DiAntonio, 2007 ). Fusion of muscle founder cells and fusion-competent myoblasts form the syncytial myotube, which develops into the embryonic and larval body wall muscles ( Dobi et al., 2015 ). Somatic mesodermal specification produces three different types of myoblasts. The Drosophila melanogaster larval body wall muscles are established during embryogenesis beginning with the invagination of the mesoderm, which spreads along the ectoderm and then forms numerous mesodermal derivatives ( Dobi et al., 2015 ). We propose that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C→A). Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and impaired T-tubule formation, phenocopying amph-null mutants. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. We validated the interaction by coimmunoprecipitation and showed direct binding between dAmph-SH3 domain and dNedd4Lo N-terminus. dAmph is a postsynaptic protein containing SH3-BAR domains and regulates muscle transverse tubule (T-tubule) formation in flies. 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). We previously showed that whereas dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. DNedd4Lo has a unique N-terminus containing a Pro-rich region. Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4-short (dNedd4S) and -long (dNedd4Lo).
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