BIOLOGICAL OVERVIEW

Amphiphysin family members are implicated in synaptic vesicle endocytosis and actin localization, and one isoform is an autoantigen in neurological autoimmune disorder; however, there has been no genetic analysis of Amphiphysin function in higher eukaryotes. Drosophila Amphiphysin is localized to actin-rich membrane domains in many cell types, including apical epithelial membranes, the intricately folded apical rhabdomere membranes of photoreceptor neurons and the postsynaptic density of glutamatergic neuromuscular junctions. Flies that lack all Amphiphysin function are viable, lack any observable endocytic defects, but have abnormal localization of the postsynaptic proteins Discs large, Lethal giant larvae and Scribbled. Flies that lack Amphiphysin function exhibit altered synaptic physiology, and behavioral defects. Misexpression of Amphiphysin outside its normal membrane domain in photoreceptor neurons results in striking morphological defects. The strong misexpression phenotype coupled with the mild mutant and the lack of phenotypes suggest that Amphiphysin acts redundantly with other proteins to organize specialized membrane domains within a diverse array of cell types. In other words, Drosophila Amphiphysin functions in membrane morphogenesis, but an additional role in endocytosis cannot yet be dismissed (Zelhof, 2001).

The creation of specialized membrane regions is important for establishing cell polarity and for the proper function of many differentiated cell types. Genetic and biochemical experiments have identified numerous proteins involved in the formation of distinct membrane domains; in neurons and yeast, one of these proteins is Amphiphysin (Amph). Vertebrate Amph I protein was discovered because of its enrichment in brain presynaptic terminal membrane preparations, whereas Amph II is more widely expressed. Both Amph I and II have an N-terminal coiled-coil domain, a central proline-rich domain and a C-terminal SH3 domain. Mutant analysis of vertebrate Amph I and II has not been described, but most evidence suggests that Amph proteins are involved in endocytosis, particularly in synaptic vesicle recycling (Wigge, 1998). Amph I is concentrated at presynaptic terminals, and expression of the SH3 domain of Amph I can block endocytosis in neurons or fibroblasts, leading to concomitant accumulation of clathrin coated pits at the plasma membrane. In addition, the Amph I central region binds the endocytic proteins alpha-Adaptin and Clathrin, while the SH3 domain interacts with the endocytic proteins Dynamin and Synaptojanin (Zelhof, 2001 and references therein).

The role of Amphiphysin family members may not be limited to endocytosis. Vertebrate Amph II does localize to presynaptic sites, however, Amph II isoforms lacking the alpha-Adaptin-binding central domain are expressed in skeletal muscle. How, or if, these isoforms modulate endocytosis is not known. A S. cerevisiae Amph homolog, rvs167, shows conservation of the coiled-coil and SH3 domains, but the central domain is not well conserved nor is it needed for function. rvs167 protein localizes to actin-rich cortical patches during G₁ phase, and then relocates to the bud site (or the leading edge) during schmoo formation. Yeast that lack rvs167 and the related rvs161 gene are viable but show abnormal cell shape, disorganization of the actin cytoskeleton during bud emergence and mating, and random bud site selection in diploid cells (Zelhof, 2001).

All known Amph family proteins contain a N-terminal coiled-coil domain and a C-terminal SH3 domain with a variable central domain. In mammalian Amph I and Amph II, the central domain provides an additional link to endocytosis via the inclusion of binding sites for clathrin and alpha-Adaptin. Although sequence comparison does not clearly indicate whether Drosophila Amph is more related to either vertebrate Amph I or Amph II, examination of expression profiles, isoform organization and inferred functions suggest that Drosophila Amph is likely to be an Amph II ortholog (Zelhof, 2001): (1) both Drosophila Amph and vertebrate Amph II have a broad tissue distribution, including postsynaptic expression in skeletal muscle; (2) both Drosophila Amph and Amph II genes have isoforms that partially or completely remove the central domain; (3) Drosophila Amph is sufficient to organize membrane morphogenesis in photoreceptor neurons and necessary for protein localization at the postsynaptic NMJ. Similarly, the putative localization of Amph II to nodes of Ranvier and the localization of yeast Rvs167 to the protruding bud and schmoo membranes, suggest that Amph II and Rvs167 may be involved in regulating membrane morphogenesis, rather than endocytosis (Zelhof, 2001).

The absence of detectable endocytic defects in synaptic vesicle recycling in Amph mutants is surprising, given the evidence linking Amph and synaptic vesicle endocytosis in vertebrate systems. The results of this study cannot completely eliminate a role of Amph in endocytosis. Amph protein is found on vesicles within cells known to be actively undergoing endo- and exocytosis, such as the hindgut, garland gland and salivary gland. A detailed characterization of the Amph phenotype in these cells may ultimately reveal a role for Amph in regulating endocytosis (Zelhof, 2001).

There are two common features of Amph expression: (1) Amph always localizes to a restricted subcellular membrane domain, typically the apical membrane in epithelial or neuronal cells; (2) Amph is detected at membranes that are undergoing 'remodeling' or morphogenesis. During cellularization, the addition of membrane occurs at defined sites and in a precise sequence. Amph localization precisely correlates with the bi-phasic insertion of new membrane: first apical and then apical-lateral. In the tubular tracheal system, Amph is localized to the inner (apical) membrane domain. It has been hypothesized that tracheal tube size is controlled by regulating surface area of the apical membrane: during tracheal tube dilation, the inner (apical) diameter of the tube increases dramatically, whereas the outer (basal) diameter shows little or no change. Thus, Amph is present on the membrane domain that undergoes regulated alteration in curvature and surface area and as such might regulate the changes observed. Similarly, the Amph-related yeast Rvs167 protein is localized to the protruding bud or schmoo membrane, another actively 'remodeled' membrane domain (Zelhof, 2001).

How does the removal of Amph result in the delocalization of Lgl, Dlg and Scrib at the synapse? One possibility is that Amph may play a role in the establishment or maintenance of postsynaptic membrane structure. One characteristic of type I synapses is that they have an extensive subsynaptic reticulum, the highly folded membrane surrounding the synapse, that is not present in type II and type III synapses. Amph mutants may have defects in the folded membrane that do not allow for proper retention of proteins at the synapse. Alternatively, Amph may act as a localized scaffold protein that recruits Lgl to the postsynaptic membrane, with Lgl being necessary for proper anchoring or targeting of Dlg and Scrib. This would be consistent with the known role of Lgl in mediating Dlg and Scrib localization in epithelia. Lgl belongs to a family of proteins that regulate polarized exocytosis and protein targeting to specific membrane domains. In addition, deletion analysis of Dlg has identified a two-step process of synaptic targeting: Dlg is first targeted to the muscle plasma membrane and then to the subsynaptic reticulum. Loss of Amph function gives a phenotype consistent with a failure in the second step of this process; however, no direct interactions between Amph and Dlg proteins in vitro have been detected. Further analysis of the physical interactions between Amph and Lgl, Dlg or Scrib may help illuminate the mechanism of postsynaptic protein localization, and the analysis of lgl and scrib synaptic phenotypes will be required to determine whether the functional defects at Amph mutant synapses (increase in quantal size) are due to the mislocalization of Lgl, Dlg or Scrib (Zelhof, 2001).

How does Amph regulate membrane morphogenesis? Amph could directly regulate the actin cytoskeleton, leading to changes in membrane topology. Alternatively, Amph could directly modify membrane structure, and only indirectly affect the actin cytoskeleton. These models are not mutually exclusive. The latter model (direct membrane bending) is supported by studies of mammalian Amph I, which show that Amph can directly bind lipid bilayers and distort them into high-curvature membranes; for example, Amph I can transform spherical liposomes into narrow tubules. The former model (actin regulation) is supported by work on yeast, Drosophila and vertebrate Amph proteins. Initially, there is the tight correlation between Amph expression and actin organization in all organisms tested: yeast bud and schmoo membrane extensions, Drosophila cellularization and rhabdomere formation, and vertebrate nodes of Ranvier are all sites of F-actin enrichment and Amph localization. Next, the delocalization of Drosophila Amph from the apical surface of photoreceptor cells, in bifocal mutants or amp overexpression mutants, results in the mislocalization of F-actin. Finally, in yeast, this idea is supported by the identification of a protein interaction between Rvs167 SH3 domain and actin binding protein Abp1 as well as synthetic lethality between rvs167 and a subset of actin act1 alleles. In Drosophila, it is currently unknown what protein(s) bind to the Amph SH3 domain; it will be interesting to determine if a similar actin-binding protein-SH3 domain interaction occurs in Drosophila. This type of biochemical analysis, as well as further genetic studies, will be necessary to understand the relationship between Amph, the actin cytoskeleton and membrane morphogenesis (Zelhof, 2001).

Given the accessibility of Drosophila photoreceptors to genetic, immunohistochemical and electron microscopic examination, whether Amph is required for the biogenesis of the rhabdomere was investigated as a model for its general role in membrane morphogenesis. Homozygous Amph mutant adults have superficially normal eyes that respond to light correctly. However, electron microscopy of Amph mutant eyes reveals that the rhabdomere membranes are unusually closely packed and occasionally fused, thus creating very little inter-rhabdomere space (Zelhof, 2001).

Each photoreceptor rhabdomere is an interconnected stack of membranes that emerges from the apical neuronal membrane. The process of rhabdomere site selection, initiation, and elaboration is poorly understood. The results indicate a role for Amph in organizing the intricately folded rhabdomere membrane. (1) Accumulation of Amph on the apical surface occurs together with an enrichment of F-actin at this site.(2) Overexpression of Amph (by targeted misexpression or in bifocal mutants) results in the delocalization of endogenous Amph and the mislocalization of both F-actin, leading to either loss or ectopic rhabdomeres. bifocal codes for a novel protein that colocalizes with actin and is necessary for photoreceptor morphogenesis (Bahri, 1997). Loss of rhabdomeres may be due to excess Amph titrating out factors necessary for the proper development of rhabdomeres; ectopic rhabdomeres may be due to ectopic Amph recruiting sufficient F-actin or other proteins to a second site in the cell and thus triggering formation of an extra rhabdomere (Zelhof, 2001).

The development or maintenance of a cellular three-dimensional structure, like a rhabdomere or synapse, not only includes the rearrangement of the membrane and actin cytoskeleton but also involves the correct targeting and anchoring of proteins to these locations. Consistent with a role in protein targeting/anchoring, in Amph null mutants multiple postsynaptic proteins are mislocalized. Dlg and Scrib are partially delocalized, and Lgl is completely undetectable at the postsynaptic membrane (although Lgl staining at the muscle M line is unaffected). These defects are not due to a general delocalization of all postsynaptic proteins, since normal localization is observed of two different epitope-tagged glutamate receptors to the postsynaptic membrane (Zelhof, 2001).

To explore the role of Amph in the elaboration and formation of the rhabdomeres, GMR-GAL4 was used to overexpress Amph in the developing photoreceptor neurons. Overexpression of Amph can produce outwardly normal eye but with a loss of all recognizable internal cell structure by electron microscopy. More informative weaker phenotypes can be generated by expressing Amph at a lower temperature (where GAL4 is less active and there is less Amph protein as confirmed by Western blots). Overexpression of intermediate levels of Amph produces two to three smaller rhabdomeres per cell, split rhabdomeres or no rhabdomeres. Despite the severe morphological defects, these photoreceptor neurons are functional, and overexpression of Amph does not affect photoreceptor neuron cell fate decisions (Zelhof, 2001).

The Amph overexpression eye phenotype (split/ectopic rhabdomeres) is similar to the loss-of-function bifocal mutant phenotype (Bahri, 1997), suggesting that Amph and bifocal may act in the same genetic pathway. Bifocal is a novel protein that is colocalized with Amph at the apical membrane of newly formed embryonic cells, as well as at the apical membrane of photoreceptor neurons. In photoreceptor neurons, Bifocal expression precedes Amph and lasts into adulthood. Amph mutants show normal Bifocal localization, but bifocal mutants show delocalization of Amph into a broad apical domain matching that of F-actin, rather than its normal tight apical crescent. The bifocal mutant phenotype can be suppressed by reducing amph gene dosage by 50%. Thus, Bifocal-mediate localization of Amph to the future rhabdomere membrane domain is essential for normal eye development (Zelhof, 2001).

To determine the developmental origin of the Amph overexpression phenotype, Amph, Bifocal and F-actin localization were assayed during rhabdomere development. Wild-type photoreceptors show an even distribution of Bifocal, Amph and F-actin at the apical surface of the cell. By contrast, photoreceptors overexpressing Amph have an abnormal punctate 'ball' of F-actin and Bifocal at the apical cortex and Amph is delocalized from the apical membrane. It is concluded that excess Amph protein leads to destabilization of the normal apical Amph localization, a mislocalization of Bifocal protein and F-actin, and the subsequent failure in rhabdomere morphogenesis (Zelhof, 2001).

These data led to the following model for Amph function. Bifocal is localized to the apical membrane of photoreceptor neurons, where it recruits Amph and other proteins. This protein complex then aids in the morphogenesis of the intricately folded rhabdomere membrane. Loss of Amph results in a mild phenotype, perhaps because Bifocal and additional proteins are still apically localized and can promote rhabdomere morphogenesis. However, when Amph is mislocalized outside of the apical membrane domain (in Amph overexpression experiments or in bifocal mutants), it can induce the formation of ectopic rhabdomere membrane domains or inhibit rhabdomere morphogenesis by relocating or titrating away the necessary protein complex to form a rhabdomere (Zelhof, 2001).

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

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. While dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, binding partners to the N-terminal unique region of dNedd4Lo were sought in larval lysates. Mass-spectrometry identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains, which regulates muscle transverse tubule (T-tubule) formation in flies. The interaction was validated by coimmunoprecipitation, and direct binding between dAmph-SH3 domain and dNedd4Lo-N-terminus was demonstrated. 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 to impaired T-tubule formation, phenocopying amph null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(C->A). It is proposed that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function (Safi, 2016).

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. Somatic mesodermal specification produces three different types of myoblasts. Fusion of muscle founder cells and fusion-competent myoblasts form the syncytial myotube, which develops into the embryonic and larval body wall muscles. After myoblast fusion, nuclei are positioned correctly throughout the myotube and form connections to surrounding tendon cells to establish the myotendinous junction, which is innervated by motorneurons in a process called neuromuscular (NM) synaptogenesis. The contractile apparatus is then assembled, and muscles begin to contract. 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. In Drosophila larva, a repeated pattern of 30 unique muscle fibers is present in each abdominal hemisegment, which are innervated by 36 motor neurons. Each muscle fiber is distinguishable by size, shape, orientation, number of nuclei, innervation, and tendon attachment sites. Throughout development, internal and external cues guide muscles to adopt specific properties that allow them to perform particular functions (Safi, 2016).

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. 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 (Safi, 2016).

Although 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. Studies have shown that the RING-family ubiquitin ligase complex Highwire inhibits synapse formation and function by inhibiting the kinase Wallenda/DLK1, the activator of JNK , whereas the deubiquitinating enzyme Fat Facet targets Liquid Facet/Epsin and promotes synaptic growth (Safi, 2016 and references therein).

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. 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). 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, 2011; Safi, 2016 and references therein).

Previous studies have shown that dNedd4S promotes NM synaptogenesis in flies by interacting and ubiquitinating Commissureless (Comm), which leads to endocytosis of Comm from the muscle surface, a step required for NM synaptogenesis. Whereas dNedd4S is essential for proper NM synaptogenesis, dNedd4Lo inhibits it (Zhong, 2011). Of importance, dNedd4Lo also inhibited normal larval locomotion (Zhong, 2011). These adverse effects of dNedd4Lo were caused by unique N-terminal and Middle regions found in dNedd4Lo (and absent from dNedd4S) and required a functional HECT domain (Zhong, 2011). Of interest, during embryonic muscle development, dNedd4Lo expression is dramatically decreased, whereas that of dNedd4S remains relatively high (Zhong, 2011; Safi, 2016 and references therein).

Because it was observed that the muscle and synaptogenesis defects of dNedd4Lo larvae were not caused by altered phosphorylation of dNedd4Lo, dNedd4Lo-mediated inhibition of catalytic activity of dNedd4S, or diminished effects of dNedd4Lo on Comm endocytosis (Zhong, 2011), it was suspected that dNedd4Lo might inhibit muscle development and/or function by interacting with other proteins via its unique regions (Safi, 2016).

This study identified, using mass spectrometry, Drosophila Amphiphysin (dAmph) as a binding partner of the unique N-terminal region of dNedd4Lo. Amphiphysins are members of the BAR-SH3 domain-containing family of proteins. Mammalian amphiphysin Amph I is involved in endocytosis and synaptic vesicle recycling during neurotransmission by interacting with clathrin/dynamin. In contrast, both mammalian Amph IIb (Bin1) and its fly orthologue, dAmph, are postsynaptic, lack binding sites for clathrin/dynamin, and are not involved in endocytosis. Instead, Bin1 and dAmph regulate transverse tubule (T-tubule) biogenesis in muscles (Safi, 2016).

This study demonstrates that the N-terminus of dNedd4Lo directly binds to dAmph-SH3 domain. In accord, dNedd4Lo and dAmph are colocalized postsynaptically at neuromuscular junctions (NMJs) and muscle transverse tubules (T-tubules). The data show that dNedd4Lo regulates the levels of dAmph postsynaptically and in muscles. Moreover, expression of dNedd4Lo in muscles results in impaired T-tubule formation, phenocopying amph-null mutants. These results demonstrate an important role of dNedd4Lo in regulating T-tubule organization of Drosophila muscles (Safi, 2016).

Previous work has shown that unlike dNedd4 (dNedd4S), muscle-specific overexpression of the dNedd4Lo isoform inhibits NM synaptogenesis and leads to impaired larval locomotion and lethality (Zhong, 2011). This effect required the catalytic activity of dNedd4Lo, since a mutant dNedd4Lo(C->A) with an inactivating mutation in the HECT domain (Cys -> Ala) was not inhibitory. This suggested that the same gene (dNedd4) can encode isoforms with opposite functions. In accord with this, it was observed that during stages of embryonic development when synaptogenesis takes place (14-24 h), dNedd4S expression remains relatively high, whereas that of the inhibitory dNedd4Lo is strongly reduced (Zhong, 2011), suggesting tight regulation of expression of these isoforms to promote muscle development at a very precise time. The inhibitory effect of dNedd4Lo is mediated by two regions unique to dNedd4Lo (N-terminus and Middle region), as deletion of these unique regions alleviated the synaptogenesis defects and increased viability. Therefore it is postulated that the unique regions of dNedd4Lo might negatively regulate NM synaptogenesis and muscle development by targeting specific substrate(s) (Safi, 2016).

This study identified dAmph as a binding partner for the unique N-terminal region of dNedd4Lo by mass spectrometry and validated the interaction in vitro and in vivo in flies. Transiently expressed dAmph coimmunoprecipitates with dNedd4Lo but not with dNedd4S in Drosophila S2 cells and demonstrated direct binding between dAmph-SH3 domain and the dNedd4Lo N-terminus. In vivo results showed that dAmph colocalizes with dNedd4Lo postsynaptically at neuromuscular junctions and muscle T-tubules, where their expression overlaps with the postsynaptic/T-tubule marker, Dlg. Of importance, it was demonstrated that dNedd4Lo expression significantly reduced the levels of dAmph in the postsynaptic region and muscles, an effect not observed in larvae expressing dNedd4S or dNedd4Lo(C->A). As expected, due to the disappearance of endogenous dAmph in larvae expressing dNedd4Lo (and the inability to 'treat' live larvae with proteasome inhibitors), it was not possible to detect ubiquitination of dAmph in these larvae. In addition to biochemical interactions, genetic interactions were also shown between dNedd4Lo and dAmph (Safi, 2016).

The reduction in dAmph levels in the dNedd4Lo-expressing muscles correlated with impaired T-tubule formation, mimicking the phenotype of the amph-null flies. These results could help explain (along with previously described NM synaptogenesis defects; Zhong, 2011) the observed locomotion defects in the dNedd4Lo-overexpressing larvae. At present, it is not possible to quantify the contribution of the T-tubule defects versus the NM synaptogenesis defects to the impaired muscle locomotion/function (Safi, 2016).

Interestingly, it was found that flies overexpressing the dAmph(ΔSH3) mutant in muscles showed reduced localization at the postsynaptic region and T-tubules and no longer colocalized with dNedd4. It is known that the SH3 domain of some membrane-associated proteins is important for their targeting to specific subcellular locations. Similarly, it was found that the SH3 domain of dAmph is also important for its location in the postsynaptic region and the muscle, since the ΔSH3 dAmph protein mislocalized to a region near the muscle plasma membrane. It is not known whether this SH3-dependent localization is related to the ability of this domain to bind dNedd4Lo or due to its interaction(s) with other molecules (Safi, 2016).

Amphiphysin has been implicated in T-tubule biogenesis due to its N-terminal amphipathic helix and BAR domain (N-BAR), which promotes membrane curvature. The BAR domain of the isoform of mammalian amphiphysin 2 (Bin1) is known to be associated with T-tubule formation in skeletal and cardiac muscles, where it induces tubular plasma membrane invaginations. Similar to Bin1, dAmph was shown to participate in plasma membrane remodeling during cleavage furrow ingression, which is required for de novo formation of cells in the Drosophila embryo; the BAR domain of dAmph is required for the formation of endocytic tubules that form at the cleavage furrow tips. This study shows that dNedd4Lo expression reduces the levels of dAmph in the muscle and significantly inhibits T-tubule formation. The degradation of dAmph by dNedd4Lo could impair T-tubule biogenesis by the BAR domain of dAmph, which could help to explain the larval locomotion defects that were observed (Safi, 2016).

Cardiac Bin1 has been implicated in calcium channel trafficking and formation of the inner membrane folds of the cardiac T-tubules. Bin1 localizes to cardiac T-tubules with the L-type calcium channel, Cav1.2, by tethering dynamic microtubules to membrane scaffolds, allowing targeted delivery of Cav1.2 to cardiac T-tubules. Knockdown of Bin1 reduces surface Cav1.2 and delays development of the calcium transient. In cardiomyopathy, decrease in Bin1 alters T-tubule morphology and can cause arrhythmia. Mice with cardiac Bin1 deletion show decreased T-tubule folding, which leads to free diffusion of local extracellular ions, prolonging action-potential duration and increasing susceptibility to arrhythmias. Bin1 is also important for maintenance of intact T-tubule structure and Ca2+ homeostasis in adult skeletal muscle. Adult mouse skeletal muscles with Bin1 knockdown display swollen T-tubule structures, alterations to intracellular Ca2+ release, and compromised coupling between the voltage-gated calcium channel, dihydropyridine receptor (DHPR), and the intracellular calcium channel, ryanodine receptor 1 (Safi, 2016).

Similar to Bin1, dAmph is also required for the organization of the excitation-contraction coupling machinery of muscle. Accordingly, dAmph mutant larvae and flies show defects in T-tubule formation, severe locomotor defects, and flight impairments, indicative of defects in muscle function. Therefore degradation of dAmph in the muscle and inhibition of T-tubule biogenesis by dNedd4Lo could have adverse effects on the localization of T-tubule-associated calcium channels and coupling between the DHPR and ryanodine receptor, as a result altering calcium signaling in muscles. Efficient intracellular Ca2+ homeostasis in skeletal muscle requires intact triad junctional complexes comprising T-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Because dNedd4Lo expression significantly reduced T-tubule projections, this would likely impair intracellular Ca2+ homeostasis and result in locomotor defects (Safi, 2016).

Although there is no direct homologue of dNedd4Lo in species other than Drosophila, the mammalian Nedd4 relative Itch has a proline-rich N-terminal region that binds the SH3 domain of Sorting Nexin 9 . In yeast, Rsp5 (the yeast orthologue of Nedd4 proteins) regulates the Amphiphysin homologue Rvs167 by monoubiquitination of lysine in the SH3 domain of Amphiphysin, demonstrating that Nedd4 family members can interact with SH3 domains, including that of amphiphysin, in other species in addition to flies (Safi, 2016).

In addition to dAmph, this study identified several other interacting partners of the N-terminal and Middle regions of dNedd4Lo that could potentially be targeted by dNedd4Lo. Similar to dAmph, two of these proteins, Syndapin (which, like dAmph, bound the unique N-terminus region of dNedd4Lo), and Sorting Nexin 9 (SH3PX1, which bound the unique Middle region of dNedd4Lo), contain BAR and SH3 domains. The SH3 domain-containing protein Cindr/CG31012 (orthologue of the mammalian Cd2ap and Cin85) was also identified as a binding partner to the unique N-terminus of dNedd4Lo. It is not known whether these proteins are bone fide substrates of dNedd4Lo or contribute to the NMJ and T-tubule defects caused by overexpression of dNedd4Lo in the muscle during development (Safi, 2016).

In conclusion, the severely reduced locomotion activity of larvae overexpressing dNedd4Lo in the muscle may be explained by both impaired neuromuscular synaptogenesis, which has been demonstrated previously (Zhong, 2011), and by impaired T-tubule formation as a result of dAmph degradation by dNedd4Lo, which is shown in this study. The defective T-tubule branching would likely impair coupling between the DHPR and ryanodine receptor, possibly by affecting the localization of calcium channels to the T-tubule network. Reduced surface calcium channels on the T-tubule network would alter calcium homeostasis and compromise excitation and contraction coupling, causing larval locomotor defects (Safi, 2016).

GENE STRUCTURE

Unlike vertebrates, the Drosophila genome contains a single Amph gene that gives rise to multiple isoforms by alternative splicing. The entire gene is contained within 18.5 kb of genomic DNA and is flanked at the 5' end by the Sin3A gene, which encodes a transcription corepressor and at the 3' end by the Galpha49B gene, which encodes an eye-specific subunit of a heterotrimeric G-protein GTPase. Amph gives rise to a 3162 bp transcript. In addition, four other predicted isoforms of Amph (2922 bp, 2862 bp, 2117 bp, and 2057 bp) have been identified by sequencing another EST (HL01753) and several positive clones isolated from a cDNA library screen. All isoforms identified arise from alternative splicing within exon 8. Additionally, the two shortest isoforms also contain a shorter 3' UTR due to polyadenylation at a cryptic site 120 bp after the stop codon. The longest isoform encodes a predicted 602 amino acid product, whereas the shorter isoforms predict proteins of 522 and 502 aa, respectively (Leventis, 2001).

PROTEIN STRUCTURE

Amino Acids - 602 (AmphA), 522 (AmphB1) and 502 (AmphB2)

Structural Domains

Overall, the predicted full-length protein of Drosophila Amph is approximately 30% identical to rat amphiphysins 1 and 2, and 24% identical to the predicted C. elegans amphiphysin. The highest degree of similarity is in the N-terminal BAR domain, which shares ~38% identity and ~60% similarity with vertebrate amphiphysin 1 and 2. This domain is predicted to form coiled-coil structures that are involved in dimerization and interaction with lipid membranes and is encoded by exons 1-6. Similarly, the C-terminal SH3 domain, which binds both dynamin and synaptojanin, is also highly conserved and is encoded by exons 9 and 10. In contrast, the central domain, which binds clathrin, endophilin, and the alpha-adaptin subunit of AP2, is poorly conserved and the consensus sequences for binding these proteins are absent. However, within this central domain, another region has been identified that is conserved between Drosophila Amph and vertebrate amphiphysin 2 (but not Bin-1 splice variants). This region lies between amino acids 363 and 398 of Drosophila Amph and is encoded by the nonvariably spliced part of exon 8. This domain starts 2 amino acid residues after the second clathrin binding domain in some forms of amphiphysin 2 and is partially conserved in amphiphysin 1 (Leventis, 2001).

A sequence similarity search of the Drosophila nucleotide database using vertebrate amphiphysin as a query identified a cDNA that encodes a Drosophila amphiphysin. The predicted protein has conserved sequence domains that should enable it to dimerise and bind to dynamin. Structural modelling suggests that the Src-homology-3 (SH3) domains of vertebrate and Drosophila amphiphysins are highly similar, supporting the putative ability of the latter to bind dynamin. However, the fly amphiphysin shows less conservation to sequences in the vertebrate amphiphysins that bind other endocytic components such as clathrin, AP-2 and endophilin. Amphiphysin is a single-copy gene that maps to position 49B on polytene chromosomes (Razzaq, 2000).

Drosophila Amph was identified in a misexpression screen for genes that alter the branching pattern of sensory neuron axonal arbors. The transposon EP(2)2175 was inserted adjacent to the Amph gene and produces a subtle disorganization of axon arborizations, which is due to the misexpression of Amph. This Amph gene is the only Amph gene in Drosophila (Lloyd, 2000; Razzaq, 2000). The three alternative splice isoforms differ in the size of their central domain. Antibodies were generated to the Amph N- and C-terminal regions, and each antibody gave the predicted pattern on Western blots, thus establishing the specificity of the antisera. In addition, both antisera detected a smaller 41 kDa band (AmphC). RT-PCR from larval mRNA was used to confirm that all four proteins, including the 41 kDa AmphC isoform, are unique splice isoforms derived from the single Amph gene. Each isoform contains the coiled-coil and SH3 domains (Zelhof, 2001).

date revised: 8 April 2017

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