Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction

Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. This study shows that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR were used to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1 (RhoGAP100F), neurexin-1, futsch, highwire, discs large, and alpha-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. These results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. It is proposed that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function (McDermott, 2014).

Localized translation is a widespread and evolutionarily ancient strategy used to temporally and spatially restrict specific proteins to their site of function and has been extensively studied during early development and in polarized cells in a variety of model systems. It is thought to be of particular importance in the regulation of neuronal development and in the plastic changes at neuronal synapses that underlie memory and learning, allowing rapid local changes in gene expression to occur independently of new transcriptional programs. The Drosophila neuromuscular junction (NMJ) is an excellent model system for studying the general molecular principles of the regulation of synaptic development and plasticity. Genetic or activity-based manipulations of synaptic translation at the NMJ has previously been shown to affect the morphological and electrophysiological plasticity of NMJ synapses. However, neither the mRNA targets nor the molecular mechanism by which such translational regulation occurs are fully understood (McDermott, 2014).

Previously work identified CG17838, the fly homolog of the mammalian RNA binding protein SYNCRIP/hnRNPQ, which was named Syncrip (Syp). Mammalian SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that contain CamKIIα, Arc, and IP3R1 mRNAs and is thought to regulate translation via an interaction with the noncoding RNA BC200/BC1, itself a translational repressor. Moreover, SYNCRIP/hnRNPQ competes with poly(A) binding proteins to inhibit translation in vitro and regulates dendritic morphology (Chen, 2012) via association with, and localization of, mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex. Drosophila Syp has a domain structure similar to its mammalian homolog, containing RRM RNA binding domains and nuclear localization signal(s), as well as encoding a number of protein isoforms. It was previously shown that Syp binds specifically to the gurken (grk) mRNA localization signal together with a number of factors previously shown to be required for grk mRNA localization and translational regulation (McDermott, 2012). Furthermore, syp loss-of-function alleles lead to patterning defects indicating that syp is required for grk and oskar (osk) mRNA localization and translational regulation in the Drosophila oocyte (McDermott, 2014).

This study shows that Syp is detected in the Drosophila third instar larval muscle nuclei and also postsynaptically at the NMJ. Syp is required for proper synaptic morphology at the NMJ, as syp loss-of-function mutants show a synaptic overgrowth phenotype, while overexpression of Syp in the muscle can suppress NMJ growth. Syp protein associates with a number of mRNAs encoding proteins with key roles in synaptic growth and function including, msp-300, syd-1, neurexin-1 (nrx-1), futsch, highwire (hiw), discs large 1 (dlg1), and α-spectrin (α-spec). The protein levels of a number of these mRNA targets, including msp-300 and dlg1, are significantly affected in syp null mutants. Furthermore, in addition to regulating MSP-300 protein levels, Syp is required for correct MSP-300 protein localization, and syp null mutants have defects in myonuclear distribution and morphology that resemble those observed in msp-300 mutants. It is proposed that Syp coordinates the protein levels from a number of transcripts with key roles in synaptic growth and is a mediator of synaptic morphology and growth at the Drosophila NMJ (McDermott, 2014).

The results demonstrate that Syp is required for the appropriate branching of the motoneurons and the number of synapses they make at the muscle. These observations are potentially explained by the finding that Syp is also required for the correct level of expression of msp-300, dlg1 and other mRNA targets. Given that it was previously shown that Syp regulates mRNA localization and localized translation in the Drosophila oocyte, and studies by others have shown that mammalian SYNCRIP/hnRNPQ inhibits translation initiation by competitively binding poly(A) sequences (Svitkin, 2013), these functions of Syp as occurring at the level of translational regulation of the mRNAs to which Syp binds. Our data are also consistent with other work in mammals showing that SYNCRIP/hnRNPQ is a component of neuronal RNA transport granulesthat can regulate dendritic morphology via the localized expression of mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex (McDermott, 2014 and references therein).

Translation at the Drosophila NMJ is thought to provide a mechanism for the rapid assembly of synaptic components and synaptic growth during larval development, in response to rapid increases in the surface area of body wall muscles or in response to changes in larval locomotion. The phenotypes observed in this study resemble, and are comparable to, those seen when subsynaptic translation is altered genetically or by increased locomotor activity. In syp null mutants, NMJ synaptic terminals are overgrown, containing more branches and synaptic boutons. Similarly, bouton numbers are increased by knocking down Syp in the muscle using RNAi. In contrast, overexpression of Syp in the muscle has the opposite phenotype, resulting in an inhibition of synaptic growth and branching. Furthermore, expressing RNAi against syp in motoneurons alone does not result in a change in NMJ morphology, indicating that Syp acts postsynaptically in muscle, but not presynaptically at the NMJ to regulate morphology. Interestingly, pan-neuronal syp knockdown or overexpression using Elav-GAL4 also results in NMJ growth defects, revealing that some of the defects observed in the syp null mutant may be attributed to Syp function in neuronal cell types other than the motoneurons, such as glial cells, which are known to influence NMJ morphology. Finally, while Syp is not required in the motoneuron to regulate synapse growth and is not detected in the motoneuron, the possibility cannot be excluded that Syp is present at low levels in the presynapse and regulates processes independent of synapse morphology. A further detailed characterization of the cell types and developmental stages in which Syp is expressed and functions is required to better understand the complex phenotypes that were observe (McDermott, 2014).

RNA binding proteins have emerged as critical regulators of both neuronal morphology and synaptic transmision, suggesting that protein production modulates synapse efficacy. Consistent with this, it has been shown in a parallel study that Syp is required for proper synaptic transmission and vesicle release and regulates the presynapse through expression of retrograde Bone Morphogenesis Protein (BMP) signals in the postsynapse. In this role, Syp may coordinate postsynaptic translation with presynaptic neurotransmitter release. These observations provide a good explanation for how Syp influences the presynapse despite being only detectable in the postsynapse. This study has shown that Syp associates with a large number of mRNAs within third instar larvae, many of which encode proteins with key roles in synaptic growth and function. Syp mRNA targets include msp-300, syd-1, nrx-1, futsch, hiw, dlg1, and α-spec. Syp negatively regulates Syd-1, Hiw, and DLG protein levels in the larval body wall but positively regulates MSP-300 and Nrx-1 protein levels. Dysregulation of these multiple mRNA targets likely accounts for the phenotypes that were observed. Postsynaptically expressed targets with key synaptic roles that could explain the synaptic phenotypes that were observed in syp alleles include MSP-300, α-Spec, and DLG. For example, mutants in dlg1 and mutants where postsynaptic DLG is destabilized or delocalized have NMJ morphology phenotypes similar to those observed upon overexpression of Syp in the muscle. Presynaptically expressed targets include syd-1, nrx-1, and hiw. However, this study has shown that syp knockdown in presynaptic motoneurons does not result in any defects in NMJ morphology. The RIP-Seq experiments were carried out using whole larvae and will, therefore, identify Syp targets in a range of different tissues and cells, the regulation of which may or may not contribute to the phenotype that were observed in syp mutants. It is, therefore, possible that Syp associates with these presynaptic targets in other neuronal cell types such as the DA neurons of the larval peripheral nervous system. It is also possible that Nrx-1 or Hiw are expressed and required postsynaptically in the muscle, but this has not been definitively determined. syp alleles may provide useful tools to examine where key synaptic genes are expressed and how they are regulated (McDermott, 2014).

The identity of localized mRNAs and the mechanism of localized translation at the NMJ are major outstanding questions in the field. To date, studies have shown that GluRIIA mRNA aggregates are distributed throughout the muscle. The Syp targets identified in this study, such as msp-300, hiw, nrx-1, α-spec, and dlg1, are now excellent candidates for localized expression at the NMJ. Ultimately, conclusive demonstration of localized translation will involve the visualization of new protein synthesis of targets during activity-dependent synaptic plasticity. Biochemical experiments will also be required to establish the precise mode of binding of Syp to its downstream mRNA targets, the basis for interaction specificity, and the molecular mechanism by which Syp differentially regulates the protein levels of its mRNA targets at the Drosophila NMJ. Despite the fact that mammalian SYNCRIP is known to associate with poly(A) tails, this study and other published work have revealed that Syp can associate with specific transcripts. How Syp associates with specific mRNAs is unknown, and future studies are needed to uncover whether the interaction of Syp with specific transcripts is dictated by direct binding of the three Syp RRM RNA binding domains or by binding to other specific mRNA binding proteins. It is also possible that specific mRNA stem–loops, similar to the gurken localization signal, are required for Syp to bind to its mRNA targets (McDermott, 2014).

This study shows that msp-300 is the most significant mRNA target of Syp. MSP-300 is the Drosophila ortholog of human Nesprin proteins. These proteins have been genetically implicated in various human myopathies. For example, Nesprin/Syne-1 or Nesprin/Syne-2 is associated with Emery-Dreifuss muscular dystrophy (EDMD) as well as severe cardiomyopathies. Moreover, Syp itself is increasingly linked with factors and targets that can cause human neurodegenerative disorders. Recent work has revealed that SYNCRIP/hnRNPQ and Fragile X mental retardation protein (FMRP) are present in the same mRNP granule, and loss of expression of FMRP or the ability of FMRP to interact with mRNA and polysomes can cause cases of Fragile X syndrome. Separate studies have also shown that SYNCRIP interacts with wild-type survival of motor neuron (SMN) protein but not the truncated or mutant forms found to cause spinal muscular atrophy, and Syp genetically interacts with Smn mutations in vivo. Understanding Syp function in the regulation of such diverse and complex targets may, therefore, provide new avenues for understanding the molecular basis of complex disease phenotypes and potentially lead to future therapeutic approaches (McDermott, 2014).

Spectrin regulates Hippo signaling by modulating cortical actomyosin activity

The Hippo pathway controls tissue growth through a core kinase cascade that impinges on the transcription of growth-regulatory genes. Understanding how this pathway is regulated in development remains a major challenge. Recent studies suggested that Hippo signaling can be modulated by cytoskeletal tension through a Rok-myosin II pathway. How cytoskeletal tension is regulated or its relationship to the other known upstream regulators of the Hippo pathway remains poorly defined. This study identifies the spectrins, α-spec, β-spec, or βH-spec contractile proteins at the cytoskeleton-membrane interface, as an upstream regulator of the Hippo signaling pathway. In contrast to canonical upstream regulators such as Crumbs, Kibra, Expanded, and Merlin, spectrin regulates Hippo signaling in a distinct way by modulating cortical actomyosin activity through non-muscle myosin II. These results uncover an essential mediator of Hippo signaling by cytoskeleton tension, providing a new entry point to dissecting how mechanical signals regulate Hippo signaling in living tissues (Deng, 2016).

Protein Interactions

The head-end associations of spectrin give rise to tetramers and make it possible for the molecule to form networks. The head-end associations of Drosophila spectrin were analyzed in vitro and in vivo. Immunoprecipitation assays using protein fragments synthesized in vitro from recombinant DNA show that interchain binding at the head end is mediated by segment 0-1 of alpha Spectrin and segment 18 of beta Spectrin. Point mutations equivalent to erythroid spectrin mutations, responsible for human hemolytic anemias, diminish Drosophila spectrin head-end interchain binding in vitro. To test the in vivo consequence of deficient head-end interchain binding, constructs expressing head-end interchain binding mutant alpha Spectrin were introduced into the Drosophila genome and tested for rescue of an alpha Spectrin null mutation (Deng, 1995).

An alpha-Spectrin minigene lacking the codons for head-end interchain binding fails to rescue the lethality of the null mutant, whereas a minigene with a point mutation in these codons overcomes the lethality of the null mutant in a temperature-dependent manner. The rescued flies are viable and fertile at 25 degrees C, but when shifted to 29 degrees C they become sterile because of defects in oogenesis. At 29 degrees C, egg chamber tissue disruption and cell shape changes are evident, even though the mutant spectrin remains stably associated with cell membranes. These results show that spectrin's capacity to form a network is a crucial aspect of its function in nonerythroid cells (Deng, 1995).

Spectrin's function as an actin-crosslinking protein and membrane skeleton component involves the tail end of the molecule, where multiple interactions between two spectrin chains and between these chains and other proteins give rise to complexes that form membrane skeleton network junctions. To determine whether the sequences that contribute to interchain binding can be distinguished from sequences that are involved in other spectrin tail end functions, the regions in each Drosophila spectrin chain that are required for interchain binding in vitro were mapped. Segments 20 and 21 of the alpha chain and 2 and 3 of the beta chain are required for binding. Binding appears to be very dependent on the lateral register of segments in the two apposed chains. Domains of the nonrepetitive segments, 22 of alpha chain and 1 of beta chain, are also involved in associating the two chains. Required sequences within these nonrepetitive segments are interspersed within domains that are known to be involved in associations with other structural proteins, such as actin, and regulatory components, such as protein 4.1 and calcium (Viel, 1994).

The self-association behavior was examined in solution of one of the repeating conformational segments of Drosophila spectrin (D-alpha-14) as well as of the two-segment unit, D-alpha-14,15. In both polypeptides, there is a reversible, moderate affinity dimerization reaction. Equilibration between monomer and dimer is kinetically limited near 5 degrees C, but occurs at a measurable rate at temperatures > or = 20 degrees C. The temperature dependence for equilibration is consistent with the requirement for extensive disruption of helix-helix packing as the reaction proceeds in either direction. Hydrodynamic studies by means of sedimentation velocity confirm that in solution the C helix in the monomer of D-alpha-14 is folded back to interact with the A and B helices, and that the form of monomeric subunit observed in the crystal structure, in which the A and B helices are continuous, does not persist in the monomer in solution. Both the dimer of D-alpha-14 and the monomer of D-alpha-14,15 appear to be twice the length of the D-alpha-14 monomer, while the frictional ration of the D-alpha-14,15 dimer is consistent with four end-to-end triple alpha-helical domains (Ralston, 1996).

A spectrin isoform termed beta H is present in Drosophila that consists of a conventional alpha spectrin subunit complexed with a novel high molecular weight beta subunit (430 kD). The native alpha beta H molecule binds actin filaments with high affinity and has a typical spectrin morphology except that it is longer than most other spectrin isoforms and includes two knoblike structures, attributed to a unique domain of the beta H subunit. Beta H is encoded by a different gene than the previously described Drosophila beta-spectrin subunit, but it shows sequence similarity to beta-spectrin as well as vertebrate dystrophin, a component of the membrane skeleton in muscle. By size and sequence similarity, dystrophin is more similar to this newly described beta-spectrin isoform (beta H) than to other members of the spectrin gene family, such as alpha-Spectrin and alpha-actinin (Dubreuil, 1990).

ß Heavy-spectrin is a unique beta-spectrin from Drosophila melanogaster that is closer in size to dystrophin than to other beta-spectrin members of the spectrin/alpha-actinin/dystrophin gene super-family. Both the subcellular localization of the beta Heavy-spectrin protein and the tissue distribution of beta Heavy-spectrin transcript accumulation change dramatically during embryonic development. Maternally loaded protein is uniformly distributed around the plasma membrane of the egg. During cellularization it is associated with the invaginating furrow canals and also with a region of the lateral membranes at the apices of the forming cells (apicolateral). During gastrulation the apicolateral staining remains and is joined by a new apical cap (or plate) of ß Heavy-spectrin in areas where morphogenetic movements occur. These locations include the ventral and cephalic furrows and the posterior midgut invagination. Thus, dynamic rearrangement of the subcellular distribution of the protein is precisely coordinated with changes in cell shape. In the developing embryo, after the germ band is fully extended, zygotic message and protein accumulate in the musculature, epidermis, hindgut, and trachea. ß Heavy-spectrin in the epidermis, hindgut, and trachea is apically localized, while the protein in the somatic and visceral musculature is not obviously polarized. The distribution of beta Heavy-spectrin suggests roles in establishing an apicolateral membrane domain that is known to be rich in intercellular junctions and in establishing a unique membrane domain associated with contractile processes (Thomas, 1994).

The Crumbs protein of Drosophila is an integral membrane protein, with 30 EGF-like and 4 laminin A G domain-like repeats in its extracellular segment, which is expressed on the apical plasma membrane of all ectodermally derived epithelia. The insertion of Crumbs into the plasma membrane is necessary and sufficient to confer apical character on a membrane domain. Overexpression of crumbs results in an enormous expansion of the apical plasma membrane and the concomitant reduction of the basolateral domain. This is followed by the redistribution of beta Heavy-spectrin, a component of the membrane cytoskeleton, and by the ectopic deposition of cuticle and other apical components into these areas. Strikingly, overexpression of the membrane-bound cytoplasmic portion of Crumbs alone is sufficient to produce this dominant phenotype. These results suggest that crumbs plays a key role in specifying the apical plasma membrane domain of ectodermal epithelial cells of Drosophila (Wodarz, A., 1995).

Oogenesis in Drosophila takes place within germline cysts that support polarized transport through ring canals interconnecting their 15 nurse cells and single oocyte. Developing cystocytes are spanned by a large cytoplasmic structure (known as the fusome) that has been postulated to help form ring canals and determine the pattern of nurse cell-oocyte interconnections. The adducin-like HTS product and alpha-Spectrin are molecular components of fusomes. A related structure has been discovered in germline stem cells, and associations between fusomes and cystocyte centrosomes have also been documented. hts mutations completely eliminate fusomes, causing the formation of abnormal cysts containing a reduced number of cells. These results imply that Drosophila fusomes are required for ovarian cyst formation and suggest that membrane skeletal proteins regulate cystocyte divisions (Lin, 1994).

The proteins encoded by polar-localized mRNAs play an important role in cell fate specification along the anteroposterior axis of the Drosophila embryo. The only maternally synthesized mRNA known previously to be localized to the anterior cortex of both the oocyte and the early embryo is the Bicoid mRNA. A second mRNA is localized to the anterior pole of the oocyte and early embryo. This mRNA encodes a Drosophila homolog of mammalian adducin, a membrane-cytoskeleton-associated protein that promotes the assembly of the spectrin-actin network. A comparison of the spatial distribution of Bicoid and Adducin-like transcripts in the maternal-effect RNA-localization mutants exuperantia, swallow, and staufen indicates different genetic requirements for proper localization of these two mRNAs to the anterior pole of the oocyte and early embryo (Ding, 1993).

Protein 4.1 functions to link transmembrane proteins with the underlying spectrin/actin cytoskeleton. Drosophila 4.1 is localized to the septate junctions of epithelial cells and is encoded by the coracle gene, a new locus whose primary mutant phenotype is a failure in dorsal closure. In addition, coracle mutations dominantly suppress Ellipse, a hypermorphic allele of the Drosophila EGF-receptor homolog. These data indicate that D4.1 is associated with the septate junction, and suggest that it may play a role in cell-cell interactions that are essential for normal development (Fehon, 1994).

Distribution of two family 4.1 proteins, Expanded and Coracle, are disrupted in discs large mutants. Loss of Discs large also affects the distribution of Fasciclin III and neuroglian, two transmembrane proteins thought to be involved in cell adhesion. These results suggest that DLG serves as a binding protein, linking cell surface receptors with the cytoskeleton via family 4.1 proteins (Woods, 1996)

Neuroglian can transmit positional information directly to Ankyrin and thereby polarize its distribution in Drosophila tissue culture cells. Ankyrin is not normally associated with the plasma membrane of S2 tissue culture cells. Upon expression of an inducible neuroglian minigene, however, cells aggregate into large clusters and Ankyrin becomes concentrated at sites of cell-cell contact. Spectrin is also recruited to sites of cell contact in response to neuroglian expression. The accumulation of Ankyrin at cell contacts requires the presence of the cytoplasmic domain of Neuroglian. Whereas Ankyrin is strictly associated with sites of cell-cell contact, Neuroglian is more broadly distributed over the cell surface. A direct interaction between Neuroglian and Ankyrin can be demonstrated using yeast two-hybrid analysis. Thus, Neuroglian appears to be activated by extracellular adhesion so that ankyrin and the membrane skeleton selectively associate with sites of contact and not with other regions of the plasma membrane (Dubreuil, 1996).

Spectrin has been proposed to function as a sorting machine that concentrates interacting proteins such as the Na,K ATPase within specialized plasma membrane domains of polarized cells. However, little direct evidence to support this model has been obtained. A genetic approach has been used to directly test the requirement for the ß subunit of the alphaß spectrin molecule in morphogenesis and function of epithelial cells in Drosophila. ß Spectrin mutations are lethal during late embryonic/early larval development and they produced subtle defects in midgut morphology and stomach acid secretion. The polarized distributions of alphaßH spectrin and ankyrin are not significantly altered in ß spectrin mutants, indicating that the two isoforms of Drosophila spectrin assemble independent of one another, and that ankyrin is upstream of alphaß spectrin in the spectrin assembly pathway. In contrast, ß spectrin mutations have a striking effect on the basolateral accumulation of the Na,K ATPase. The results establish a role for ß spectrin in determining the subcellular distribution of the Na,K ATPase and, unexpectedly, this role is independent of alpha spectrin (Dubreuil, 2000).

The cellular consequences of the ß spectrin mutations were analyzed in epithelial cells of the larval middle midgut. The copper cells in particular require alpha spectrin for their normal differentiation and function in stomach acid secretion. These cells have a peculiar invaginated morphology in which the apical cell surface is tucked within the cell body. The invagination is connected to the gut lumen through a pore formed by neighboring interstitial cells. Smooth septate junctions occupy the apicolateral contact region between copper cells and interstitial cells, forming a collar that surrounds the pore. Despite their unusual morphology, copper cells exhibit many of the properties of conventional epithelia. The apical surface, extending inward from the collar, displays densely packed microvilli toward the gut lumen. The basolateral domain, including the apicolateral collar, is the site of contact with neighboring cells in the epithelial sheet. All plasma membrane markers that have been examined so far are segregated within either the apical or the basolateral domain (Dubreuil, 2000).

Double-label immunofluorescent staining was used to compare the relative distributions of ankyrin and ß spectrin within the basolateral membrane domain of copper cells. ß spectrin, encoded in this study by an epitope-tagged transgene, is detected throughout the basolateral region in first instar larvae, and is especially concentrated in the apicolateral collar. Ankyrin is also concentrated at the collar, with only faint staining visible in the rest of the basolateral domain. Ankyrin staining appears as comma shapes on either side of the entrance to the apical invagination in favorable optical sections. As larvae grow and copper cells increase in size, ankyrin staining becomes visible throughout the basolateral domain, although ankyrin remains relatively concentrated at the apicolateral contacts. These results are consistent with a role for ankyrin in attaching alphaß spectrin to the plasma membrane, both at the apicolateral contact region and throughout the rest of the basolateral domain of copper cells. However, ankyrin staining outside of the apicolateral collar is relatively weak and near the threshold of detection in first instar larvae (Dubreuil, 2000).

Ankyrin staining was used to monitor the effect of ß spectrin mutations on cell pattern in the middle midgut epithelium. The en face pattern of ankyrin staining in the first instar middle midgut provides a convenient map of cell outlines in the epithelial sheet. The apicolateral contacts between wild-type copper cells and interstitial cells appear as small rings interconnected by lines that represent contacts between adjacent interstitial cells. Ankyrin staining reveals the same overall pattern of cell contacts in ß-specem6 and ß-specem21 male first instar larvae, indicating that development of the cell pattern is normal in the mutants and that the association of ankyrin with the plasma membrane is independent of ß spectrin. However, whereas the ring-shaped profiles are consistently small in the posterior region of the wild-type middle midgut, the rings from ß spectrin mutants are large and irregular. In some cases, the diameter of the pore remains relatively small, whereas the zone of ankyrin staining is broadened into a wide collar. In other cases, the thickness of the ring of ankyrin staining remains narrow, but the pore size is expanded as in most anterior copper cells of the wild-type middle midgut. Thus, the size and shape of the apicolateral contact between copper cells and interstitial cells is dependent on ß spectrin function (Dubreuil, 2000).

The effects of ß spectrin mutations on alpha and ßH spectrin assembly were also examined by immunofluorescence. ß spectrin appears to be required for efficient basolateral targeting of the alpha subunit, but not for the apical assembly of alphaßH spectrin (Dubreuil, 2000).

The effect of ß spectrin mutations on plasma membrane polarity was monitored by staining for the Na,K ATPase, which is normally concentrated in the basolateral membrane domain of copper cells. In wild-type larvae, the en face Na,K ATPase pattern appears as rings representing the copper cell basolateral domain. Optical sections through the central region of the gut have revealed that the basolateral staining of copper cells extends up to the point of apicolateral contact with interstitial cells. A fine reticular pattern of cytoplasmic staining is also observed, but most of the signal is associated with the plasma membrane. A striking change in the distribution of Na,K ATPase staining is observed in ß-specem6 mutants. The nature of the change is dependent on the region of the gut examined. The most anterior copper cells exhibit occasional plasma membrane staining, although in most cells the Na,K ATPase is associated with intracellular compartments. In the most posterior cells, Na,K ATPase staining is typically punctate and irregular. The large puncta of staining are often closely apposed to the nucleus, indicating that the Na,K ATPase is intracellular rather than clumped at the plasma membrane. Copper cells in between these two regions exhibit very weak staining that is not obviously associated with the plasma membrane. Thus, it appears that there are different fates of the Na,K ATPase within copper cell subpopulations in the ß spectrin mutants. However, in all cases, the normal accumulation of Na,K ATPase at the plasma membrane is severely perturbed by the loss of ß spectrin function (Dubreuil, 2000).

The physiological role of copper cells is to secrete stomach acid. Acid secretion is easily monitored by feeding larvae with yeast paste containing bromphenol blue. The dye changes from a brilliant blue color (pH > 4) to a bright yellow color (pH < 2.35) in the copper cell region of wild-type larvae. In between these pH ranges, the dye exhibits a variable green color. alpha Spectrin mutants lack detectable midgut acidification, presumably because of defects within the apical or basolateral domain, or both, of copper cells. Most control larvae exhibit strong (pH < 2.3) midgut acidification. A significant fraction of the ß-specem6 and ß-specem15 mutant larvae also exhibit acidification below pH 2.3. Interestingly, the ß-specem12 and ß-specem21 mutants, which express large truncated fragments of ß spectrin, are less efficient in acid secretion than the mutants that altogether lack detectable ß spectrin. Nevertheless, the effect on midgut acidification in ß spectrin mutants is small compared with the previously described null alpha spectrin mutants. Based on these results it is concluded that the ß spectrin mutations have little effect on plasma membrane integrity or the activity of copper cells, despite their effects on cell morphology and Na,K ATPase localization (Dubreuil, 2000).

Lava lamp (Lva) is nostalgically named for the apical/basal movements observed in the Golgi bodies of Lva mutants during the process of cellularization, reminiscent of the motion of droplets in a lava lamp (Sisson, 2000). Drosophila cellularization and animal cell cytokinesis rely on the coordinated functions of the microfilament and microtubule cytoskeletal systems. To identify new proteins involved in cellularization and cytokinesis, a biochemical screen was conducted for microfilament/microtubule-associated proteins (MMAPs). 17 MMAPs were identified; seven have been previously implicated in cellularization and/or cytokinesis, including KLP3A, Anillin, Septins, and Dynamin. A novel MMAP, Lava Lamp is also required for cellularization. Lva is a coiled-coil protein and, unlike other proteins previously implicated in cellularization or cytokinesis, it is Golgi associated. Functional analysis shows that cellularization is dramatically inhibited upon injecting embryos with anti-Lva antibodies (IgG and Fab). In addition, brefeldin A, a potent inhibitor of membrane trafficking, also inhibits cellularization. Biochemical analysis demonstrates that Lva physically interacts with the MMAPs Spectrin and CLIP190. It is suggested that Lva and Spectrin may form a Golgi-based scaffold that mediates the interaction of Golgi bodies with microtubules and facilitates Golgi-derived membrane secretion required for the formation of furrows during cellularization. These results are consistent with the idea that animal cell cytokinesis depends on both actomyosin-based contraction and Golgi-derived membrane secretion (Sisson, 2000).

Blastoderm embryos were treated with fixatives that efficiently preserve both cortical F-actin and MTs and prepared for immunofluorescence. In syncytial and cellularizing blastoderms, Lva, alpha-Spectrin, and CLIP190 colocalize to large cytoplasmic puncta, some of which are found closely apposed to the furrow front in cellularizing blastoderms. Additional CLIP190-specific puncta are also observed at furrow tips. While most Lva is associated with puncta, low levels are seen throughout the cortical cytoplasm. alpha-Spectrin and CLIP190 are also observed elsewhere within the cortical cytoplasm. Although alpha-Spectrin forms stable complexes with ß- and ßH-Spectrin, punctate ß- or ßH-Spectrin localization is not observed. Instead, these proteins are found throughout the cortical cytoplasm. Minimal Spectrin localization is seen along the PM (Sisson, 2000).

Because the punctate colocalization pattern of Lva, alpha-Spectrin, and CLIP190 is reminiscent of that observed for two cis-Golgi markers, ß-coatomer (ß-COP) and a 120-kD integral membrane protein (p120), their distribution was examined relative to the three MMAPs. Indeed, double immunofluorescence shows that p120 and Lva colocalize. alpha-Spectrin and CLIP190 colocalize with the Golgi markers, as with Lva (Sisson, 2000).

Although a special isoform of mammalian ß-Spectrin (ßIsigma-Spectrin) has been shown to associate with Golgi, Golgi-associated Spectrin has not been previously described in Drosophila, where Spectrins have been shown primarily at the PM. To rule out fixation artifacts, three different preparative conditions were tested. In each case, the punctate localization for alpha-Spectrin, Lva, and the Golgi markers was clearly observed, while very weak Spectrin localization was found at the PM. Together, these results suggest that Spectrins reside on both the PM and Golgi bodies of cellularizing blastoderms, and that each Spectrin population is differentially sensitive to the immunofluorescence preparative conditions used (Sisson, 2000).

To assess whether Lva interacts with other proteins, the native size of Lva was compared with other microfilament/microtubule-associated proteins (MMAPs). The S100 and the final protein (MMAP) fraction were passed separately over a gel filtration column, and fractions were assayed by immunoblot. alpha-Spectrin has been previously shown to copurify with ß- and ßH-Spectrin in two stable heterotetrameric complexes (alpha2ß2 and alpha2ßH2, respectively) and coimmunoprecipitates with ß- and ßH-Spectrin, but information on association of Lva with alpha-Spectrin only is presented for simplicity. Immunoblots show that in both the S100 and the MMAP fraction, Lva, CLIP190, alphaß-, and alphaßH-Spectrin coelute from the column with native molecular weights larger then their predicted molecular weights, indicating that each protein exists in large, stable complexes (Sisson, 2000).

Lva, CLIP190, alphaß-, and alphaßH-Spectrin also cofractionate over two consecutive F-actin affinity columns, indicating that each protein is associated with a stable F-actin-binding activity. S100 was passed over an F-actin column; ABPs were eluted as before, dialyzed against F-actin-binding buffer, and the soluble protein was passed over a second F-actin column. Immunoblots show that Lva, alphaßH-Spectrin, CLIP190, and Anillin each efficiently rebind the second column, while KLP3A does not. The initial binding and subsequent rebinding of alphaß-Spectrin to F-actin is relatively weak. Lva, Spectrins, and CLIP190 elute with a common peak in fractions (Sisson, 2000).

Because Lva, CLIP190, and Spectrins, cofractionate in the above experiments, an assessment was made of whether they interact by immunoprecipitation (IP). Anti-Lva antibody efficiently precipitates Lva protein, and co-IPs alphaßH- and alphaß-Spectrin, as well as CLIP190. Although the anti-alpha-Spectrin and anti-CLIP190 antibodies are inefficient at precipitating their respective antigens, both corroborate the co-IPs obtained with the anti-Lva antibody. Because antibodies to alpha-Spectrin and CLIP190 do not co-IP one another, it is likely that Lva associates with Spectrins and CLIP190 separately (Sisson, 2000).

Trans-synaptic Teneurin signalling in neuromuscular synapse organization and target choice

Synapse assembly requires trans-synaptic signals between the pre- and postsynapse, but understanding of the essential organizational molecules involved in this process remains incomplete. Teneurin proteins are conserved, epidermal growth factor (EGF)-repeat-containing transmembrane proteins with large extracellular domains. This study shows that two Drosophila Teneurins, Ten-m and Ten-a, are required for neuromuscular synapse organization and target selection. Ten-a is presynaptic whereas Ten-m is mostly postsynaptic; neuronal Ten-a and muscle Ten-m form a complex in vivo. Pre- or postsynaptic Teneurin perturbations cause severe synapse loss and impair many facets of organization trans-synaptically and cell autonomously. These include defects in active zone apposition, release sites, membrane and vesicle organization, and synaptic transmission. Moreover, the presynaptic microtubule and postsynaptic spectrin cytoskeletons are severely disrupted, suggesting a mechanism whereby Teneurins organize the cytoskeleton, which in turn affects other aspects of synapse development. Supporting this, Ten-m physically interacts with alpha-Spectrin. Genetic analyses of teneurin and neuroligin reveal that they have differential roles that synergize to promote synapse assembly. Finally, at elevated endogenous levels, Ten-m regulates target selection between specific motor neurons and muscles. This study identifies the Teneurins as a key bi-directional trans-synaptic signal involved in general synapse organization, and demonstrates that proteins such as these can also regulate target selection (Mosca, 2012).

Vertebrate Teneurins are enriched in the developing brain, localized to synapses in culture and pattern visual connections. Both Drosophila Teneurins, Ten-m and Ten-a, function in olfactory synaptic partner matching and were further identified in neuromuscular junction (NMJ) defect screens, with Ten-m also affecting motor axon guidance (Zheng, 2011). This study examined their roles and underlying mechanisms in synapse development (Mosca, 2012).

Both Teneurins are enriched at the larval NMJ. Ten-a was detected at neuronal membranes: this staining was undetectable beyond background in ten-a null mutants and barely detectable following neuronal ten-a RNAi, indicating that Ten-a is predominantly presynaptic. Partial colocalization has been observed between Ten-a and the periactive zone marker Fasciclin II as well as the active zone marker Bruchpilot, suggesting a localization between these regions. Ten-m appeared strongly postsynaptic and surrounded each bouton. Muscle-specific ten-m RNAi eliminated the postsynaptic staining, but uncovered weak presynaptic staining that ubiquitous ten-m RNAi eliminated. Thus, the Ten-m signal was specific and, while partly presynaptic, enriched postsynaptically. Consistently, muscle Ten-m colocalized extensively with Dlg and completely with α-spectrin and is thus, likely coincident with all postsynaptic membranes (Mosca, 2012).

The localization of Ten-a and Ten-m suggested their transsynaptic interaction. To examine this, myc-tagged Ten-a was co-expressed in nerves using the Q system, and HA-tagged Ten-m was expressed in muscles using GAL4. Muscle Ten-m was able to co-immunoprecipitate nerve Ten-a from larval synaptosomes, suggesting that the Teneurins form a heterophilic transsynaptic receptor pair at the NMJ (Mosca, 2012).

To determine Teneurin function at the NMJ, the ten-a null allele and larvae with neuron or muscle RNAi of ten-a and/or ten-m were examined. Following such perturbations, bouton number and size were altered: the quantity was reduced by 55% and the incidence of large boutons markedly increased. Both elements represent impaired synaptic morphogenesis. The reduction in bouton number was likely cumulative through development, as it was visible in first instar ten-a mutants and persisted. In the ten-a mutant, bouton morphogenesis was rescued by restoring Ten-a expression in neurons, but not muscles. Neuronal Ten-m overexpression could not substitute for the lack of Ten-a, revealing their nonequivalence. Neuronal knockdown of Ten-a or Ten-m both showed an impairment, indicating presynaptic function for both, though presynaptic Ten-a plays a more significant role. Moreover, knocking down postsynaptic Ten-m in the ten-a mutant did not enhance the phenotype. Thus, presynaptic Ten-a (and to a lesser extent, Ten-m) and postsynaptic Ten-m are required for synapse development (Mosca, 2012).

Perturbation of teneurins also caused defects in the apposition between presynaptic active zones (release sites) and postsynaptic glutamate receptor clusters: up to 15% of the active zones/receptor clusters lacked their partner compared to 1.8% in controls. Under electron microscopy, active zones are marked by electron dense membranes and single presynaptic specializations called T-bars, which enable synapse assembly, vesicle release and Ca2+ channel clustering. Teneurin disruption caused defects in T-bar ultrastructure, membrane organization and apposition to contractile tissue. Teneurin perturbation also impaired postsynaptic densities while increasing membrane ruffling, further indicating organizational impairment. These phenotypes resemble mutants with adhesion and T-bar biogenesis defects, suggesting a role for Teneurins in synaptic adhesion and stability. Synaptic vesicle populations similarly required Teneurins for clustering at the bouton perimeter and proper density. As these effects are not synonymous with active zone disruption, Teneurins are also required for synaptic vesicle organization (Mosca, 2012).

Synapses lacking teneurin were also functionally impaired. The mean amplitude of evoked excitatory postsynaptic potentials (EPSP) in larvae was decreased by 28% in the ten-a mutant . Spontaneous miniature EPSPs (mEPSPs) showed a 20% decrease in amplitude, a 46% decrease in frequency and an altered amplitude distribution compared with control). These defects resulted in a 20% reduction in quantal content, which could be partly due to fewer boutons and release sites. However, release probability may also be reduced, as suggested by an increased paired pulse ratio in ten-a mutants. The decay kinetics of responses were faster in ten-a mutants, suggesting additional postsynaptic effects on glutamate receptors and/or intrinsic membrane properties. Further, FM1-43 dye loading revealed markedly defective vesicle cycling in ten-a mutants. Consistent with physiological impairment, teneurin-perturbed larvae exhibited profound locomotor defects. In summary, Teneurins are required for multiple aspects of NMJ organization and function (Mosca, 2012).

As a potential mechanism for synaptic disorganization following teneurin perturbation, the pre- and postsynaptic cytoskeletons were examined. In the presynaptic terminal, organized microtubules contain Futsch (a microtubule-binding protein)-positive “loops” while disorganized microtubules possess punctate, “unbundled” Futsch. Each classification normally represented ~10% (often distal) of boutons. Following teneurin perturbation, many more boutons had unbundled Futsch while those with looped microtubules were decreased by 62%–95%. Therefore, proper microtubule organization requires pre- and postsynaptic Teneurins. In contrast to mild active zone/glutamate receptor apposition defects, most boutons displayed microtubule organizational defects (Mosca, 2012).

Removal of teneurins also severely disrupted the postsynaptic spectrin cytoskeleton, with which Ten-m colocalized. Postsynaptic α-spectrin normally surrounds the bouton. Perturbing neuronal or muscle Teneurins markedly reduced postsynaptic α-spectrin without affecting Dlg. Postsynaptic β-spectrin, Adducin and Wsp were similarly affected. In the muscle, α-spectrin is coincident with and essential for the integrity of the membranous subsynaptic reticulum (SSR). Consistent with this, teneurin disruption reduced SSR width up to 70% and increased the frequency of 'ghost' boutons, which are failures of postsynaptic membrane organization). Thus, Teneurins are involved in the organization of the pre- and postsynaptic cytoskeletons and postsynaptic membranes. Further, endogenous α-spectrin co-immunoprecipitates with muscle-expressed, FLAG-tagged Ten-m, suggesting that Ten-m physically links the synaptic membrane to the cytoskeleton (Mosca, 2012).

As the most severe defects following teneurin perturbation were cytoskeletal, it is proposed that Teneurins primarily organize the presynaptic microtubule and postsynaptic spectrin-based cytoskeletons. However, such a solitary role cannot fully explain the observed phenotypes. The bouton number defects associated with cytoskeletal disruption are milder than those following teneurin disruption. Also, while active zone dynamics are affected by cytoskeletal perturbation, defects in apposition are not. Moreover, the T-bar structural defects more closely resemble synapse adhesion and active zone formation defects. Thus, Teneurins may regulate release site organization and synaptic adhesion independent of the cytoskeleton (Mosca, 2012).

These data also indicate that Teneurins act bi-directionally across the synaptic cleft. Ten-a acts predominantly neuronally as evidenced by localization, phenotypes caused by neuronal (but not muscle) knockdown, and mutant rescue by neuronal (but not muscle) expression. Yet, in addition to the presynaptic phenotypes, many others were postsynaptic, including reduced muscle spectrin, SSR and membrane apposition. Similarly, although Ten-m is present both pre-and postsynaptically, muscle knockdown resulted in presynaptic defects, including microtubule and vesicle disorganization, reduced active zone apposition, and T-bar defects. Thus, Teneurins function in bi-directional transsynaptic signaling to organize neuromuscular synapses. This may involve downstream pathways or simply establish an organizational framework by the receptors themselves. Moreover, as the single disruptions of neuronal ten-a or muscle ten-m arevsimilarly severe and not enhanced by combination, they likely function in the same pathway. The finding that Ten-a and Ten-m co-immunoprecipitate from different cells in vitro (Hong, 2012) and across the NMJ in vivo further suggests a signal via trans-synaptic complex. Teneurin function, however, may not be solely transsynaptic. In some cases (vesicle density, SSR width), cell-autonomous knockdown showed stronger phenotypes than knocking down in synaptic partners. This suggests additional cell-autonomous roles unrelated to transsynaptic Teneurin signaling (Mosca, 2012).

Signaling involving the transmembrane proteins Neurexin and Neuroligin also mediates synapse development (Craig, 2007). In Drosophila, Neurexin (dnrx) and Neuroligin1 (dnlg1) mutations cause phenotypes similar to teneurin perturbation: reduced boutons, active zone organization, transmission and SSR. dnlg1 and dnrx mutations do not enhance each other, suggesting their function in the same pathway. Consistently, this study found that dnrx and dnlg1 mutants exhibited largely similar phenotypes. To investigate the relationship between the teneurins and dnrx/dnlg1, focus was placed on the dnlg1 null mutant. Both Ten-m and DNlg1-eGFP occupy similar postsynaptic space. teneurin and dnlg1 loss-of-function also displayed similar bouton number reductions, vesicle disorganization and ghost bouton frequencies. Other phenotypes showed notable differences in severity. In dnlg1 mutants, there was a 29% failure of active zone/glutamate receptor apposition, compared to 15% for the strongest teneurin perturbation. For the cytoskeleton, dnlg1 mutants were mildly impaired compared to teneurin perturbations (Mosca, 2012).

To further examine their interplay, ten-a dnlg1 double mutants were analyzed. Both single mutants were viable, despite their synaptic defects. Double mutants, however, were larval lethal. Rare escapers were obtained that displayed a 72% reduction in boutons, compared to a 50%–55% decrease in single mutants. Active zone apposition in double mutants was enhanced synergistically over either single mutant. Cytoskeletal defects in the double mutant resembled the ten-a mutant. These data suggest that teneurins and dnrx/dnlg1 act in partially overlapping pathways, cooperating to properly organize synapses, with Teneurins contributing more to cytoskeletal organization and Neurexin/Neuroligin to active zone apposition (Mosca, 2012).

In the accompanying manuscript (Hong, 2012), it was shown that while the basal Teneurins are broadly expressed in the Drosophila antennal lobe, elevated expression in select glomeruli mediates olfactory neuron partner matching. At the NMJ, this basal level mediates synapse organization. Analogous to the antennal lobe, elevated ten-m expression was found at muscles 3 and 8 using the ten-m-GAL4 enhancer tra. This was confirmed for endogenous ten-m, and it was determined to be contributed by elevated Ten-m expression in both nerves and muscles. Indeed, ten-m-GAL4 is highly expressed in select motoneurons, including MN3-Ib, which innervates muscle 3. This elevated larval expression also varied along the anterior-posterior axis, and was specific for Ten-m as Ten-a expression did not differ within or between segments (Mosca, 2012).

To test whether elevated Ten-m expression in muscle 3 and MN3-Ib affects neuromuscular connectivity, ten-m RNAi was expressed using ten-m-GAL4. Wild-type muscle 3 was almost always innervated. However, following ten-m knockdown, muscle 3 innervation failed in 11% of hemisegments. This required Ten-m on both sides of the synapse, as the targeting phenotype persisted following neuronal or muscle RNAi suppression using tissue-specific GAL80 transgenes. ten-a RNAi did not show this phenotype, consistent with homophilic target selection via Ten-m. The phenotype was specific to muscle 3, as innervation onto the immediately proximal or distal muscle was unchanged. The low penetrance is likely due to redundant target selection mechanisms. Where innervation did occur, the terminal displayed similarly severe phenotypes to other NMJs. Thus, in addition to generally mediating synaptic organization, Ten-m also contributes to correct target selection at a specific NMJ (Mosca, 2012).

To determine whether Ten-m overexpression could alter connectivity, Ten-m was expressed in muscle 6 (but not the adjacent muscle 7), and the motoneurons innervating both muscles using H94-GAL4. Normally, 60% of the boutons at muscles 6/7 are present on muscle 6 with 40% on muscle 7 . Ten-m overexpression caused a shift whereby 81% of boutons synapsed onto muscle 6 and only 19% onto muscle 7. This shift also required both neuronal and muscle Ten-m as neuronal or muscle GAL80 abrogated it. The effect was specific as Ten-a overexpression did not alter this synaptic balance, nor was it secondary to altered bouton number, which is unchanged. Therefore, elevated Ten-m on both sides of the NMJ can bias target choice. This, combined with evidence that Ten-m can engage homophilic interaction in vitro, supports a transsynaptic homophilic attraction model at the NMJ as in the olfactory system (Mosca, 2012).

In summary, this study has identified a two-tier mechanism for Teneurin function in synapse development at the Drosophila NMJ. At the basal level, Teneurins are expressed at all synapses and engage in hetero- and homophilic bi-directional transsynaptic signaling to properly organize synapses. Supporting this, the Teneurins can mediate homo-and heterophilic interactions in vitro and heterophilic interactions in vivo. At the synapse, Teneurins organize the cytoskeleton, interact with α-spectrin, and enable proper adhesion and release site formation. Further, elevated Ten-m expression regulates target selection in specific motoneurons and muscles via homophilic matching and functions with additional molecules to mediate precise neuromuscular connectivity. Teneurin-mediated target selection at the NMJ is analogous to its role in olfactory synaptic partner matching (Hong, 2012). As the Teneurins are expressed broadly throughout the antennal lobe, it remains an attractive possibility that they also regulate central synapse organization (Mosca, 2012).

Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction

Synaptic communication requires precise alignment of presynaptic active zones with postsynaptic receptors to enable rapid and efficient neurotransmitter release. How transsynaptic signaling between connected partners organizes this synaptic apparatus is poorly understood. To further define the mechanisms that mediate synapse assembly, a chemical mutagenesis screen was carried out in Drosophila to identify mutants defective in the alignment of active zones with postsynaptic glutamate receptor fields at the larval neuromuscular junction. From this screen a mutation was identified in Actin 57B that disrupted synaptic morphology and presynaptic active zone organization. Actin 57B, one of six actin genes in Drosophila, is expressed within the postsynaptic bodywall musculature. The isolated allele, actE84K, harbors a point mutation in a highly conserved glutamate residue in subdomain 1 that binds members of the Calponin Homology protein family, including spectrin. Homozygous actE84K mutants show impaired alignment and spacing of presynaptic active zones, as well as defects in apposition of active zones to postsynaptic glutamate receptor fields. actE84K mutants have disrupted postsynaptic actin networks surrounding presynaptic boutons, with the formation of aberrant actin swirls previously observed following disruption of postsynaptic spectrin. Consistent with a disruption of the postsynaptic actin cytoskeleton, spectrin, adducin and the PSD-95 homolog Discs-Large are all mislocalized in actE84K mutants. Genetic interactions between actE84K and neurexin mutants suggest that the postsynaptic actin cytoskeleton may function together with the Neurexin-Neuroligin transsynaptic signaling complex to mediate normal synapse development and presynaptic active zone organization (Blunk, 2014).

Spectrin tetramer formation is not required for viable development in Drosophila

The dominant paradigm for spectrin function is that (αβ)2-spectrin tetramers or higher order oligomers form membrane associated two-dimensional networks in association with F-actin to reinforce the plasma membrane. Tetramerization is an essential event in such structures. This study characterized the tetramerization interaction between α-spectrin and β-spectrins in Drosophila. Wild-type α-spectrin binds to both β- and βH-chains with high affinity, resembling other non-erythroid spectrins. However, α-specR22S, a tetramerization site mutant homologous to the pathological α-specR28S allele in humans, eliminates detectable binding to β-spectrin and reduces binding to βH-spectrin ~1000 fold. Even though spectrins are essential proteins, α-specR22S rescues α-spectrin mutants to adulthood with only minor phenotypes indicating that tetramerization, and thus conventional network formation, is not the essential function of non-erythroid spectrin. These data provide the first rigorous test for the general requirement for tetramer-based non-erythroid spectrin networks throughout an organism and find that they have very limited roles, in direct contrast to the current paradigm (Khanna, 2015).

alpha Spectrin: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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