hu-li tai shao


Genetic requirements for anterior RNA localization revealed by the distribution of Adducin-like transcripts during Drosophila oogenesis

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 whose localization is required to generate a homeodomain protein gradient that specifies position along the anteroposterior embryonic axis. A second mRNA has been identified and characterized that 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; full text of article).

Role of Adducin-like (hu-li tai shao) mRNA and protein localization in regulating cytoskeletal structure and function during Drosophila oogenesis and early embryogenesis

Adducin is a cytoskeletal protein that can function in vitro to bundle F-actin and to control the assembly of the F-actin/spectrin cytoskeletal network. The Drosophila Adducin-like (Add) locus, also referred to as hu-li tai shao encodes two adducin-related protein isoforms: a 95 x 10(3) Mr form (ADD-95) and an 87 x 10(3) Mr form (ADD-87). ADD-87 protein is present throughout the oocyte cortex at stages 9 and 10 of oogenesis but is restricted to its anterior pole from stage 11 onward. This ADD-87 protein localization is preceded by localization of Add-hts mRNA first to the cortex and then to the anterior pole of the oocyte. Mutation of the swallow gene results in delocalization of Add-hts mRNA and ADD-87 protein from the cortex of stage 9 and 10 oocytes, and from the anterior pole of later stage oocytes. Early embryos produced by swallow or Add-hts mutant females have severe defects in the distribution of F-actin and spectrin as well as abnormalities in nuclear division, nuclear migration, and cellularization. In addition to their cytoskeletal defects, embryos produced by swallow females have an abnormal anterior pattern because bicoid mRNA is delocalized from the anterior pole. In contrast, bicoid mRNA is still found at the anterior of embryos produced by Add-hts mothers. Thus swallow functions to restrict bicoid mRNA and Add-hts mRNA to the cortex of the oocyte. Cortical restriction of Add-hts mRNA and protein is required for the normal structure and function of the early embryonic F-actin/spectrin cytoskeleton. A defective embryonic cytoskeleton can be induced in either of two ways: (1) by delocalization of functional ADD from the oocyte cortex (as in swallow mutants), or (2) by reduction of ADD function while retaining its normal cortical localization during oogenesis (as in Add-hts mutants) (Zaccai, 1996b).

Formation of the Drosophila ovarian ring canal inner rim depends on cheerio

In Drosophila oogenesis, the development of a mature oocyte depends on having properly developed ring canals that allow cytoplasm transport from the nurse cells to the oocyte. Ring canal assembly is a step-wise process that transforms an arrested cleavage furrow into a stable intercellular bridge by the addition of several proteins. A gene is described, cheerio, that provides a critical function for ring canal assembly. Mutants in cheerio fail to localize ring canal inner rim proteins including filamentous actin, the ring canal-associated products from the hu-li tai shao (hts) gene, and kelch. Since Hts and Kelch are present but unlocalized in cheerio mutant cells, cheerio is likely to function upstream from each of them. Examination of mutants in cheerio places it in the pathway of ring canal assembly between cleavage furrow arrest and localization of hts and actin filaments. Furthermore, this mutant reveals that the inner rim cytoskeleton is required for expansion of the ring canal opening and for plasma membrane stabilization (Robinson, 1997; full text of article).

Different 3' untranslated regions target alternatively processed hu-li tai shao transcripts to distinct cytoplasmic locations during Drosophila oogenesis

Cytoplasmic mRNA localization is one method by which protein production is restricted to a particular intracellular site. A novel mechanism is described for localization of transcripts encoding distinct protein isoforms to different destinations. Alternative processing of transcripts produced in the Drosophila ovary by the hu-li tai shao locus introduces distinct 3' untranslated regions (3'UTRs) that differentially localize the mRNAs. Three classes of hts mRNA (R2, N32 and N4) are synthesized in the germ line nurse cells and encode proteins with adducin-homologous amino-terminal regions but divergent carboxy-terminal domains. The R2 and N32 classes of mRNA remain in the nurse cells and are not transported into the oocyte. In contrast, the N4 class of transcripts is transported from the nurse cells into the oocyte starting at stage 1, is subsequently localized to the oocyte cortex at stage 8 and then to the anterior pole from stage 9 on. All aspects of N4 transcript transport and localization are directed by the 345-nucleotide(nt)-long 3' untranslated region (3'UTR). The organization of localization elements in the N4 3'UTR is modular: a 150 nt core is sufficient to direct transport and localization throughout oogenesis. Additional 3'UTR elements function additively together with this core region at later stages of oogenesis to maintain or enhance anterior transcript anchoring. The swallow locus is required to maintain hts transcripts at the anterior pole of the oocyte and functions through the N4 3'UTR. In addition to the three classes of germ line-expressed hts transcripts, a fourth class (R1) is expressed in the somatic follicle cells that surround the germ line cells. This transcript class encodes the Drosophila orthologue of mammalian adducin (Whittaker, 1999; full text of article).

The Drosophila RNA-binding protein Lark is required for the organization of the actin cytoskeleton and Hu-li tai shao localization during oogenesis

Elimination of maternal expression of the Drosophila RNA-binding protein Lark results in female sterility. This is due to a requirement during oogenesis. Developing oocytes from lark mutatn germline clones (GLCs) are often smaller than normal due to defects in nurse cell cytoplasmic 'dumping.' Late-stage egg chambers from lark mutant GLCs contain low levels of cortical and ring canal associated actin and completely lack nurse cell cytoplasmic F-actin bundles, suggesting the 'dumping' phenotype is due to a defect in the actin cytoskeleton. Localization of Hu-li tai shao (Hts) protein, a component of ring canals, is also disrupted in these mutants. In addition to the dumpless phenotype, a buildup of late-stage egg chambers is observed, a phenotype that correlates with the decrease in egg-laying observed in the mutants. It is postulated that this phenotype is due to defects in the cytoskeletal integrity of eggs since retained and oviposited eggs are fragile and often deflated. These mutant phenotypes are likely due to disruption of an RNA-binding function of Lark since similar phenotypes were observed in flies carrying specific RNA-binding domain mutations. It is proposed that Lark functions during oogenesis as an RNA-binding protein, regulating mRNAs required for nurse cell transport or apoptosis (McNeil, 2004).

Rho-kinase (DRok) is required for tissue morphogenesis in diverse compartments of the egg chamber during oogenesis

The Rho-kinases are widely utilized downstream targets of the activated Rho GTPase that have been directly implicated in many aspects of Rho-dependent effects on F-actin assembly, acto-myosin contractility, and microtubule stability, and consequently play an essential role in regulating cell shape, migration, polarity, and division. The single closely related Drosophila Rho-kinase ortholog, DRok, is required for several aspects of oogenesis, including maintaining the integrity of the oocyte cortex, actin-mediated tethering of nurse cell nuclei, 'dumping' of nurse cell contents into the oocyte, establishment of oocyte polarity, and the trafficking of oocyte yolk granules. These defects are associated with abnormalities in DRok-dependent actin dynamics and appear to be mediated by multiple downstream effectors of activated DRok that have previously been implicated in oogenesis. DRok regulates at least one of these targets, the membrane cytoskeletal cross-linker DMoesin, via a direct phosphorylation that is required to promote localization of DMoesin to the oocyte cortex. The collective oogenesis defects associated with DRok deficiency reveal its essential role in multiple aspects of proper oocyte formation and suggest that DRok defines a novel class of oogenesis determinants that function as key regulators of several distinct actin-dependent processes required for proper tissue morphogenesis (Verdier, 2006a).

The observation of dumpless-like oversized nurse cells in most of Drok2 GLCs supports a role for DRok in the rapid phase of cytoplasmic transport at stages 10B–11 of oogenesis. Unlike other classes of dumpless mutants including chickadee, singed or quail, failure of rapid cytoplasmic transport from the Drok2 mutant nurse cells to the oocyte does not result from the obstruction of the ring canals by unanchored nurse cell nuclei, suggesting that Drok constitutes a distinct class of dumpless-like mutants. In addition, in sqhAX3 GLCs, dumpless nurse cells are associated with a lack of acto-myosin contractility by nurse cells, as revealed by mislocalization of myosin II and by absence of the perinuclear organization of actin filaments bundles in the nurse cells. Therefore, sqhAX3 mutant nurse cells cannot contract properly to expulse their cytoplasm through otherwise weakly damaged ring canals. Drok2 and sqhAX3 mutant nurse cells do not share the same actin filament phenotype; Drok2 mutant nurse cells exhibit a more dramatic phenotype associated with absence of radial filaments and disorganization of cortical actin. It is, however, likely that DRok and Sqh are part of the same signaling pathway that regulates acto-myosin contractility in nurse cells; it has already been shown that DRok phosphorylates Sqh in Drosophila development. Moreover, the severity of the Drok2 mutant F-actin phenotypes may reflect DRok's potential to engage multiple distinct downstream substrates, of which Sqh is only one. Significantly, the actin-binding protein, adducin, is also reportedly a direct substrate for mammalian Rho-kinases, and the Drosophila Adducin ortholog, Hts, is a major component of ring canals. Thus, it is possible that the observed defects in ring canal morphology in Drok2 GLCs involve abnormal regulation of adducin by DRok. However, it is difficult to determine whether this ring canal phenotype contributes to the dumpless-like nurse cell phenotype observed in Drok2 GLCs (Verdier, 2006a).

The observation that nurse cell nuclei are substantially increased in size in Drok2 GLCs suggests a possible involvement of DRok in increased endoreplication of the nurse cells. The Rho-related Rac and Cdc42 GTPases have previously been associated with endoreplication in porcine aortic endothelial (PAE) cells, although Rho has not been implicated thus far. Interestingly, this nurse cell nuclei phenotype has not been observed in other previously described GLC mutants of other actin cytoskeleton-regulating signaling components that exhibit oogenesis defects. Thus, chic as well as sqhAX3 GLCs reveal cytokinesis defects associated with the presence of multinucleated nurse cells. In addition, the majority of sqhAX3 mutant egg chambers harbor less than 15 nurse cells (64% of sqhAX3 mutant egg chambers have less than 7 nurse cells), a phenotype that is not shared by Drok2 mutant nurse cells. These findings also suggest that Drok2 defines a new category of oogenesis mutants that affect the actin cytoskeleton (Verdier, 2006a).

Both Dmoe and Drok2 GLCs exhibit similar actin defects in the oocyte, associated with a loose uneven cortical actin distribution and the presence of actin clumps in the ooplasm and near the cortex. Moreover, phospho-DMoesin levels are decreased at the cortex or mislocalized within the ooplasm of Drok2 GLCs and the conserved kinase domain of Rho-kinase phosphorylates DMoesin on threonine 559 in vitro. A potential mechanism for the DRok-DMoesin signal in this setting is that DRok controls actin reorganization through phosphorylation of DMoesin, which has been previously shown to cross-link actin to the plasma membrane when phosphorylated on T559 at the oocyte cortex. However, the detection of some phospho-DMoesin in the Drok2 GLCs indicates that the critical T559 residue can be phosphorylated by other kinases in the oocyte. Indeed, direct phosphorylation of T559 of mammalian Moesin by protein kinase C (PKC)-θ has been shown in vitro. In addition, mammalian Rho-kinase and PAK have been reported to both phosphorylate the very conserved T508 residue of LIM-kinase in vitro. Therefore, phosphorylation of the conserved T559 residue of Moesin by additional kinases might also occur in Drosophila, highlighting the complexity of cross-talk within developmental signaling pathways (Verdier, 2006a).

The observation that Drok2 mutant oocytes are morphologically more affected than Dmoe mutant oocytes with regard to the deformed plasma membrane also suggests that to exert its functions at the oocyte cortex, DRok is not only signaling to DMoesin but probably also to additional downstream targets that cooperate with DMoesin in the maintenance of the cortical actin cytoskeleton. The strong phenotype associated with the deformed oocyte plasma membrane, which separates dramatically from the apical plasma membranes of the follicle cell layer in most Drok2 GLCs, raises an intriguing question about DRok's apparent role in an adhesive process. That specific phenotype has not been previously reported in studies of other oogenesis mutants associated with defective adhesion between the oocyte and the surrounding follicle cells. Previous reports regarding such adhesion largely address cross-signaling between the apical Notch receptor and the germline-derived putative secreted and transmembrane proteins, Brainiac and Egghead, respectively, in which germline loss of either Brainiac or Egghead results in loss of epithelial apico-basal polarity and accumulation of follicular epithelial cells in multiple layers around the oocyte, but does not lead to a physical separation between the oocyte and the follicle cells membranes. The unique phenotype of Drok2 GLCs could reflect a role for DRok in mediating a distinct signaling pathway from the oocyte to regulate its shape and its adherence to the surrounding follicle cells. Alternatively, the aberrant morphology of the nurse cells, which appear to 'push' against the oocyte without contracting, might produce a mechanical stress on the oocyte itself that prevents it from remaining apposed to the follicle cell layer. Notably, it was found that the follicle cells themselves also appear to require DRok function for the maintenance of their shape, and it is possible that their ability to signal to the oocyte is also affected by DRok deficiency (Verdier, 2006a).

In summary, the single closely related Drosophila Rho-kinase ortholog, DRok, is required for several aspects of oogenesis, including maintaining the integrity of the oocyte cortex, actin-dependent tethering of nurse cell nuclei, 'dumping' of nurse cell contents into the oocyte, establishment of oocyte polarity, and the trafficking of oocyte yolk granules. It is likely that several previously identified direct phosphorylation targets of DRok, including DMoesin, Sqh (myosin light chain), and Hts (adducin), which have each been implicated in various aspects of oogenesis, mediate at least some of the functions of DRok in developing egg chambers. These findings indicate an essential role for Rho-DRok signaling via multiple DRok effectors in several distinct aspects of oogenesis (Verdier, 2006a).

Rho-kinase regulates tissue morphogenesis via non-muscle myosin and LIM-kinase during Drosophila development

The Rho-kinases (ROCKs) are major effector targets of the activated Rho GTPase that have been implicated in many of the Rho-mediated effects on cell shape and movement via their ability to affect acto-myosin contractility. The role of ROCKs in cell shape change and motility suggests a potentially important role for Rho-ROCK signaling in tissue morphogenesis during development. Indeed, in Drosophila, a single ROCK ortholog, DRok, has been identified and has been found to be required for establishing planar cell polarity. A potential role for DRok in additional aspects of tissue morphogenesis was examined using an activated form of the protein in transgenic flies. The findings demonstrate that DRok activity can influence multiple morphogenetic processes, including eye and wing development. Furthermore, genetic studies reveal that Drok interacts with multiple downstream effectors of the Rho GTPase signaling pathway, including non-muscle myosin heavy chain, adducin, and Diaphanous in those developmental processes. Finally, in overexpression studies, it was determined that Drok and Drosophila Lim-kinase interact in the developing nervous system. These findings indicate widespread diverse roles for DRok in tissue morphogenesis during Drosophila development, in which multiple DRok substrates appear to be required (Verdier, 2006b; full text of article).

Ovhts-RC is necessary for actin localization to ring canals

The ability to rescue RCs with Ovhts transgenes provided an opportunity to investigate the function of Ovhts-RC. To determine when full-length Ovhts needs to be expressed for RC localization, stage-specific induction of Ovhts expression was done. Wild-type flies carrying a P{UASH-Ovhts::GFP} transgene were crossed to two different Gal4 lines: MTD-Gal4 that induces strong expression throughout oogenesis or P{bam-Gal4} that induces expression only in Region 1 of the germarium. In ovaries from P{UASH-Ovhts::GFP}, MTD-Gal4 flies, Ovhts::GFP was on RCs in all stages of oogenesis. However, in ovaries from P{UASH-Ovhts::GFP}; P{bam-Gal4}, Ovhts::GFP was only in the germaria, and on rare occasions on RCs in stage 2 egg chambers. Thus, continuous expression of Ovhts-RC protein is needed to maintain localization to RCs (Petrella, 2007).

The continual expression and localization of Ovhts-RC throughout oogenesis indicates that it may be important for actin maintenance at the RC. To test this possibility, P{UASH-Ovhts::GFP} was expressed in htsDeltaG flies using the P{nos-GAL4} driver whose expression is high in the germarium, low during stages 2-6, and then high again starting approximately at stage 7. This allowed rescue of RCs when they form in the germaria of mutants, followed by about a day where little or no new Ovhts protein is produced. As expected Ovhts::GFP was present on RCs in the germaria, absent in mid-stage egg chambers, and present again in later egg chambers within the same ovariole. Both the amount of F-actin and its organization at RCs mirrored the presence of Ovhts::GFP. When Ovhts::GFP was present, RCs appeared wild type. In egg chambers lacking Ovhts::GFP, there were no clear F-actin-containing RC rims. There were, however, actin-rich areas that may be disintegrating RC rims. Thus, continued expression of Ovhts is needed for the recruitment and/or maintenance of F-actin on RCs (Petrella, 2007).

Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. This study found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and this study show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and evidence is presented that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation (Wang, 2011).

Dlg is a Drosophila member of a family of MAGUK scaffolding proteins that has four mammalian members, SAP97/hDlg, PSD-93/Chapsyn-110, PSD-95/SAP90 and SAP102/NE-Dlg, and which has important developmental and regulatory roles in the nervous system and in epithelia. Phosphorylation is emerging as a mechanism for the regulation of the localization and function of the Dlg family. The post-synaptic targeting of Dlg to the Drosophila NMJ is inhibited by phosphorylation of the first PDZ domain by CaMKII and of the GUK domain by PAR-1. In mammalian neurons, CaMKII phosphorylation of PDZ1 of PSD-95 terminates long term potentiation-induced spine growth by inducing translocation of PSD-95 out of the active spine. Furthermore, phosphorylation of PDZ1 of either PSD-95 or SAP97/hDlg by CaMKII regulates their interaction with NMDA subunits, and additional cell culture studies in epithelial cells demonstrate effects of SAP97/hDlg phosphorylation by various kinases (Wang, 2011).

An important function of Dlg at the NMJ is involvement in synaptic plasticity during muscle growth, at least in part by controlling the localization at the pre-synaptic and post-synaptic membranes of Fas2, a homophilic cell adhesion molecule of the immunoglobulin superfamily that binds Dlg. The breaking and restoration of Dlg/Fas2-mediated adhesion between the pre- and post-synaptic membranes is likely critical to synaptic growth during development. This study has shown that Hts exists in a complex with Dlg, probably at both the post-synaptic membrane of the NMJ and at the lateral membrane in epithelia, where the two proteins are likely brought into close proximity by a shared association with the spectrin-actin junction. Hts is therefore positioned to locally regulate Dlg and participate in synaptic plasticity at sites of association with the membrane cytoskeleton, and it positively contributes to phosphorylation of Dlg on its GUK domain. This site of phosphorylation is a target for PAR-1, and consistent with this it was found that Hts can regulate the levels of PAR-1. Phosphorylation of Dlg impedes its targeting to the post-synaptic membrane and it is proposed that Hts regulates this targeting at the synapse by controlling the levels of PAR-1 at the post-synaptic membrane. This could occur through Hts acting as a scaffold participating in localized stabilization or translation of PAR-1 at the post-synaptic membrane and, consistent with the latter mechanism, localized post-synaptic translation of NMJ proteins has been reported in Drosophila. Over-expression of Hts permits synaptic overgrowth, which may in part be due to effects on Dlg targeting to the post-synaptic membrane, but unpublished results indicating additional routes of action. Furthermore, if Hts were acting mainly by elevating PAR-1 levels, synaptic undergrowth would be expected rather than overgrowth with Hts over-expression, as the post-synaptic over-expression of PAR-1 leads to decreased bouton number and an oversimplified synapse. CaMKII also regulates synaptic growth and Dlg targeting at the NMJ through phosphorylation. There is currently no antibody available to examine phosphorylation of Dlg by CaMKII in vivo, but it was determined that Hts controls the levels of this kinase similarly to PAR-1, suggesting that Hts may also be regulating Dlg targeting via CaMKII-dependent phosphorylation (Wang, 2011).

Two other studies address Hts function in neurons, one describing a role in photoreceptor axon guidance and the other characterizing pre-synaptic Hts function at the NMJ (Ohler, 2011; Pielage, 2011). The latter study reported a phenotype of synaptic retraction at the hts mutant NMJ, revealed by the absence of the pre-synaptic marker Bruchpilot from extensive stretches of the NMJ, which is consistent with the observation of small synapses in hts mutants stained for the synaptic vesicle marker CSP-2 (Pielage, 2011). However, in addition to retraction, this group observed overgrowth of the hts mutant NMJ, visible with anti-HRP staining. Although no quantification was performed using anti-HRP, qualitative examination of hts mutant muscle 6/7 NMJs in the current study did not indicate an overgrowth phenotype. A possible explanation for this discrepancy is the choice of the muscle 6/7 NMJ for analysis in contrast to Pielage, who focused on muscle 4. Unlike most other larval muscles, muscles 6 and 7 are not innervated by type II boutons. A component of the synaptic overgrowth reported by Pielage is the extension of thin, actin-rich extensions somewhat similar in appearance to type II and type III processes and, accordingly, growth of these processes is impaired with pre-synaptic over-expression of Hts (Pielage, 2011). Pielage proposes that pre-synaptic Hts restricts synaptic growth through its function as an actin-capping protein. At the muscle 6/7 NMJ this role may not be so prevalent and the post-synaptic growth-promoting function of Hts prevails. By examining NMJs other than muscle 6/7 in hts mutant larvae it was possible to confirm the overgrowth phenotype observed by Pielage (Wang, 2011).

Synaptic plasticity at the Drosophila NMJ has parallels with the structural changes seen at synapses in cellular models of learning such as long-term facilitation (LTF) in Aplysia and long-term potentiation in the mammalian hippocampus, and several studies indicate that the current results with hts at the NMJ may be relevant to these other systems. Phosphorylation of the conserved serine residue in the MARCKS domain of adducin is increased during LTF in Aplysia, and mice lacking β-adducin exhibit impaired synaptic plasticity and learning (Bednarek, 2011; Gruenbaum, 2003; Porro, 2010; Rabenstein, 2005; Ruediger, 2011; Wang, 2011 and references therein).

Similar to the plasticity exhibited by the NMJ during development, the morphogenesis of epithelia involves what has been referred to as 'epithelial plasticity' in which cell-cell adhesions are disassembled and cells become motile, for example in epithelial-mesenchymal transitions. The acquisition of motility by epithelial cells is also involved in tumor cell invasion and metastasis. The Drosophila follicular epithelium is a model for studying both developmental and pathological epithelial plasticity, and recent observations indicate that Dlg and Fas2 collaborate to prevent inappropriate invasion of follicle cells between neighboring germ cells. Furthermore, border cell migration, an invasion of a subset of follicle cells between germ cells occurring during normal oogenesis, requires downregulation of Fas2 expression in the border cells. PAR-1 is required for detachment of border cells from the follicular epithelium and it is interesting to speculate that this might involve regulation of Dlg localization. These various results indicate that regulation of the Dlg/Fas2 complex is important for the epithelial plasticity exhibited by follicle cells and this mechanism likely applies to epithelia in the developing embryo. As in muscle, Hts over-expression causes elevated levels of PAR-1 and CaMKII in epithelial cells. While Hts over-expression does not cause discernible Dlg delocalization in the epidermis, it disrupts the membrane localization of Dlg in amnioserosa cells. Dlg in the amnioserosa might be particularly susceptible to Hts function, as it not incorporated into septate junctions in this tissue, unlike in the epidermis. Furthermore, if the delocalization of Dlg by Hts is CaMKII-dependent, it would be more pronounced in the amnioserosa than the epidermis as Hts can only weakly promote CaMKII accumulation in the epidermis. In addition to showing that Hts can regulate Dlg localization in the amnioserosa, this study has determined that proper cortical localization of Hts in amnioserosa cells in the early stages of dorsal closure is dependent on Dlg. This result suggests that where Dlg and Hts are together in a complex, Dlg stabilizes the membrane localization of Hts. This stabilization may occur in cells other than the amnioserosa but might not be readily detectable. Hts localizes all around the epithelial membrane, with much of it not co-localizing with Dlg. In the very flat, squamous cells of the amnioserosa early in dorsal closure, the proportion of lateral membrane Hts dependent on Dlg for stabilization may be greater than in more columnar epithelial cells such as those in the epidermis (Wang, 2011).

Consistent with the effects that they have on each other's localization, the frequency of cuticle defects seen in dlg hts zygotic double mutant embryos suggests that Hts and Dlg co-operate in epithelial development, and it is of interest that mammalian adducin has recently been implicated in the stabilization and remodeling of epithelial junctions (Naydenov, 2010). Embryos deficient in Hts likely have a diminished ability to delocalize Dlg, effectively reducing the pool of Dlg available for de novo junction formation, and this situation would be worsened by reducing Dlg levels with a dlg allele. Conversely, reducing Dlg in an embryo deficient in Hts may further compromise Hts function through effects on Hts membrane localization. One of the phenotypes seen in hts mutants, the frequency of which is increased by additionally reducing Dlg, is that of embryos which secrete only small pieces of cuticle. This phenotype is characteristic of maternal zygotic dlg mutant embryos (Wang, 2011).

The interaction of Hts with Dlg suggests that adducin could be a regulator of the septate junction and it will be of interest to examine adducin effects on septate junction morphology in the nervous system. In the Drosophila nervous system septate junctions are formed between glial cells, whereas in mammals they are found between glial and neuronal membranes at the paranodal junction. Interestingly, β-adducin was recently identified as a paranodal junction protein localized to the neuronal membrane where it co-localizes with NCP1, the vertebrate homolog of the Drosophila septate junction protein Neurexin IV (Ogawa and Rasband, 2009) (Wang, 2011).

Hts, the Drosophila homologue of adducin, physically interacts with the transmembrane receptor golden goal to guide photoreceptor axons

Neurons steer their axons towards their proper targets during development. Molecularly, a number of guidance receptors have been identified. The transmembrane protein Golden Goal (Gogo) was reported previously to guide photoreceptor (R) axons in the Drosophila visual system. This study shows that Hts, the Drosophila homologue of Adducin, physically interacts with Gogo's cytoplasmic domain via its head-neck domain. hts null mutants show similar defects in R axon guidance as do gogo mutants. Rescue experiments suggest that the C-terminal tail but not the MARCKS homology domain of Hts is required. Overexpression of either gogo or hts causes abnormally thick swellings of R8 axons in the medulla, but if both are co-overexpressed, R8 axons appear normal and the amount of excessive Hts is reduced. The results fit with a model where Gogo both positively and negatively regulates Hts that affects the Actin-Spectrin cytoskeleton in growth cone filopodia, thereby guiding R axons (Ohler, 2011).

This study show that the axon guidance receptor Gogo physically interacts with the cytoskeletal protein Hts. The loss-of-function phenotypes of hts and gogo mutants are qualitatively very similar, albeit gogo null mutants show a phenotype slightly more severe than the htsnull mutant. This suggests that Gogo and Hts collaborate in a functional complex to guide R7 and R8 axons to their correct targets in the medulla (Ohler, 2011).

However, evidence is also shown for an antagonistic interaction between Hts and Gogo. Strong overexpression of Gogo causes abnormally thick swellings of R8 axons at layers M1 and M3. Strong overexpression of Add1 causes a different but similar phenotype leading to abnormal swellings that are restricted to layer M1. If both Gogo and Add1 are overexpressed, no abnormally thick swellings occur and R8 axons do not look different from wild type R8 axons. Moreover, in flies lacking one copy of the hts locus, the effect of excessive Gogo is enhanced. This indicates that Hts and Gogo antagonize each other and need to be in balance for the correct formation of axons. Direct evidence for an antagonistic interaction between Gogo and Hts comes from the observation that an increase in axonal Gogo protein level reduces the amount of Add1 protein in the axon. Moreover, the fact that the Add1 protein level is regulated by Gogo strongly suggests that gogo acts upstream of hts (Ohler, 2011).

How can these superficially conflicting observations be explained and reconciled? Below, a hypothetical model is discussed that explains how the Gogo-Hts complex could function to guide R axons:
Axons find their correct targets by means of the growth cone that is equipped with guidance receptors reading guidance cues provided by the growth cone's environment. The growth cone translates this guidance information into rearrangements of its cytoskeleton, which leads to directed growth of the axon. The two main components of the growth cone cytoskeleton are F-Actin (appearing as filopodia or lamellipodia) and microtubules. Within a filopodium, F-Actin is organized as parallel bundles, which requires the action of F-Actin bundling proteins like α-actinin. The barbed ends of the F-Actin bundles point distally, so that F-Actin assembly takes place at the very tip of the filopodium. This produces a force on the F-Actin bundle that moves the bundle rearwards (retrograde flow) and a force on the plasma membrane that extends the filopodium. F-Actin capping proteins influence the rate of Actin assembly, whereas the rate of retrograde F-Actin flow has been suggested to be regulated by a 'clutch' that links the cytoskeleton via transmembrane proteins to the extracellular matrix and thereby countervails the retrograde F-Actin flow (Ohler, 2011).

Adducin bundles Actin filaments and caps the barbed Actin filament ends in vitro. Assuming that its Drosophila homologue Hts serves the same molecular functions, Hts is an attractive candidate for a protein that is involved in the proper organization of filopodial F-Actin during axon guidance. To see the effect of Add1 on axonal F-Actin, medullae of flies overexpressing either Add1 or Gogo were stained with phalloidin. Compared to the control flies that have only the GMR-Gal4 driver, but no UAS-target, F-Actin seems to be stabilized in axons that feature an excessive amount of Add1 or a reduced amount of Add1 caused by excessive Gogo. In both cases, R7/R8 axons in the medulla are stained more clearly than in the control. This indicates that Add1 indeed affects the F-Actin in R axons in some way. Due to some analogies to the L1-Ankyrin system, which has been shown to function as a molecular clutch, especially a possible involvement in the regulation of retrograde F-Actin flow immediately comes to mind (Ohler, 2011).

Like Adducin, the peripheral membrane protein Ankyrin is a part of the Actin-Spectrin cytoskeleton. Ankyrin binds to the cell adhesion molecule L1, especially when L1 is homophilically bound to another L1 molecule in trans. The physical link of Ankyrin via L1 to the substratum exerts a pulling force on the filopodial F-Actin during the outgrowth of neurites, which is dependent on the binding of Ankyrin to Spectrin (Ohler, 2011).

The similar loss-of-function phenotypes of hts and Spectrin mutants indicate an intimate link between Hts and Spectrin. It is proposed that Hts links the filopodial F-Actin in a Spectrin-dependent manner via Gogo or another transmembrane protein to the substratum, which inhibits retrograde Actin flow and lets the growth cone steer straight forward towards the side where Hts is activ. When Gogo is bound to its as yet unidentified ligand, it could act as a repulsive receptor and remove Hts from the lateral filopodia, thereby assuring the proper spacing of single R8 axons. This fits with the suggested R8-R8 repulsion mediated by Gogo. Additionally, Gogo has been shown to have another adhesive function, which fits with the proposed adhesive function of Hts mediated by Gogo. This could also explain the thickening of R8 axons caused by excessive Hts, as the result of an excessive anchoring of the growth cone (Ohler, 2011).

Moreover, like Adducin, Hts may also serve as an Actin-capping protein and the abnormal swellings caused by an excess of Gogo may be the consequence of increased Actin polymerization due to an abnormally low level of Hts. When both Hts and Gogo are overexpressed, abnormally thick swellings are not observed. Although the anchoring of R8 growth cones should be abnormally strong in this situation, the Hts-antagonizing function of Gogo could also be increased and counteract this elevated adhesive force (Ohler, 2011).

An interesting finding from this work is that the MARCKS domain seems not to be required for the functions of Hts during axon guidance. Expression of both Add1, the Hts isoform including the MARCKS domain that is most closely related to mammalian Adducin, and of isoform HtsPD, an isoform lacking the MARCKS domain, in Rs rescues the defective phenotype caused by the loss of hts. This is consistent with the observation that homozygous htsΔG mutant flies, which have only truncated Hts protein lacking the MARCKS-related domain, do not show defects in the medulla. Both were surprising, since the MARCKS-related domain of Adducin is required for its activity in promoting association of Spectrin with Actin filaments as well as the Actin capping and Actin binding activity of Adducin in vitro. How can it be that the MARCKS domain is not required for the function of Hts during axon guidance, although the MARCKS domain has been shown to be required for the in vitro functions of Adducin including Actin binding, Actin capping, and Spectrin recruiting (Ohler, 2011)?

A possible explanation is that the function of Hts during axon guidance is indeed independent of Actin and Spectrin and that Hts serves a completely novel function here. However, for several reasons it is thought more likely that the Drosophila Hts can interact with Actin and Spectrin as the mammalian Adducin does, but does not strictly require its MARCKS domain in order to do so. There are several reasons for this assumption:
First, in the Drosophila germ line the Hts isoforms ShAdd and Ovhts are exclusively expressed, both lacking the MARCKS domain. Nevertheless, in hts mutants, the fusome, a Spectrin-based cytoskeletal structure in the germarium, is disorganized. This indicates that these MARCKS domain lacking proteins are required for the proper assembly of the Spectrin cytoskeleton (Ohler, 2011).

Second, again the similar phenotype of hts and Spectrin mutants suggest that Hts and Spectrin are functionally linked during R axon guidance. Since the hts mutant phenotype can be rescued by Hts lacking MARCKS, this indicates that the interaction with Spectrin does not require the MARCKS domain (Ohler, 2011).

Third, it has not been shown directly that mammalian Adducin binds Spectrin via its MARCKS domain. The MARCKS domain is the target of many regulatory processes. It contains phosphorylation sites for PKA and PKC and it binds calmodulin in a Ca2+-dependent manner. The MARCKS domain could function merely in a regulatory manner, regulating the binding of Spectrin to another part of Adducin. Indeed, the neck domain is also required for Adducin binding to Spectrin and may, therefore, contain the actual binding site (Ohler, 2011).

Although the MARCKS-related domain is dispensable for the function of Hts in R axon guidance, some part of the remaining tail domain seems to be essential, as ShAdd, the Hts isoform that does not contain the tail domain, fails to rescue flies from the defects caused by hts mutations. Moreover, ShAdd can not be detected in R axons in the medulla. It was not ascertain if this absence is due to reduced translation, degradation, impaired transport to, or efficient removal from the axon. Since ShAdd appears to be expressed and stable in Schneider cells and larval eye-brain complexes, the idea is favored that the tail domain is required for the localization of Hts to the axon. In any case, the absence of ShAdd protein from the axon could account for the failure of ShAdd to rescue the defects caused by hts (Ohler, 2011).

This work has demonstrated that the axon guidance receptor Gogo physically interacts with the cytoskeletal protein Hts to guide Drosophila R axons to their correct target in the medulla. Although there are some indications for a synergistic interaction, it was shown that Hts and Gogo also antagonize each other. This points to a highly sophisticated mechanism of the Gogo-Hts complex. Further work will be required to dissect the different aspects of this intricate complex and will shed light not only on its role in Drosophila R axon guidance, but also on the role of Hts/Adducin and, in succession, the Spectrin cytoskeleton in other systems (Ohler, 2011).

Distinct roles for hu li tai shao and swallow in cytoskeletal organization during Drosophila oogenesis

Cytoskeletal organization is essential for localization of developmentally significant molecules during Drosophila oogenesis. Swallow (Swa) and an isoform of Hu li tai shao (Ovhts-RC) have been implicated in the organization of actin filaments in developing oocytes but their precise roles have been obscured by the dependence of hts RNA localization on swa function. The functional significance of hts RNA localization in the oocyte has not been established. This study examined Ovhts-RC distribution and cytoskeletal organization under conditions in which Swa protein and/or hts RNA localization are perturbed. Swa was found to be required for overall actin organization and for the maintenance of a distinct subset of microtubules in the oocyte. hts RNA localization modulates the distribution of Ovhts-RC in the oocyte and in turn, local actin filament proliferation. These results support separate contributions of Swa and hts RNA localization to actin organization during oogenesis. Swa is crucial for the organization of actin networks that lead to the formation of a specialized microtubule population, while Ovhts-RC acts to modulate spatially restricted actin filament growth at the oocyte cortex. This suggests RNA localization can lead to modifications of both the actin and microtubule cytoskeletons at specific subcellular locales (Pokrywka, 2014).

Proper regulation of the cytoskeleton is critical for successful oogenesis in Drosophila, and the cytoskeleton undergoes a number of stage-specific changes during this multiday process. This study investigated the relative contributions of two proteins, Swa and Ovhts-RC, to cytoskeletal organization during mid-oogenesis. Available evidence suggests that Swa participates in RNA localization indirectly, most likely by regulating some features of the actin and microtubule cytoskeletons. Ovhts-RC is a component of actin-rich ring canals but no other role for it in oogenesis has been reported. This study presents evidence that both Swallow and Ovhts-RC modulate cytoskeletal organization in the oocyte during late oogenesis (Pokrywka, 2014).

hts is the Drosophila homolog of adducin, and its germline RNA isoform, htsN4, is localized to the anterior cortex of the oocyte. The htsN4 transcript produces two distinct proteins, termed Ovhts-fus and Ovhts-RC, but only Ovhts-RC is present during egg chamber development. Previous reports have identified Ovhts- RC as an actin-binding protein that is a major component of ring canals (Pokrywka, 2014).

Analysis of Ovhts-RC protein distribution in oocytes suggests that the localization of htsN4 RNA has functional relevance for oocyte organization. Ovhts- RC protein was found to be present in the oocyte during late oogenesis where its distribution mirrors the density of actin filaments. Ovhts-RC is found in a shallow but reproducible gradient, with highest concentrations found at the anterior cortex and much lower concentrations associated with the posterior cortex. Overall, the amount of Ovhts-RC correlates with the extent of actin filaments at a given location. This gradient is dependent on microtubules and Swa protein, and is abolished when hts RNA localization is defective, consistent with the idea that htsN4 RNA localization underlies the distribution of Ovhts-RC protein. It is proposed that htsN4 RNA localization leads to higher amounts of Hts-RC protein at the anterior pole, and this shapes the gradient of cortical actin seen in egg chambers during mid-oogenesis, where actin filaments are longer and more densely packed at the anterior cortex of the oocyte (Pokrywka, 2014).

Ovhts-RC is found in the nurse cells as well, and although it is actin associated in both cell types, it is concentrated primarily at the pointed ends of actin filaments. In nurse cells, particularly high concentrations of Ovhts-RC surround the nurse cell nuclei, and similar observations have recently been reported in another study. Speculations about the function of Ovhts-RC are difficult despite the observation that hts is the Drosophila homolog of adducin, a well-characterized actin-binding protein. Drosophila hts encodes a variety of isoforms, but the Ovhts-RC isoform is a novel protein with no similarities to other adducins. Thus, Ovhts-RC is a novel actin-binding protein with a preference for the barbed ends of actin filaments (Pokrywka, 2014).

This study has produced new evidence that swa mutations are associated with defects in several microtubule-dependent processes; in addition to its previously reported role in RNA localization, swa mutations affect oocyte nucleus positioning and microtubule organization during stage 10- 12 of oogenesis, but do not affect ooplasmic streaming. Others have implicated a group of γ- TuRC components (γTub37C, Dgrip75, and Dgrip128) in the formation of anterior/lateral microtubules required for RNA localization. The anterior/lateral microtubule defects this study sees in swa mutants are similar to those reported for mutations in Dgrip128, and it is proposed that anterior/lateral microtubules are required for positioning of the oocyte nucleus. Previous study have not analyzed oocyte nuclear positioning in their mutants, and this study looked for nuclear positioning defects in γTub37C mutants and found that slightly more than half (22 of 40) late stage oocytes had a mis-positioned oocyte nucleus. Overall then, the phenotypes caused by mutations in swa and γTub37C are consistent, suggesting a role for swa in regulation or organization of anterior/lateral microtubules associated with γTub37C, and by association Dgrip75 and Dgrip128. Among this group of genes, only swa mutations result in ectopic F-actin aggregates, and so it is proposed that swa influences these specialized γ-TuRCs by participating in actin organization during mid-oogenesis, at a step that is upstream of the aforementioned MTOC components. Support for this model also comes from work by a previous study show that swa is membrane associated, and that cytochalasin D treatment can phenocopy swa effects on bcd RNA localization (Pokrywka, 2014).

In recent years, evidence for the interplay between actin and microtubules in the oocyte has been steadily mounting, and the interaction between actin and microtubules in the oocyte is likely to be complex. Indeed, this study found that in addition to swa mutations, a variety of conditions that interfere with microtubule organization also give rise to actin defects at low frequencies, highlighting the interplay of actin and microtubules during stages 10-12. These actin defects share similarities with those seen in swa mutants, and under these conditions, the distribution of Swa protein is also disrupted. Thus, at least some of the actin defects arising from microtubule disruption may be due to a loss of Swa protein function. The picture becomes still more complex when it is considered that microtubule disruptors also interfere with proper localization of htsN4 RNA, meaning that these treatments potentially represent actin defects stemming from a combination of Swa protein malfunction and htsN4 RNA mis-localization (Pokrywka, 2014).

Both swa and hts appear to influence actin organization in oocytes, and in this project conditions were devised in which the actions of swa and hts were genetically separable. γTub37C mutants have failures in htsN4 RNA localization and allowed altering of the Ovhts- RC protein gradient in the presence of normal Swa function. In these flies, a marked increase is seen in the proliferation and length of cortical actin filaments at all positions along the anterior-posterior axis. These alterations in actin filaments are distinct from the phenotype associated with swa mutations or microtubule disruption. A similar actin overgrowth phenotype was generated in flies expressing the htsΔ100 construct, whose modified htsN4 RNA is not properly localized. Overall this suggests that higher levels of hts RNA and protein lead to increased growth of actin filaments, perhaps via modification of the pointed ends of actin filaments. In conditions where hts RNA is not localized, locally higher than normal levels of Ovhts-RC protein result in longer, more abundant actin filaments. Further support for this conclusion comes from flies that are expressing two wild type copies of hts and two copies of htsΔ100. In these oocytes, very long actin filaments are observed along the anterior cortex, where combined levels of both types of hts RNA are extremely high (Pokrywka, 2014).

Neither γTub37C mutants nor flies expressing the htsΔ100 construct exhibit ectopic actin structures in the oocyte. This is in stark contrast with swa mutants and to a lesser extent msps mutants and colchicine-fed flies. A reasonable explanation for the ectopic actin phenotype is that it is a direct result of defects in Swa protein function, since all the aforementioned conditions interfere with Swa protein localization. Unfortunately, it is not possible to generate oocytes in which swa is non-functional but htsN4 RNA localization is wild type, and so the action of swa can only be inferred indirectly. It is noted that the actin overgrowth phenotype is absent in swa mutants, and propose swa is required for a step upstream of hts function (Pokrywka, 2014).

It is possible that ectopic actin aggregates and spheres seen in swa mutants are the result of excessive actin polymerization that is not membrane-associated. In this scenario, actin overgrowth occurs but the resulting structures are not anchored at the cortex (possibly due to a loss of swa function), and so form aggregates and spheres in deeper ooplasm that also contain Ovhts-RC. A number of protein candidates were investiged to test the possibility that the actin spheres in swa mutants contain membranes. The spheres do not stain with common Drosophila membrane markers such as calmodulin, syntaxin, or Gilgamesh, or with membrane dyes such as FM1-46, or with lectins such as WGA. Thus it is unlikely they represent vesicles. The actin spheres do stain with antibodies to Enabled, indicating they are composed of multiple short actin filaments (Pokrywka, 2014).

Why then do actin defects in swa mutants commonly manifest as spheres? One possibility is that Ovhts-RC is involved in the formation of cage-like actin structures, or interactions between obliquely-oriented actin filaments. A number of observations are consistent with this idea. First, Ovhts-RC is an essential component of circular ring canals, and may contribute to their unusual arrangement of actin filaments. Ovhts-RC also associates with nurse cell nuclei as filamentous structures that are oriented obliquely to actin cables but parallel to the surface of the nucleus, forming a cage around the nuclei. Two populations of actin filaments in nurse cells have been proposed: the actin cables extending from the cell periphery, and a second group of actin filaments that surround the nucleus. The data is consistent with a role for Ovhts- RC in mediating interactions between these two groups. Moreover, in swa mutants the appearance of actin spheres beginning at stage 10, as well as their abrupt disappearance at the end of stage 12 mirrors the timeline of hts RNA defects and Ovhts-RC association with actin in oocytes. Similar structures have been described in an analysis of Bicaudal C and trailer hitch mutants. In these mutants both the oocyte cytoskeleton and hts RNA localization are disrupted at earlier stages of oogenesis. The timing of the hts RNA localization defects in Bicaudal C and trailer hitch mutants corresponds to the appearance of actin cages that also contain Ovhts-RC. Finally, a recent report describes actin rings in neurons that are associated with adducin. These rings are likely to be composed of short, capped actin filaments. Available evidence indicates the actin spheres in swa mutants are also composed of multiple short filaments, based on their association with both Enabled and Ovhts-RC. Experiments to further probe the relationship of Ovhts-RC with actin organization are currently underway (Pokrywka, 2014).

In summary, both swa and hts are involved in shaping the three dimensional architecture of the cytoskeleton. The data are consistent with a model in which Swa acts to organize actin filaments at the anterior cortex, leading to the establishment of anterior/lateral microtubules that are utilized to localize RNAs and the oocyte nucleus during stage 10-12 of oogenesis. Ovhts-RC contributes to actin filament growth and the polarization of the oocyte cortical actin cytoskeleton along the anterior-posterior axis, possibly via interactions with the pointed end of actin filaments (Pokrywka, 2014).

Phospho-regulated Drosophila adducin is a determinant of synaptic plasticity in a complex with Dlg and PIP2 at the larval neuromuscular junction

Adducin is a ubiquitously expressed actin- and spectrin-binding protein involved in cytoskeleton organization, and is regulated through phosphorylation of the myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain by Protein kinase C (PKC). The Drosophila adducin, Hu-li tai shao (Hts), has been shown to play a role in larval neuromuscular junction (NMJ) growth. This study finds that the predominant isoforms of Hts at the NMJ contain the MARCKS-homology domain, which is important for interactions with Discs large (Dlg) and phosphatidylinositol 4,5-bisphosphate (PIP2). Through the use of Proximity Ligation Assay (PLA), this study shows that the adducin-like Hts isoforms are in complexes with Dlg and PIP2 at the NMJ. Evidence is provided that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle. It was also shown that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain. These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila (Wang, 2014: PubMed ID).

The Drosophila neuromuscular junction (NMJ) is the site of contact between motor neuron and muscle, and is stably maintained but remodelled during the growth and development of the fly. To permit these differing functions, the NMJ uses an actin- and spectrin-based cytoskeleton both pre- and post-synaptically, where a number of synaptic proteins modify the cytoskeleton dynamically. One such protein involved in the dynamic responses of the synapse to stimuli in vertebrates is the actin- and spectrin-binding protein adducin, a heteromeric protein composed of α, β and γ subunits that is widely expressed in many cell types including neurons and myocytes. The adducins are composed of a globular N-terminal head domain, a neck domain and a C-terminal myristoylated alanine-rich C-terminal kinase (MARCKS)-homology domain containing an RTPS-serine residue which is a major phosphorylation site for protein kinase C (PKC), as well as cAMP-dependent protein kinase (PKA). Phosphorylation of adducin in the MARCKS-homology domain inhibits adducin-mediated promotion of actin-spectrin interactions, resulting in cytoskeletal reorganization (Wang, 2014).

Multiple studies have demonstrated that the mammalian MARCKS protein, or more specifically its MARCKS effector domain, can bind to and sequester the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), in artificial lipid vesicles. This interaction has been linked to the regulation of the actin cytoskeleton during the growth and branching of dendrites in rat brains, as well as the directed migration of bovine aortic endothelial cells in wound healing assays. Notably, it has been proposed that aberrant MARCKS regulation of PIP2 signaling may be implicated in the formation of amyloid plaques in Alzheimer's disease. A recent study has also provided evidence that reduced hippocampal levels of MARCKS, and thus PIP2, in mice contributes to age-related cognitive loss (Wang, 2014).

MARCKS binds to PIP2 as the MARCKS effector domain carries basic residue clusters that can interact with acidic lipids in the inner leaflet of the cell membrane. By analogy to other MARCKS-homology domain-containing proteins, it is hypothesized that phosphorylation of adducin at the RTPS-serine may alter the electrostatic interaction between adducin and phosphoinositides, thus reversing the binding between them and causing translocation of adducin from the membrane to the cytosol. In this way, adducin might act as a molecular switch in its regulation of synaptic plasticity, with its localization at the synapse controlled by phosphorylation (Wang, 2014).

In Drosophila, orthologs of adducin are encoded by the hu-li tai shao (hts) locus, and the Hts protein is present at both the pre- and post-synaptic sides of the larval NMJ where it regulates synaptic development. Previous studies have shown that Hts interacts with the scaffolding protein Discs large (Dlg), and regulates Dlg localization at the postsynaptic membrane by promoting its phosphorylation through Partitioning-defective 1 (PAR-1) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), two known regulators of Dlg postsynaptic targeting. Dlg is an important regulator of synaptic plasticity, and likely constitutes a major route by which Hts controls NMJ development. This study found that the main isoforms of Hts at the NMJ are the MARCKS-homology domain-containing isoforms, Add1 and/or Add2. There, the adducin-like isoforms form complexes with Dlg and PIP2, interactions that were identified through Proximity Ligation Assay (PLA). Evidence is provided that Hts promotes the phosphorylation, and thus delocalization, of Dlg at the postsynaptic membrane by regulating the re-distribution of par-1 and camkII transcripts from the muscle nucleus to the cytoplasm. It was also shown that these Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain, further establishing that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila (Wang, 2014).

Through the use of PLA, this study has shown that Hts forms complexes with Dlg and PIP2 at the postsynaptic region of the larval NMJ, with its ability to associate with these proteins being negatively regulated through phosphorylation of the MARCKS-homology domain. Studies on mammalian adducin have demonstrated that phosphorylation of the MARCKS-homology domain impedes its actin-binding and spectrin-recruiting functions, reduces its affinity for these cytoskeletal components and the membrane, and targets it for proteolysis. It is proposed that phosphorylation of the MARCKS-homology domain in the Add1/Add2 isoforms of Hts in response to upstream signaling events at the synapse reduces their affinity for spectrin-actin junctions and Dlg at the NMJ, but may also hinder their interactions with PIP2 and other phosphoinositides in line with the electrostatic switch model for phosphoinositide binding by the MARCKS-homology domain (Wang, 2014).

It was proposed previously that Hts regulates Dlg localization at the NMJ by controlling the protein levels of PAR-1 and CaMKII, which phosphorylate Dlg and disrupt its postsynaptic targeting. This study now shows that regulation of these kinases appears to occur at the level of transcript processing, with Hts promoting the accumulation of par-1 and camkII transcripts in the muscle cytoplasm. Cytoplasmic accumulation of the transcripts would then presumably lead to higher levels of PAR-1 and CaMKII protein. How is Hts achieving this mode of regulation when it is residing with Dlg at the postsynaptic membrane? One possibility is that Hts at the NMJ is activating a signaling pathway that promotes the transcription and/or stability of par-1 and camkII transcripts, as well as their transport out of the nucleus. Another possibility is that Hts itself, which contains predicted NLS and NES sequences, translocates to the nucleus in response to events at the NMJ, similar to the way that mammalian α-adducin translocates to the nucleus upon loss of cell-cell adhesion in epithelia. This study was unable to detect endogenous Hts in muscle nuclei, however, nuclear Hts levels might be tightly restricted and undetectable under wild-type conditions. Over-expressed wild-type Hts, on the other hand, is readily observable in the nucleus, though not its phosphorylated form - a result also seen with α-adducin. Whatever the mechanism may be, the presence of Hts in a complex with Dlg may allow it to evaluate the status of Dlg and the synapse, and execute a response in the form of regulating Dlg localization through PAR-1 and CaMKII mediated phosphorylation (Wang, 2014).

A recent study has uncovered a novel nuclear envelope budding mechanism that can export select transcripts from muscle nuclei during larval NMJ development, and involves Lamin C (LamC) and the Wnt receptor, DFrizzled2 (DFzz2) (Speese, 2012). Interestingly, camkII, but not dlg, transcripts are regulated by this process, which is consistent with the findings that CaMKII, but not Dlg, expression is regulated by Hts. Future work will determine whether Hts is involved in this LamC/DFzz2-dependent mechanism (Wang, 2014).

Two papers have underscored the importance of phosphoinositides in synaptic development at the Drosophila NMJ (Forrest, 2013; Khuong, 2010). Binding of Hts to PIP2 and probably other phosphoinositides at the NMJ, as seen with other MARCKS-homology domain-containing proteins, may affect the availability of these lipids for processes such as signal transduction, thus affecting synaptic development. Conversely, the localization of Hts at the NMJ may be regulated by the distribution of phosphoinositides. In line with this, postsynaptic knockdown of the phosphoinositide phosphatase Sac1 via transgenic RNAi expression disrupts Hts localization at the NMJ (Wang, 2014).

The observations reported in this study may have important implications for understanding diseases that affect synaptic function in humans and other mammals. Many neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), a disorder characterized by the progressive loss of motor neurons, have been assumed until recently to be a consequence of neuronal death within the central nervous system. However, there is substantial recent evidence indicating that neuron pathology in ALS and other neurodegenerative diseases is due to a degenerative process that begins in the presynaptic terminal, NMJ or distal axon. This may also be the case in normal aging (Wang, 2014).

Initial interested in adducin arose when elevated levels of phospho-adducin protein was found in the spinal cord tissue of patients who died with ALS, compared to individuals who died without neurological disease. Similar observations were also made in mSOD-expressing mice, a transgenic animal model of ALS. Multiple studies have shown that adducin plays important roles in synaptic plasticity, and that mice mutant for β-adducin display defects in memory, learning and motor coordination. It is clear that modulation of Hts expression and phosphorylation can affect synaptic development. This study provides evidence here that phosphorylation of Hts impedes its function at the larval NMJ, a result that is consistent with the mammalian adducins. In addition, overexpression of phospho-mimetic Hts has dominant negative effects over endogenous Hts. Thus, loss of adducin function through aberrant phosphorylation of the MARCKS-homology domain may be a contributing factor for human neurodegenerative diseases (Wang, 2014).


Drosophila females bearing mutations in a previously undescribed gene, hu-li tai shao [(hts) too little nursing], produced egg chambers that contained fewer than the normal 15 nurse cells and that usually lacked an oocyte. The cytoplasmic bridges (ring canals) interconnecting nurse cells and the oocyte appeared abnormal, and lacked associated actin rings. The hts locus was found to encode a homolog of the mammalian membrane skeletal protein adducin. During oogenesis, hts mRNA became localized at the anterior of the oocyte and was subsequently expressed in a variety of embryonic tissues. These studies suggested that Drosophila adducin is needed to assemble actin at specialized regions of cell-cell contact in developing egg chambers and may also function at other times during the Drosophila life cycle (Yue, 1992; full text of article).

The structure of cytoplasmic bridges called ring canals were analyzed in Drosophila egg chambers. Two mutations, hu-li tai shao (hts) and kelch, disrupt normal ring canal development. Antibodies were raised against the carboxy-terminal tail of hts and it was found that they recognize a protein that localizes specifically to ring canals very early in ring canal assembly. Accumulation of filamentous actin on ring canals coincides with the appearance of the Hts protein. Kelch, which is localized to the ring canals hours after Hts and actin, is necessary for maintaining a highly ordered ring canal rim since kelch mutant egg chambers have ring canals that are obstructed by disordered actin and hts. Anti-phosphotyrosine antibodies immunostain ring canals beginning early in the germarium before Hts and actin and throughout egg chamber development. The use of antibody reagents to analyze the structure of wild-type and mutant ring canals has shown that ring canal development is a dynamic process of cytoskeletal protein assembly, possibly regulated by tyrosine phosphorylation of some ring canal components (Robinson, 1994; full text of article).

Adducin is a cytoskeletal protein that can function in vitro to bundle F-actin and to control the assembly of the F-actin/spectrin cytoskeletal network. The Drosophila Adducin-like (Add) locus (also referred to as hu-li tai shao) encodes a family of proteins of which several are homologous to mammalian adducin. Two novel adducin isoforms have been identified: a 95 x 10(3) Mr form (ADD-95) and an 87 x 10(3) Mr form (ADD-87). A detailed analysis of the distribution patterns of ADD-95 and ADD-87 during oogenesis and embryogenesis is presented. The isoforms are co-expressed in several cell- and tissue-types; however, only ADD-87 is present in mid- to late-stage oocytes. ADD-87 is present throughout the oocyte cortex at stages 9 and 10 of oogenesis but is detectable only at the anterior pole from stage 11 onward, correlated with localisation of Add-hts mRNA first to the cortex and then to the anterior pole of the oocyte. ADD-87 co-localises with F-actin and spectrin in the cortex of the oocyte through stage 10 of oogenesis, consistent with a possible role in cytoskeletal assembly or function predicted by mammalian studies (Zaccai, 1996a).

Because previous examination of Hts protein localization was done before there was a complete understanding of the hts locus, Hts antibody localization was characterized in more detail. Four antibodies directed against different Hts protein domains were used. htsF (Lin, 1994; Robinson, 1997) recognizes ShAdd, Add1 and Add2 (Add1/2) and Ovhts. In germaria, htsF antibody labels the fusome in the germline and plasma membranes in follicle cells. 1B1 antibody (Zaccai, 1996b), which recognizes Ovhts and Add1/2, has an identical germarium labeling pattern to htsF. htsM antibody, which recognizes only Add1/2, labeled follicle cell membranes and shows no labeling of the germline. In later-stage egg chambers, htsF, 1B1 and htsM antibodies continued to show specific labeling of lateral follicle cell membranes but no germline labeling. Consistent with RNA in-situ data (Whittaker, 1999), Ovhts is germline-specific and Add1/2 are follicle-cell-specific. As shadd mRNA is also exclusively found in the germline, and the htsF antibody only labels the fusome in the germline, ShAdd is likely a fusome component. Although both shadd and ovhts mRNAs are expressed in the germline throughout oogenesis (Whittaker, 1999), their protein products detected with 1B1 and htsF antibodies are only present in the germarium. This suggests that the proteins are either not translated or are not stable once egg chambers are formed (Petrella, 2007).

Because it was not possible able to make a useful peptide antibody specific for ShAdd, a ShAdd transgene was produced expressing ShAdd fused to Venus, a modified fluorescent protein (EYFP). When ShAdd::Ven was expressed with the ovarian tumor (otu) promoter in the germline of wild-type flies, it localized specifically to spectrosomes and fusomes. Since the fusome began to degrade in Region 2, the localization of ShAdd::Ven also became more dispersed. Thus, ShAdd::Ven provided additional evidence that ShAdd is a fusome protein (Petrella, 2007).

HtsRC antibody (Robinson, 1994), which recognizes the C-terminus of Ovhts, has a completely different localization pattern. Starting in Region 2a of germaria, htsRC labeled discrete puncta, which resolve into RCs in Region 2. htsRC antibody labels RCs throughout the rest of oogenesis (Petrella, 2007).

In order to verify the different labeling patterns of antibodies against the N- and C-termini of Ovhts, Ovhts transgenes were made that were expressed specifically in the germline. In separate constructs containing the native ovhts UTRs, the N-terminus of Ovhts was tagged with Cerulean, a modified ECFP, and the C-terminus was tagged with GFP. The Cer::Ovhts transgene did not produce a fluorescent product; however, upon labeling with anti-GFP antibodies, Cer::Ovhts was detected on the fusomes in germaria. Like the N-terminus of Ovhts as seen by antibody labeling, Cer::Ovhts localizes to both spectrosomes and branched fusomes. Co-staining with 1B1 showed that as the fusome begins to break down in Region 2, Cer::Ovhts becomes dispersed and looses colocalization with 1B1 (Petrella, 2007).

GFP fluorescence from Ovhts::GFP localizes specifically to RCs. As with htsRC antibody, protein is first detected in Region 2a as puncta that appear to be near, although not within the fusome. By Region 2b Ovhts::GFP is in rings. Ovhts::GFP is seen on RCs in all subsequent stages until stage 13. Thus, localization of tagged Ovhts transgenes recapitulates antibody labeling, with the N-terminus present on fusomes and the C-terminus localizing to RCs (Petrella, 2007).

Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis.

While Prostaglandins (PGs), lipid signals produced downstream of cyclooxygenase (COX) enzymes, regulate actin dynamics in cell culture and platelets, their roles during development are largely unknown. This study definee a new role for Pxt, the Drosophila COX-like enzyme, in regulating the actin cytoskeleton-temporal restriction of actin remodeling during oogenesis. PGs are required for actin filament bundle formation during stage 10B (S10B). Additionally, loss of Pxt results in early actin remodeling, including extensive actin filaments and aggregates, within the posterior nurse cells of stage 9 (S9) follicles; wild-type follicles exhibit similar structures at a low frequency. Hu li tai shao (Hts), the homolog of Adducin, and Villin (Quail), an actin bundler, localize to all early actin structures, while Enabled (Ena), an actin elongation factor, preferentially localizes to those in pxt mutants. Reduced Ena levels strongly suppress early actin remodeling in pxt mutants. Furthermore, loss of Pxt results in reduced Ena localization to the sites of bundle formation during S10B. Together these data lead to the model that PGs temporally regulate actin remodeling during Drosophila oogenesis by controlling Ena localization/activity, such that in S9, PG signaling inhibits, while at S10B, it promotes Ena-dependent actin remodeling (Spracklen, 2013).

Effects of Mutation or Deletion

hts mutation eliminate fusomes

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 have been identified as molecular components of fusomes, a related structure has been identified in germline stem cells, and regular associations between fusomes and cystocyte centrosomes have been documented. hts mutations completely eliminated fusomes, causing abnormal cysts containing a reduced number of cells to form. These results imply that Drosophila fusomes are required for ovarian cyst formation and suggest that membrane skeletal proteins regulate cystocyte divisions (Lin, 1994; full text of article).

Centrosome inheritance in the male germ line of Drosophila requires hu-li tai-shao function

Cytokinesis partitions a centrosome to each daughter cell at cell division that will duplicate and assemble a bipolar spindle in the subsequent M phase. Cytokinesis is incomplete in proliferating germ cells in Drosophila and cytoplasmic channels connect sibling germ cells. Although centrosomes are essential to male fertility, the molecular mechanism that retains centrosomes in parental germ cells is not known. Cortical cytoplasmic structures known as fusomes extend through ring canals and connect cells within the cyst. Fusome assembly in males requires function of hu-li tai-shao (hts), an adducin like protein found in fusomes and in the cortical membrane cytoskeleton of somatic cells. This work used immunological and cytological methods to place hts mutants in an allelic series. Male fertile hts mutants express Hts protein and generate apparently normal or fragmented fusomes. A male sterile allele does not express Hts protein or show fusome structures. Gonial cells in all hts mutants showed 2 centrosomes and mitotic spindles were bipolar. Yet, primary spermatocytes, with and without fusome structures, frequently contained too many or too few centrosomes. Although spindle structures were not found in spermatocytes without centrosomes, meiotic spermatocytes with centrosomes generated bipolar, monopolar, and multipolar spindles. Collectively, these results indicate that hts function is necessary for centrosome inheritance in spermatocytes as well as for male fertility (Wilson, 2005).

Characterization of new hts alleles

In order to elucidate the functions of the individual hts proteins, new alleles of hts were characterized. All previously described alleles of hts were P-element insertions or imprecise excisions that reduce expression of all hts transcripts. Two new EMS-induced hts alleles were examined. htsW532X contains a single nonsense mutation, W532X, in the tail domain. DNA sequencing of htsDeltaG showed a deletion of a single G in the last part of the Tail domain (G2346 of the ovhts transcript). This results in a frame shift followed by six novel amino acids and a stop codon. Conceptual translation of htsDeltaG results in a truncated protein that does not contain any of the normal C-terminal domains. These mutations are downstream of the entire ShAdd coding sequence (Petrella, 2007).

The phenotypes of these truncation alleles are indistinguishable from the P-element alleles. Both are female sterile and show a loss of oocyte specification, too few nurse cells, and no actin on RCs. However, labeling of htsDeltaG and htsW532X with Hts antibodies and western analysis showed a distinct difference between the alleles. Even though both truncation alleles should encode the epitope for the htsF antibody, protein was detected only in htsDeltaG. Western analysis showed that whereas htsDeltaG expressed a single truncation product, htsW532X produced no detectable protein and is therefore a null allele. Additionally in htsDeltaG, antibodies 1B1 and htsF labeled a cytoplasmic protein that persisted in egg chambers after they emerged from the germarium, which is never seen in wild type. Therefore, the mutant truncated protein is aberrantly stable in germline cells that normally do not have Ovhts-Fus. Mutant follicle cells labeled with 1B1 antibody show a significant, but not complete loss of Add1/2 localization to lateral membranes (Petrella, 2007).

To determine the functional requirements of the different Hts proteins in the germline, tagged hts transgenes expressed from the otu promoter were crossed into both htsDeltaG and htsW532X mutant backgrounds for rescue experiments. Both P{Ovhts::GFP} and P{Cer::Ovhts} rescued recruitment of Ovhts-RC and actin on RCs. However, other hts phenotypes were not rescued. Labeling with htsF, 1B1 or alpha-spectrin antibodies showed no fusome-like structure. Anti-GFP labeling in mutants expressing P{Cer::Ovhts} showed only cytoplasmic labeling. Additionally, the egg chambers still have too few cells and degenerate. Whether the addition of ShAdd would improve rescuing activity was tested. When P{ShAdd-Ven} is expressed alone in htsDeltaG, Venus fluorescence was diffuse in the cytoplasm, and hts phenotypes were not rescued. Expression of P{ShAdd::Ven} with either P{Cer::Ovhts} or P{Ovhts::GFP} in a htsDeltaG background showed the same phenotype as the single rescue: only RCs were rescued, but not the fusome or any of the phenotypes resulting from the loss of the fusome (Petrella, 2007).

Recent work has shown that the fusome precursor, the spectrosome, first begins to form during stage 11 of embryogenesis (Wawersik, 2005). Since the fusome develops from the spectrosome, it is possible that the otu promoter does not provide Ovhts at an early enough stage. However, earlier expression of Ovhts by driving P{UASH-ovhts::GFP} with either P{nos-GAL4} or P{tub-GAL4} produces the same result as the otu promoter (Petrella, 2007).

To determine whether there is a somatic contribution to the hts phenotype that results in the inability to rescue the fusome, clonal analysis was performed with htsDeltaG. Germline clones showed the same phenotype as homozygous mutants, whereas egg chambers that had only follicle cell clones were normal. Therefore, the loss of the fusome and RCs is caused solely by the loss of full-length Ovhts in the germline (Petrella, 2007).

During the rescue experiments with P{Ovhts::GFP}, it was noticed that localization of Ovhts-RC::GFP to RCs was delayed. In wild-type flies expressing this transgene, GFP is always present in Region 2 of germaria. In contrast, in either htsDeltaG or htsW532X flies expressing P{Ovhts::GFP}, GFP is often absent from germaria and only appears later. Quantitation of this phenotype revealed that in htsW532X there is a delay in 30% of germaria, and in htsDeltaG there is a delay in 91% of germaria. Thus, in hts mutants the recruitment of Ovhts-RC and actin, and therefore the establishment of RCs, does not occur at the correct developmental stage, suggesting that the fusome is necessary for the timing of RC development (Petrella, 2007).

Advantage of the ability of S2 cells to cleave Ovhts to identify amino acids necessary for its cleavage. A series of small in-frame deletion mutations were made in the region of the predicted cleavage site, and expressed in S2 cells. The Delta1 deletion, which removes 20 amino acids in the Tail domain, is cleaved at a wild-type level. Deletion Delta2 removed the last 20 amino acids of the Tail domain, and Delta3 removed the last nine amino acids of the Tail and the first 11 amino acids of the RC domain. The Delta2 protein was cleaved, although less efficiently than wild-type protein, whereas the Delta3 protein was not cleaved. This result demonstrates that the first 11 amino acids (ALVSQLAQKYA) of the RC domain are required for cleavage (Petrella, 2007).

To study the effect of uncleavable Ovhts in the ovary, a P{Ovhts-Delta3::GFP} transgene was made that was expressed from the otu promoter. Except for the 20 amino acid deletion, this transgene was identical to the P{Ovhts::GFP} transgene. Western analysis of Ovhts-Delta3::GFP from ovary extracts demonstrated that, as in S2 cells, this protein is not cleaved. When expressed in wild-type flies, Ovhts-Delta3::GFP was present not only on RCs, but also on the fusome. Additionally, the 1B1 antibody, which normally only labels the fusome, now also labeled RCs. Therefore, the uncleaved protein localized to both structures where the cleavage products are normally found (Petrella, 2007).

Although wild-type flies expressing Ovhts-Delta3::GFP are fertile and produce apparently normal egg chambers, uncleaved Ovhts does cause a subtle, but completely penetrant dominant defect in the disappearance of the fusome. In wild-type germaria, the fusome begins to disappear where the RCs are starting to form. This results in unobstructed RCs with fragmented fusome material between them, but not through them. In flies expressing P{Ovhts-Delta3::GFP}, fusome material is present within the RCs as late as stage 2 and 3 egg chambers. In some cases, GFP-positive rings are occluded with GFP-negative fusome material that can be visualized with 1B1 antibody. Therefore, at least some of the aberrant fusome contains only wild-type Ovhts-Fus protein and not the N-terminal portion of P{Ovhts- Delta3::GFP}. Additionally, RC rims were thicker than normal, less organized and misshapen. These results suggest that the cleavage and proper maintenance of the Ovhts domains may play a role in the transition from a fusome to RCs (Petrella, 2007).

Whether the P{Ovhts-Delta3::GFP} transgene could rescue hts mutants, htsDeltaG and htsW532X, was also tested. As with expression of P{Ovhts::GFP}, P{Ovhts-Delta3::GFP} rescues RCs but not the fusome. The rescued RCs were also labeled with 1B1 antibody indicating that the N-terminus of Ovhts was present (Petrella, 2007).

β-Adducin is required for stable assembly of new synapses and improved memory upon environmental enrichment

Learning is correlated with the assembly of new synapses, but the roles of synaptogenesis processes in memory are poorly understood. This study shows that mice lacking β-Adducin fail to assemble new synapses upon enhanced plasticity and exhibit diminished long-term hippocampal memory upon environmental enrichment. Enrichment enhanced the disassembly and assembly of dynamic subpopulations of synapses. Upon enrichment, stable assembly of new synapses depends on the presence of β-Adducin, disassembly involves β-Adducin phosphorylation through PKC, and both are required for augmented learning. In the absence of β-Adducin, enrichment still leads to an increase in spine structures, but the assembly of synapses at those spines is compromised. Virus-mediated re-expression of β-Adducin in hippocampal granule cells of β-Adducin-/- mice rescues new synapse assembly and learning upon enrichment. These results provide evidence that synapse disassembly and the establishment of new synapses are both critically important for augmented long-term learning and memory upon environmental enrichment (Bednarek, 2011).

Hts/Adducin controls synaptic elaboration and elimination

Neural development requires both synapse elaboration and elimination, yet relatively little is known about how these opposing activities are coordinated. This study provides evidence Hts/Adducin can serve this function. Drosophila Hts/Adducin is enriched both pre- and post-synaptically at the NMJ. Presynaptic Hts/Adducin is necessary and sufficient to control two opposing processes associated with synapse remodeling: (1) synapse stabilization as determined by light level and ultrastructural and electrophysiological assays and (2) the elaboration of actin-based, filopodia-like protrusions that drive synaptogenesis and growth. Synapse remodeling is sensitive to Hts/Adducin levels, and evidence is provided that the synaptic localization of Hts/Adducin is controlled via phosphorylation. Mechanistically, Drosophila Hts/Adducin protein has actin-capping activity. It is proposed that phosphorylation-dependent regulation of Hts/Adducin controls the level, localization, and activity of Hts/Adducin, influencing actin-based synapse elaboration and spectrin-based synapse stabilization. Hts/Adducin may define a mechanism to switch between synapse stability and dynamics (Pielage, 2011).

This study provides evidence that Hts/Adducin is an important player in the mechanisms that control both the stability and growth of the NMJ. hts mutations cause a profound destabilization of the presynaptic nerve terminal. These data are consistent with the well-established function of Adducin as a spectrin-binding protein that participates in the stabilization of the submembranous spectrin-actin skeleton. Remarkably, hts mutations also promote the growth and elaboration of new processes at the NMJ. Indeed, the elaboration of new processes and increased growth overcome the effects of synapse destabilization such that, on average, the NMJ is significantly larger in the hts mutant animals compared to wild-type. Process elaboration is accompanied by the extension of small-caliber, actin-rich protrusions that contain the necessary machinery for synaptic transmission including essential components of the active zone, postsynaptic glutamate receptors, and homophilic cell-adhesion molecules. This phenotype has not been observed in animals lacking presynaptic α-/β-Spectrin or Ankyrin2, indicating that Hts/Adducin has a specific activity relevant to the formation of these new synaptic processes (Pielage, 2011).

Biochemical insight is provided into how Hts/Adducin might control new process formation at the NMJ. Drosophila Hts-M has actin-capping activity similar to its vertebrate homolog. Based on recent work in other systems, loss of actin-capping activity at the plasma membrane could reasonably favor the formation of actin-based filopodia that might promote the elaboration of small-caliber synaptic protrusions. Consistent with such a model, overexpression of Hts/Adducin, thereby increasing the amount of actin-capping protein at the NMJ, is sufficient to inhibit the growth and elaboration of small-caliber type II and type III nerve terminals. Finally, this study demonstrates that synaptic localization of Hts/Adducin is controlled via phosphorylation of a conserved serine residue in the C-terminal MARCKS domain. Based on these and additional data, a model is presented in which Hts/Adducin functions as a molecular keystone, stabilizing the submembranous spectrin skeleton to achieve synapse stability and simultaneously capping actin filaments at the plasma membrane to influence the shape and growth potential of the presynaptic nerve terminal. Modulation of Adducin activity, either through changes in protein abundance or phosphoregulation, might then influence the balance of growth versus stability (Pielage, 2011).

Synapse retraction at the Drosophila NMJ occurs without cell death implying a local degenerative process. Mechanistically, withdrawal of target-derived BMP signaling causes retraction while overexpression of BMPs can suppress retractions, suggesting similarities with developmental remodeling. However, this study has identified several mutations that cause NMJ retraction that are linked to neuromuscular degeneration in human. Furthermore, overexpression of a WldS (Wallarian degeneration slowed) transgene is able to significantly suppress synapse retraction at the NMJ, implying a degenerative mechanism similar to that observed in mammalian motoneurons. Based upon these data, it is hypothesized that synapse retraction at the Drosophila NMJ is driven by local, degenerative processes that are similar to those observed during neural development and the early stages of neurodegeneration in other systems (Pielage, 2011).

Data demonstrating the involvement of Hts/Adducin in both NMJ degeneration and nerve terminal sprouting/growth is quite unique. It may be possible to partition these functions to the spectrin-binding and actin-capping activities of Adducin. It is particularly intriguing that the subcellular distribution of Adducin can be regulated by phosphorylation. Might changes in the local concentration of Adducin be involved in developmental pruning and neuromuscular synapse elimination or degeneration in other systems? If so, the data seem to highlight an increasingly opaque distinction between degenerative and developmental mechanisms (Pielage, 2011).

Two general phenotypes of synaptic overgrowth have been previously documented at the Drosophila NMJ. The first phenotype involves a uniform and dramatic expansion of the NMJ. The second phenotype involves the formation of highly ramified clusters of synaptic boutons that have been termed satellite boutons. This phenotype has been linked to disruption of endocytic proteins such as Dap160/Intersectin, Dynamin, and Endophilin as well as mutations that disrupt actin regulatory molecules including Wasp, Arp2/3, and Nervous Wreck (Pielage, 2011).

By contrast, the phenotypes documented in hts/adducin are quite different from any previously reported mutation. The NMJ is transformed into a hybrid structure consisting of normal type Ib boutons that support the extension of long, small-diameter synaptic protrusions. This phenotype is robust and highly penetrant. Adducin has two prominent functions. It participates in the stabilization of the spectrin-ankyrin skeleton and it caps actin filaments. By comparison of the current data with prior genetic analyses of α-/β-spectrin and ankyrin2L, these two functions of hts/adducin can be partitioned. α-/β-Spectrin and Ankyrin2L are necessary for NMJ stability but do not influence NMJ growth. Thus, it is proposed that Adducin is required to stabilize the nerve terminal through a well-established association with the spectrin/ankyrin skeleton. By extension, it is propose that the actin-capping activity of Adducin regulates NMJ growth. One possibility is that loss of actin-capping at the nerve terminal membrane promotes filopodia formation and this drives the extension of the observed small-caliber protrusions. However, the loss of actin-capping activity alone may not be sufficient since it occurs in the presence of an impaired spectrin/ankyrin/adducin submembranous skeleton. An alternative possibility is that loss of Adducin causes two simultaneous effects. First, it relieves a constraining influence of the spectrin skeleton. Second, in this relaxed context, increased filopodia formation is able to efficiently drive new nerve-terminal extension. Such a model could explain why the htsδG mutation does not have prominent protrusions despite showing increased growth. If the htsδG mutation retains some actin-binding activity, as suggested by in vitro data, and retains some stabilizing activity, then the combined effect might be sufficient to suppress protrusion formation while allowing enhanced synaptic growth. Interestingly, the association of Adducin with the submembranous spectrin skeleton can be controlled via phosphorylation downstream of growth factor signaling in other systems (Pielage, 2011).

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).


Search PubMed for articles about Drosophila Hts

Aoshima, R., Hiraoka, R., Shimada, N. and Kawata, T. (2006). Analysis of a homologue of the adducin head gene which is a potential target for the Dictyostelium STAT protein Dd-STATa. Int. J. Dev. Biol. 50(6): 523-32. Medline abstract: 16741867

Barkalow, K. L., et al. (2003). Alpha-adducin dissociates from F-actin and spectrin during platelet activation. J. Cell Biol. 161(3): 557-70. Medline abstract: 12743105

Bednarek, E. and Caroni, P. (2011). β-Adducin is required for stable assembly of new synapses and improved memory upon environmental enrichment. Neuron. 69(6): 1132-46. PubMed Citation: 21435558

Bennett, V., Gardner, K. and Steiner, J. P. (1988). Brain adducin: a protein kinase C substrate that may mediate site-directed assembly at the spectrin-actin junction. J. Biol. Chem. 263: 5860-5869. Medline abstract: 2451672

Blunk, A. D., Akbergenova, Y., Cho, R. W., Lee, J., Walldorf, U., Xu, K., Zhong, G., Zhuang, X. and Littleton, J. T. (2014). Postsynaptic actin regulates active zone spacing and glutamate receptor apposition at the Drosophila neuromuscular junction. Mol Cell Neurosci. PubMed ID: 25066865

Chen, C.-L., Hsieh, Y.-T., Chen, H.-C. (2007). Phosphorylation of adducin by protein kinase C{delta} promotes cell motility. J. Cell Sci. 120: 1157-1167. Medline abstract: 17341583

Cordes, K. R., et al. (2009). miR-145 and miR-143 regulate smooth muscle cell fate and plasticity. Nature 460: 705-710. PubMed Citation: 19578358

Costessi, L., Devescovi, G., Baralle, F. E. and Muro, A. F. (2006). Brain-specific promoter and polyadenylation sites of the beta-adducin pre-mRNA generate an unusually long 3'-UTR . Nucleic Acids Res. 34(1): 243-53. Medline abstract: 16414955

Deacon, D. C., et al. (2010). The miR-143-adducin3 pathway is essential for cardiac chamber morphogenesis. Development 137(11): 1887-96. PubMed Citation: 20460367

Ding, D., Parkhurst, S. M. and Lipshitz, H. D. (1993). Different genetic requirements for anterior RNA localization revealed by the distribution of Adducin-like transcripts during Drosophila oogenesis. Proc. Natl. Acad. Sci. 90: 2512-2516. Medline abstract: 7681599

Falet, H., et al. (2002). Importance of free actin filament barbed ends for Arp 2/3 complex function in platelets and fibroblasts. Proc. Natl. Acad. Sci. 99: 16782-16787. Medline abstract: 12464680

Forrest, S., Chai, A., Sanhueza, M., Marescotti, M., Parry, K., Georgiev, A., Sahota, V., Mendez-Castro, R. and Pennetta, G. (2013). Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis. Hum Mol Genet 22: 2689-2704. PubMed ID: 23492670

Fukata, Y., et al. (1999). Phosphorylation of adducin by rho-kinase plays a crucial role in cell motility. J. Cell Biol. 145: 347-361. Medline abstract: 10209029

Gardner, K., and Bennett, V. (1987). Modulation of spectrin-actin assembly by erythrocyte adducin. Nature. 328:359-362. Medline abstract: 3600811

Gilligan, D., et al. (1999). Targeted disruption of the beta-adducin gene (Add2) causes red blood cell spherocytosis in mice. Proc. Natl. Acad. Sci. 96: 10717-10722. Medline abstract: 10485892

Gilligan, D., Sarid, R. and Weese, J. (2002). Adducin in platelets: activation-induced phosphorylation by PKC and proteolysis by calpain. Blood. 99: 2418-2426. Medline abstract: 11895774

Gruenbaum, L. M., et al. (2003). Identification and characterization of Aplysia adducin, an Aplysia cytoskeletal protein homologous to mammalian adducins: increased phosphorylation at a protein kinase C consensus site during long-term synaptic facilitation. J. Neurosci. 23: 2675-2685. PubMed Citation: 12684453

Hughes, C., and Bennett, V. (1995). Adducin: a physical model with implications for function in assembly of spectrin-actin complexes. J. Biol. Chem. 270: 18990-18996. Medline abstract: 7642559

Ichetovkin, I., W. Grant, and J. Condeelis. 2002. Cofilin produces newly polymerized actin filaments that are preferred for dendritic nucleation by Arp2/3 complex. Curr. Biol. 12: 79-84. Medline abstract: 11790308

Khuong, T. M., Habets, R. L., Slabbaert, J. R. and Verstreken, P. (2010). WASP is activated by phosphatidylinositol-4,5-bisphosphate to restrict synapse growth in a pathway parallel to bone morphogenetic protein signaling. Proc Natl Acad Sci U S A 107: 17379-17384. PubMed ID: 20844206

Kimura, K., et al. (1998). Regulation of the association of adducin with actin filaments by Rho-associated kinase (Rho-kinase) and myosin phosphatase. J. Biol. Chem. 273: 5542-5548. Medline abstract: 9488679

Kuhlman, P., Hughes, C., Bennett, B. and Fowler, V. (1996). A new function for adducin. Calcium/calmodulin-regulated capping of the barbed ends of actin filaments. J. Biol. Chem. 271: 7986-7991. Medline abstract: 8626479

Li, X., Matsuoka, Y. and Bennett. V. (1998). Adducin preferentially recruits spectrin to the fast growing ends of actin filaments in a complex requiring the MARCKS-related domain and a newly defined oligomerization domain. J. Biol. Chem. 273: 19329-19338. Medline abstract: 9668123

Lin, H., Yue, L. and Spradling, A. C. (1994). The Drosophila fusome, a germline-specific organelle, contains membrane skeletal proteins and functions in cyst formation. Development 120: 947-956. Medline abstract: 7600970

Matsuoka, Y., Li, X. and Bennett, V. (1998). Adducin is an in vivo substrate for protein kinase C: phosphorylation in the MARCKS-related domain inhibits activity in promoting spectrin-actin complexes and occurs in many cells, including dendritic spines of neurons. J. Cell Biol. 142(2): 485-97. Medline abstract: 9679146

McNeil, G. P., Smith, F. and Galioto, R. (2004). The Drosophila RNA-binding protein Lark is required for the organization of the actin cytoskeleton and Hu-li tai shao localization during oogenesis. Genesis 40(2): 90-100. Medline abstract: 15452872

Naydenov, N. G. and Ivanov, A. I. (2010). Adducins regulate remodeling of apical junctions in human epithelial cells. Mol. Biol. Cell 21: 3506-3517. PubMed Citation: 20810786

Ohler, S., Hakeda-Suzuki, S. and Suzuki, T. (2011). Hts, the Drosophila homologue of Adducin, physically interacts with the transmembrane receptor Golden goal to guide photoreceptor axons. Dev. Dyn. 240: 135-148. PubMed Citation: 21128303

Pariser, H., Herradon, G., Ezquerra, L., Perez-Pinera, P. and Deuel, T. F. (2005). Pleiotrophin regulates serine phosphorylation and the cellular distribution of beta-adducin through activation of protein kinase C. Proc. Natl. Acad. Sci. 102(35): 12407-12. Medline abstract: 16116087

Petrella, L. N., Smith-Leiker, T. and Cooley, L. (2007). The Ovhts polyprotein is cleaved to produce fusome and ring canal proteins required for Drosophila oogenesis. Development 134(4): 703-12. Medline abstract: 17215303

Pielage, J., et al. (2011). Hts/Adducin controls synaptic elaboration and elimination. Neuron 69: 1114-1131. PubMed Citation: 21435557

Pokrywka, N. J., Zhang, H. and Raley-Susman, K. (2014). Distinct roles for hu li tai shao and swallow in cytoskeletal organization during Drosophila oogenesis. Dev Dyn [Epub ahead of print]. PubMed ID: 24677508

Porro, F., et al. (2010). β-adducin (Add2) KO mice show synaptic plasticity, motor coordination and behavioral deficits accompanied by changes in the expression and phosphorylation levels of the α- and γ-adducin subunits. Genes Brain Behav. 9: 84-96. PubMed Citation: 19900187

Rabenstein, R. L., et al. (2005). Impaired synaptic plasticity and learning in mice lacking beta-adducin, an actin-regulating protein. J. Neurosci. 25(8): 2138-45. Medline abstract: 15728854

Robinson, D. N., Cant, K. and Cooley, L. (1994). Morphogenesis of Drosophila ovarian ring canals. Development 120: 2015-2025. Medline abstract: 7925006

Robinson, D. N., Smith-Leiker, T. A., Sokol, N. S., Hudson, A. M. and Cooley, L. (1997). Formation of the Drosophila ovarian ring canal inner rim depends on cheerio. Genetics 145: 1063-1072. Medline abstract: 9093858

Ruediger, S. et al. (2011). Learning-related feedforward inhibitory connectivity growth required for memory precision. Nature 473: 514-518. PubMed Citation: 21532590

Shan, X., Hu, J. H., Cayabyab, F. S. and Krieger, C. (2005). Increased phospho-adducin immunoreactivity in a murine model of amyotrophic lateral sclerosis. Neuroscience. 134(3): 833-46. Medline abstract: 15994023

Shima, T., et al. (2001). Interaction of the SH2 domain of Fyn with a cytoskeletal protein, beta-adducin. J. Biol. Chem. 276(45): 42233-40. Epub 2001 Aug 28. Medline abstract: 11526103

Speese, S. D., Ashley, J., Jokhi, V., Nunnari, J., Barria, R., Li, Y., Ataman, B., Koon, A., Chang, Y. T., Li, Q., Moore, M. J. and Budnik, V. (2012). Nuclear envelope budding enables large ribonucleoprotein particle export during synaptic Wnt signaling. Cell 149: 832-846. PubMed ID: 22579286

Spracklen, A. J., Kelpsch, D. J., Chen, X., Spracklen, C. N. and Tootle, T. L. (2013). Prostaglandins temporally regulate cytoplasmic actin bundle formation during Drosophila oogenesis. Mol Biol Cell 25(3): 397-411. PubMed ID: 24284900

Verdier, V., et al. (2006a). Drosophila Rho-kinase (DRok) is required for tissue morphogenesis in diverse compartments of the egg chamber during oogenesis. Dev. Biol. 297(2): 417-32. Medline abstract: 16887114

Verdier, V., Guang-Chao-Chena and Settleman, J. (2006b). Rho-kinase regulates tissue morphogenesis via non-muscle myosin and LIM-kinase during Drosophila development. BMC Dev. Biol. 6: 38. Medline abstract: 16882341

Wang, S., et al. (2011). Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane. Dev. Biol. 357(2): 392-403. PubMed Citation: 21791202

Wang, S. J., Tsai, A., Wang, M., Yoo, S., Kim, H. Y., Yoo, B., Chui, V., Kisiel, M., Stewart, B., Parkhouse, W., Harden, N. and Krieger, C. (2014). Phospho-regulated Drosophila adducin is a determinant of synaptic plasticity in a complex with Dlg and PIP2 at the larval neuromuscular junction. Biol Open 3(12): 1196-206. PubMed ID: 25416060

Wawersik, M. and Van Doren, M. (2005). nanos is required for formation of the spectrosome, a germ cell-specific organelle. Dev. Dyn. 234: 22-27. Medline abstract: 16028275

Wilson, P. G. (2005). Centrosome inheritance in the male germ line of Drosophila requires hu-li tai-shao function. Cell Biol. Int. 29(5): 360-9. Medline abstract: 15993631

Whittaker, K. L., Ding, D., Fisher, W. W. and Lipshitz, H. D. (1999). Different 3' untranslated regions target alternatively processed hu-li tai shao (hts) transcripts to distinct cytoplasmic locations during Drosophila oogenesis. J. Cell Sci. 112: 3385-98. Medline abstract: 10504343

Xin M., et al. (2009). MicroRNAs miR-143 and miR-145 modulate cytoskeletal dynamics and responsiveness of smooth muscle cells to injury. Genes Dev. 23: 2166-2178. PubMed Citation: 19720868

Yue, L. and Spradling, A. C. (1992). hu-li tai shao, a gene required for ring canal formation during Drosophila oogenesis, encodes a homolog of adducin. Genes Dev. 6(12B): 2443-54. Medline abstract: 1340461

Zaccai, M. and Lipshitz, H. D. (1996a). Differential distributions of two adducin-like protein isoforms in the Drosophila ovary and early embryo. Zygote 4(2): 159-66. Medline abstract: 8913030

Zaccai, M. and Lipshitz, H. D. (1996b). Role of Adducin-like (hu-li tai shao) mRNA and protein localization in regulating cytoskeletal structure and function during Drosophila oogenesis and early embryogenesis. Dev. Genet. 19(3): 249-57. Medline abstract: 8952067

hu-li tai shao: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 October 2014

Home page: The Interactive Fly © 2006 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.