Drosophila filamin encoded by the cheerio locus is a component of ovarian ring canals.

BACKGROUND: The ring canals in the ovary of the fruit fly Drosophila provide a versatile system in which to study the assembly and regulation of membrane-associated actin structures. Derived from arrested cleavage furrows, ring canals allow direct communication between cells. The robust inner rim of filamentous actin that attaches to the ring-canal plasma membrane contains cytoskeletal proteins encoded by the hu-li-tao shao (hts) and kelch genes, and is regulated by the Src64 and Tec29 tyrosine kinases. Female sterile cheerio mutants fail to recruit actin to ring canals, disrupting the flow of cytoplasm to oocytes. RESULTS: We have cloned cheerio and found that it encodes a member of the Filamin/ABP-280 family of actin-binding proteins, known to bind transmembrane proteins and crosslink actin filaments into parallel or orthogonal arrays. Antibodies to Drosophila Filamin revealed that Filamin is an abundant ring-canal protein and the first known component of both the outer and inner rims of the ring canal. The cheerio gene also encodes a new Filamin isoform that lacks the actin-binding domain. CONCLUSIONS: Localization of Filamin to nascent ring canals is necessary for the recruitment of actin filaments. We propose that Filamin links filamentous actin to the plasma membrane of the ring canal. Although loss of Filamin in human cells supports a role for Filamin in organizing orthogonal actin arrays at the cell cortex, the cheerio mutant provides the first evidence that Filamin is required in membrane-associated parallel actin bundles, such as those found in ring canals, contractile rings and stress fibers.     

Src64 is involved in fusome development and karyosome formation during Drosophila oogenesis

Src family tyrosine kinases respond to a variety of signals by regulating the organization of the actin cytoskeleton. Here, we show that during early oogenesis Src64 mutations lead to uneven accumulation of cortical actin, defects in fusome formation, mislocalization of septins, defective transport of Orb protein into the oocyte, and possible defects in cell division. Similar mutant phenotypes suggest that Src64, the Tec29 tyrosine kinase, and the actin crosslinking protein Kelch act together to regulate actin crosslinking, much as they do later during ring canal growth. Condensation of the oocyte chromatin into a compact karyosome is also defective in Src64, Tec29, and kelch mutants and in mutants for spire and chickadee (profilin), genes that regulate actin polymerization. These data, along with changes in G-actin accumulation in the oocyte nucleus, suggest that Src64 is involved in a nuclear actin function during karyosome condensation. Our results indicate that Src64 regulates actin dynamics at multiple stages of oogenesis.     

Domains of Importin-alpha2 required for ring canal assembly during Drosophila oogenesis

Null-mutation in Drosophila importin-alpha2, such as the deficiency imp-alpha2(D14), causes recessive female sterility with the formation of dumpless eggs. In imp-alpha2(D14) the transfer of nurse cell components to the oocyte is interrupted and the Kelch protein, an oligomeric ring canal actin organizer, is normally produced but fails to associate with the ring canals resulting in their occlusion. To define domains regulating Kelch deposition on ring canals we performed site-directed mutagenesis on protein binding domains and putative phosphorylation sites of Imp-alpha2. Phenotypic analysis of the mutant transgenes in imp-alpha2(D14) revealed that mutations affecting the Imp-beta binding-domain, the dimerization domain, and specific serine residues of putative phosphorylation sites led to a normal or nearly normal oogenesis but arrested early embryonic development, whereas mutations in the nuclear localization signal (NLS) and CAS/exportin binding domains resulted in ring canal occlusion and a drastic nuclear accumulation of the mutant proteins. Deletion of the Imp-beta binding domain also gave rise to a nuclear localization of the mutant protein, which partially retained its function in ring canal assembly. Thus, we propose that mutations in NLS and CAS binding domains affect the deposition of Kelch onto the ring canals and prevent the association of Imp-alpha2 with a negative regulator of Kelch function.     

Drosophila Kelch Is an Oligomeric Ring Canal Actin Organizer

Drosophila kelch has four protein domains, two of which are found in kelch-family proteins and in numerous nonkelch proteins. In Drosophila, kelch is required to maintain ring canal organization during oogenesis. We have performed a structure–function analysis to study the function of Drosophila kelch. The amino-terminal region (NTR) regulates the timing of kelch localization to the ring canals. Without the NTR, the protein localizes precociously and destabilizes the ring canals and the germ cell membranes, leading to dominant sterility. The amino half of the protein including the BTB domain mediates dimerization. Oligomerization through the amino half of kelch might allow cross-linking of ring canal actin filaments, organizing the inner rim cytoskeleton. The kelch repeat domain is necessary and sufficient for ring canal localization and likely mediates an additional interaction, possibly with actin.     

Importin-alpha 2 is critically required for the assembly of ring canals during Drosophila oogenesis

The interstitial deletion D14 affecting the importin-alpha 2 gene of Drosophila, or imp-alpha 2(D14), causes recessive female sterility characterized by a block of nurse cell-oocyte transport during oogenesis. In wild-type egg chambers, the Imp-alpha 2 protein is uniformly distributed in the nurse cell cytoplasm with a moderate accumulation along the oocyte cortex. Cytochalasin D treatment of wild-type egg chambers disrupts the in vivo association of Imp-alpha 2 with F-actin and results in its release from the oocyte cortex and its transfer into nurse cell nuclei. Binding assay shows that the interaction of Imp-alpha 2 with F-actin, albeit not monomeric actin, requires the occurrence of NLS peptides. Phenotypic analysis of imp-alpha 2(D14) ovaries reveals that the block of nurse cell-oocyte transport results from the occlusion of the ring canals that constitute cytoplasmic bridges between the nurse cells and the oocyte. Immunohistochemistry shows that, although the Imp-alpha2 protein cannot be detected on the ring canals, the Kelch protein, a known ring canal component, fails to bind to ring canals in imp-alpha 2(D14) egg chambers. Since loss-of-function mutations of kelch results in a similar dumpless phenotype, we propose that the Imp-alpha 2 protein plays a critical role in Kelch function by regulating its deposition on ring canals during their assembly.     

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. Here we describe a new gene we named 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.     

An A-kinase anchoring protein is required for protein kinase A regulatory subunit localization and morphology of actin structures during oogenesis in Drosophila

Protein kinase A (PKA) holoenzyme is anchored to specific subcellular regions by interactions between regulatory subunits (Pka-R) and A-kinase anchoring proteins (AKAPs). We examine the functional importance of PKA anchoring during Drosophila oogenesis by analyzing membrane integrity and actin structures in mutants with disruptions in Akap200, an AKAP. In wild-type ovaries, Pka-RII and Akap200 localized to membranes and to the outer rim of ring canals, actin-rich structures that connect germline cells. In Akap200 mutant ovaries, Pka-RII membrane localization decreased, leading to a destabilization of membrane structures and the formation of binucleate nurse cells. Defects in membrane integrity could be mimicked by expressing a constitutively active PKA catalytic subunit (Pka-C) throughout germline cells. Unexpectedly, nurse cells in Akap200 mutant ovaries also had enlarged, thin ring canals. In contrast, overexpressing Akap200 in the germline resulted in thicker, smaller ring canals. To investigate the role of Akap200 in regulating ring canal growth, we examined genetic interactions with other genes that are known to regulate ring canal morphology. Akap200 mutations suppressed the small ring canal phenotype produced by Src64B mutants, linking Akap200 with the non-receptor tyrosine kinase pathway. Together, these results provide the first evidence that PKA localization is required for morphogenesis of actin structures in an intact organism.     

Localization of Tec29 to ring canals is mediated by Src64 and PtdIns(3,4,5)P3-dependent mechanisms

Two tyrosine kinases, Src64 and Tec29, regulate the growth of actin rich-ring canals in the Drosophila ovary. We have shown previously that Src64 directs the localization of Tec29 to ring canals, but the mechanism underlying this process was unknown. Here, we show that Tec29 localizes to ring canals via its Src homology 3 (SH3) and Src homology 2 (SH2) domains. Tec29 activity is required for its own ring canal localization, suggesting that a phosphotyrosine ligand for the SH2 domain is generated by Tec29 itself. Src64 regulates this process by phosphorylating Y677 within the kinase domain of Tec29, an event required for Tec29 activation. We also show that the pleckstrin homology (PH) domain of Tec29 has dual functions in mediating Src64 regulation. In the absence of Src64, the PH domain prevents Tec29 ring canal localization. In the presence of Src64, it enhances membrane targeting of Tec29 by a PI(3,4,5)P(3)-mediated mechanism. In the absence of its PH domain, Tec29 constitutively localizes to ring canals, but still requires Src64 for full activation.     

hDKIR, a human homologue of the Drosophila kelch protein, involved in a ring-like structure

We have previously purified and cloned an apoptosis-inducing protein (AIP) derived from fish infected with the anisakis simplex. Recently, we identified a series of AIP-responsive genes in the HL-60 cell line using a subtractive hybridization method. Here we report the molecular cloning and characterization of one of these genes, which encodes a novel human kelch protein containing 568 amino acid residues, termed hDKIR. The Drosophila Kelch protein localizes to a ring canal structure, which is required for oocyte development. When hDKIR was expressed in cultured-mammalian cells, hDKIR localized to a ring-like structure. Furthermore, when coexpressed with Mayven or Keap1, hDKIR bound to Mayven and recruited Mayven into ring-like structures perfectly. This indicates that kelch homologues can interact with each other in a specific manner and such interaction can affect the subcellular localization of kelch proteins. Finally, domain analysis revealed that both the N-terminal POZ (poxviruses and zinc fingers) and intervening region (IVR) domains of hDKIR are essential for ring-like structure activity, suggesting that the development of the ring-like structure is independent of the ability to bind actin.     

Formation of actin filament bundles in the ring canals of developing Drosophila follicles

Growing the intracellular bridges that connect nurse cells with each o ther and to the developing oocyte is vital for egg development. These ring canals increase from 0.5 microns in diameter at stage 2 to 10 microns in diameter at stage 11. Thin sections cut horizontally as you would cut a bagel, show that there is a layer of circumferentially oriented actin filaments attached to the plasma membrane at the periphery of each canal. By decoration with subfragment 1 of myosin we find actin filaments of mixed polarities in the ring such as found in the "contractile ring" formed during cytokinesis. In vertical sections through the canal the actin filaments appear as dense dots. At stage 2 there are 82 actin filaments in the ring, by stage 6 there are 717 and by stage 10 there are 726. Taking into account the diameter, this indicates that there is 170 microns of actin filaments/canal at stage 2 (pi x 0.5 microns x 82), 14,000 microns at stage 9 and approximately 23,000 microns at stage 11 or one inch of actin filament! The density of actin filaments remains unchanged throughout development. What is particularly striking is that by stages 4-5, the ring of actin filaments has achieved its maximum thickness, even though the diameter has not yet increased significantly. Thereafter, the diameter increases. Throughout development, stages 2-11, the canal length also increases. Although the density (number of actin filaments/micron2) through a canal remains constant from stage 5 on, the actin filaments appear as a net of interconnected bundles. Further information on this net of bundles comes from studying mutant animals that lack kelch, a protein located in the ring canal that has homology to the actin binding protein, scruin. In this mutant, the actin filaments form normally but individual bundles that comprise the fibers of the net are not bound tightly together. Some bundles enter into the ring canal lumen but do not completely occlude the lumen. all these observations lay the groundwork for our understanding of how a noncontractile ring increases in thickness, diameter, and length during development.     

Csk differentially regulates Src64 during distinct morphological events in Drosophila germ cells

The Src family protein tyrosine kinases (SFKs) are crucial regulators of cellular morphology. In Drosophila, Src64 controls complex morphological events that occur during oogenesis. Recent studies have identified key Src64-dependent mechanisms that regulate actin cytoskeletal dynamics during the growth of actin-rich ring canals, which act as intercellular bridges between germ cells. By contrast, the molecular mechanisms that regulate Src64 activity levels and potential roles for Src64 in additional morphological events in the ovary have not been defined. In this report, we demonstrate that regulation of Src64 by Drosophila C-terminal-Src Kinase (Csk) contributes to the packaging of germline cysts by overlying somatic follicle cells during egg chamber formation. These results uncover novel roles for both Csk and Src64 in a dynamic event that involves adhesion, communication between cell types and control of cell motility. Strikingly, Src64 and Csk function in the germline to control packaging, not in migrating follicle cells, suggesting novel functions for this signaling cassette in regulating dynamic adhesion. In contrast to the role played by Csk in the regulation of Src64 activity during packaging, Csk is dispensable for ring canal growth control, indicating that distinct mechanisms control Src64 activity during different morphological events.     

Characterization of Mayven, a Novel Actin-binding Protein Predominantly Expressed in Brain

The cytoskeleton plays an important role in neuronal morphogenesis. We have identified and characterized a novel actin-binding protein, termed Mayven, predominantly expressed in brain. Mayven contains a BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain-like structure in the predicted N terminus and "kelch repeats" in the predicted C-terminal domain. Mayven shares 63% identity (77% similarity) with the Drosophila ring canal ("kelch") protein. Somatic cell-hybrid analysis indicated that the human Mayven gene is located on chromosome 4q21.2, whereas the murine homolog gene is located on chromosome 8. The BTB/POZ domain of Mayven can self-dimerize in vitro, which might be important for its interaction with other BTB/POZ-containing proteins. Confocal microscopic studies of endogenous Mayven protein revealed a highly dynamic localization pattern of the protein. In U373-MG astrocytoma/glioblastoma cells, Mayven colocalized with actin filaments in stress fibers and in patchy cortical actin-rich regions of the cell margins. In primary rat hippocampal neurons, Mayven is highly expressed in the cell body and in neurite processes. Binding assays and far Western blotting analysis demonstrated association of Mayven with actin. This association is mediated through the "kelch repeats" within the C terminus of Mayven. Depolarization of primary hippocampal neurons with KCl enhanced the association of Mayven with actin. This increased association resulted in dynamic changes in Mayven distribution from uniform to punctate localization along neuronal processes. These results suggest that Mayven functions as an actin-binding protein that may be translocated along axonal processes and might be involved in the dynamic organization of the actin cytoskeleton in brain cells.     

Actomyosin contraction during cellularization is regulated in part by Src64 control of Actin 5C protein levels

Src64 is required for actomyosin contraction during cellularization of the Drosophila embryonic blastoderm. The mechanism of actomyosin ring constriction is poorly understood even though a number of cytoskeletal regulators have been implicated in the assembly, organization, and contraction of these microfilament rings. How these cytoskeletal processes are regulated during development is even less well understood. To investigate the role of Src64 as an upstream regulator of actomyosin contraction, we conducted a proteomics screen to identify proteins whose expression levels are controlled by src64. Global levels of actin are reduced in src64 mutant embryos. Furthermore, we show that reduction of the actin isoform Actin 5C causes defects in actomyosin contraction during cellularization similar to those caused by src64 mutation, indicating that a relatively high level of Actin 5C is required for normal actomyosin contraction and furrow canal structure. However, reduction of Actin 5C levels only slows down actomyosin ring constriction rather than preventing it, suggesting that src64 acts not only to modulate actin levels, but also to regulate the actomyosin cytoskeleton by other means.     

Btk29A-Mediated Tyrosine Phosphorylation of Armadillo/β-Catenin Promotes Ring Canal Growth in Drosophila Oogenesis

Drosophila Btk29A is the ortholog of mammalian Btk, a Tec family nonreceptor tyrosine kinase whose deficit causes X-linked agammaglobulinemia in humans. The Btk29A^ficP mutation induces multiple abnormalities in oogenesis, including the growth arrest of ring canals, large intercellular bridges that allow the flow of cytoplasm carrying maternal products essential for embryonic development from the nurse cells to the oocyte during oogenesis. In this study, inactivation of Parcas, a negative regulator of Btk29A, was found to promote Btk29A accumulation on ring canals with a concomitant increase in the ring canal diameter, counteracting the Btk29A^ficP mutation. This mutation markedly reduced the accumulation of phosphotyrosine on ring canals and in the regions of cell-cell contact, where adhesion-supporting proteins such as DE-cadherin and β-catenin ortholog Armadillo (Arm) are located. Our previous in vitro and in vivo analyses revealed that Btk29A directly phosphorylates Arm, leading to its release from DE-cadherin. In the present experiments, immunohistological analysis revealed that phosphorylation at tyrosine 150 (Y150) and Y667 of Arm was diminished in Btk29A^ficP mutant ring canals. Overexpression of an Arm mutant with unphosphorylatable Y150 inhibited ring canal growth. Thus Btk29A-induced Y150 phosphorylation is necessary for the normal growth of ring canals. We suggest that the dissociation of tyrosine-phosphorylated Arm from DE-cadherin allows dynamic actin to reorganize, leading to ring canal expansion and cell shape changes during the course of oogenesis.     

F-actin rings are associated with the ring canals of the Drosophila egg chamber

Staining of Drosophila egg chambers with rhodaminyl-lysine-phallotoxin (RLP), a specific stain for F-actin, has demonstrated the presence of dense F-actin rings associated with the inner surfaces of the ring canals. They were first observed in the distal part of the germarium where rings of four different size classes were found, differing in diameter by up to twofold. The ring sizes are considered to correspond to the ring canals formed at each of four successive incomplete cleavages. During the growth of the egg chamber the actin rings were found to increase in diameter from less than 1 micron to approx. 10 micron. Concomitantly a secondary outer ring of more diffuse material is built up in association with the cell membranes. A well developed array of microfilament bundles was also associated with the nurse cell plasmalemma. In stages where the transfer of the bulk of the nurse cell cytoplasm into the oocyte was occurring the rings came closer together in a central area. In late stage chambers the F-actin rings and the microfilament bundles appeared to be incorporated into large irregular masses of actin, which subsequently disappeared as the mature oocyte formed. The F-actin rings are suggested to act as mechanical strengthening elements for the canal plasmalemma, whilst cytoplasmic transport occurs through the ring canals.     

Drosophila Kelch functions with Cullin-3 to organize the ring canal actin cytoskeleton

Drosophila melanogaster Kelch (KEL) is the founding member of a diverse protein family defined by a repeated sequence motif known as the KEL repeat (KREP). Several KREP proteins, including Drosophila KEL, bind filamentous actin (F-actin) and contribute to its organization. Recently, a subset of KREP proteins has been shown to function as substrate adaptor proteins for cullin-RING (really interesting new gene) ubiquitin E3 ligases. In this study, we demonstrate that association of Drosophila KEL with Cullin-3, likely in a cullin-RING ligase, is essential for the growth of Drosophila female germline ring canals. These results suggest a role for protein ubiquitylation in the remodeling of a complex F-actin cytoskeletal structure.     

A subset of dynamic actin rearrangements in Drosophila requires the Arp2/3 complex

The Arp2/3 complex has been shown to dramatically increase the slow spontaneous rate of actin filament nucleation in vitro, and it is known to be important for remodeling the actin cytoskeleton in vivo. We isolated and characterized loss of function mutations in genes encoding two subunits of the Drosophila Arp2/3 complex: Arpc1, which encodes the homologue of the p40 subunit, and Arp3, encoding one of the two actin-related proteins. We used these mutations to study how the Arp2/3 complex contributes to well-characterized actin structures in the ovary and the pupal epithelium. We found that the Arp2/3 complex is required for ring canal expansion during oogenesis but not for the formation of parallel actin bundles in nurse cell cytoplasm and bristle shaft cells. The requirement for Arp2/3 in ring canals indicates that the polymerization of actin filaments at the ring canal plasma membrane is important for driving ring canal growth.     

Molecular characterization of KLHL3, a human homologue of the Drosophila kelch gene

The Drosophila kelch protein is a structural component of ring canals and is required for oocyte maturation. Here, we report the cloning and genomic structure of a new human homologue of kelch, KLHL3. At the amino acid level, KLHL3 shares 77% similarity with Drosophila kelch and 89% similarity with Mayven (KLHL2), another human kelch homolog. The approximately 6.5-kb mRNA has a single open reading frame encoding a protein of 587 amino acids with a predicted molecular mass of 650 kDa. Like kelch and KLHL2, the KLHL3 protein contains a poxvirus and zinc finger domain at the N-terminus and six tandem repeats (kelch repeats) at the C-terminus. At least three isoforms, which differ in the length of the N-terminus, are produced and may be the result of alternative promoter usage. We also identified alternative polyadenylation sites and alternative splicing; thus, as many as 12 mRNA variants and six putative protein isoforms could be produced. The KLHL3 gene is mapped to human chromosome 5, band q31, contains 17 exons, and spans approximately 120 kb of genomic DNA. KLHL3 maps within the smallest commonly deleted segment in myeloid leukemias characterized by a deletion of 5q; however, we detected no inactivating mutations of KLHL3 in malignant myeloid disorders with loss of 5q.     

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. Here we show that this is due to a requirement during oogenesis. Developing oocytes from lark(1) germline clones (GLCs) are often smaller than normal due to defects in nurse cell cytoplasmic "dumping." Late-stage egg chambers from lark(1) 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, we observed a buildup of late-stage egg chambers, a phenotype that correlates with the decrease in egg-laying observed in the mutants. We postulate 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 as similar phenotypes were observed in flies carrying specific RNA-binding domain mutations. We propose that Lark functions during oogenesis as an RNA-binding protein, regulating mRNAs required for nurse cell transport or apoptosis.