Drosophila Kelch regulates actin organization via Src64-dependent tyrosine phosphorylation

The Drosophila kelch gene encodes a member of a protein superfamily defined by the presence of kelch repeats. In Drosophila, Kelch is required to maintain actin organization in ovarian ring canals. We set out to study the actin cross-linking activity of Kelch and how Kelch function is regulated. Biochemical studies using purified, recombinant Kelch protein showed that full-length Kelch bundles actin filaments, and kelch repeat 5 contains the actin binding site. Two-dimensional electrophoresis demonstrated that Kelch is tyrosine phosphorylated in a src64-dependent pathway. Site-directed mutagenesis determined that tyrosine residue 627 is phosphorylated. A Kelch mutant with tyrosine 627 changed to alanine (KelY627A) rescued the actin disorganization phenotype of kelch mutant ring canals, but failed to produce wild-type ring canals. Electron microscopy demonstrated that phosphorylation of Kelch is critical for the proper morphogenesis of actin during ring canal growth, and presence of the nonphosphorylatable KelY627A protein phenocopied src64 ring canals. KelY627A protein in ring canals also dramatically reduced the rate of actin monomer exchange. The phenotypes caused by src64 mutants and KelY627A expression suggest that a major function of Src64 signaling in the ring canal is the negative regulation of actin cross-linking by Kelch.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Protein Phosphatase 1? Limits Ring Canal Constriction during Drosophila Germline Cyst Formation

Germline cyst formation is essential for the propagation of many organisms including humans and flies. The cytoplasm of germline cyst cells communicate with each other directly via large intercellular bridges called ring canals. Ring canals are often derived from arrested contractile rings during incomplete cytokinesis. However how ring canal formation, maintenance and growth are regulated remains unclear. To better understand this process, we carried out an unbiased genetic screen in Drosophila melanogaster germ cells and identified multiple alleles of flapwing (flw), a conserved serine/threonine-specific protein phosphatase. Flw had previously been reported to be unnecessary for early D. melanogaster oogenesis using a hypomorphic allele. We found that loss of Flw leads to over-constricted nascent ring canals and subsequently tiny mature ring canals, through which cytoplasmic transfer from nurse cells to the oocyte is impaired, resulting in small, non-functional eggs. Flw is expressed in germ cells undergoing incomplete cytokinesis, completely colocalized with the Drosophila myosin binding subunit of myosin phosphatase (DMYPT). This colocalization, together with genetic interaction studies, suggests that Flw functions together with DMYPT to negatively regulate myosin activity during ring canal formation. The identification of two subunits of the tripartite myosin phosphatase as the first two main players required for ring canal constriction indicates that tight regulation of myosin activity is essential for germline cyst formation and reproduction in D. melanogaster and probably other species as well.     

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.     

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.     

The PACT domain, a conserved centrosomal targeting motif in the coiled-coil proteins AKAP450 and pericentrin

AKAP450 (also known as AKAP350, CG-NAP or Hyperion) and pericentrin are large coiled-coil proteins found in mammalian centrosomes that serve to recruit structural and regulatory components including dynein and protein kinase A. We find that these proteins share a well conserved 90 amino acid domain near their C-termini that is also found in coiled-coil proteins of unknown function from Drosophila and fission yeast. Fusion of the C-terminal region from either protein to a reporter protein confers a centrosomal localization, and overexpression of the domain from AKAP450 displaces endogenous pericentrin, suggesting recruitment to a shared site. When isolated from transfected cells the C-terminal domain of AKAP450 was associated with calmodulin, suggesting that this protein could contribute to centrosome assembly.     

Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm

The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin.     

Orbit/CLASP Is Required for Germline Cyst Formation through Its Developmental Control of Fusomes and Ring Canals in Drosophila Males

Orbit, a Drosophila ortholog of microtubule plus-end enriched protein CLASP, plays an important role in many developmental processes involved in microtubule dynamics. Previous studies have shown that Orbit is required for asymmetric stem cell division and cystocyte divisions in germline cysts and for the development of microtubule networks that interconnect oocyte and nurse cells during oogenesis. Here, we examined the cellular localization of Orbit and its role in cyst formation during spermatogenesis. In male germline stem cells, distinct localization of Orbit was first observed on the spectrosome, which is a spherical precursor of the germline-specific cytoskeleton known as the fusome. In dividing stem cells and spermatogonia, Orbit was localized around centrosomes and on kinetochores and spindle microtubules. After cytokinesis, Orbit remained localized on ring canals, which are cytoplasmic bridges between the cells. Thereafter, it was found along fusomes, extending through the ring canal toward all spermatogonia in a cyst. Fusome localization of Orbit was not affected by microtubule depolymerization. Instead, our fluorescence resonance energy transfer experiments suggested that Orbit is closely associated with F-actin, which is abundantly found in fusomes. Surprisingly, F-actin depolymerization influenced neither fusome organization nor Orbit localization on the germline-specific cytoskeleton. We revealed that two conserved regions of Orbit are required for fusome localization. Using orbit hypomorphic mutants, we showed that the protein is required for ring canal formation and for fusome elongation mediated by the interaction of newly generated fusome plugs with the pre-existing fusome. The orbit mutation also disrupted ring canal clustering, which is essential for folding of the spermatogonia after cytokinesis. Orbit accumulates around centrosomes at the onset of spermatogonial mitosis and is required for the capture of one of the duplicated centrosomes onto the fusome. Moreover, Orbit is involved in the proper orientation of spindles towards fusomes during synchronous mitosis of spermatogonial cysts.     

cdc12p, a Protein Required for Cytokinesis in Fission Yeast, Is a Component of the Cell Division Ring and Interacts with Profilin

As in many other eukaryotic cells, cell division in fission yeast depends on the assembly of an actin ring that circumscribes the middle of the cell. Schizosaccharomyces pombe cdc12 is an essential gene necessary for actin ring assembly and septum formation. Here we show that cdc12p is a member of a family of proteins including Drosophila diaphanous, Saccharomyces cerevisiae BNI1, and S. pombe fus1, which are involved in cytokinesis or other actin-mediated processes. Using indirect immunofluorescence, we show that cdc12p is located in the cell division ring and not in other actin structures. When overexpressed, cdc12p is located at a medial spot in interphase that anticipates the future ring site. cdc12p localization is altered in actin ring mutants. cdc8 (tropomyosin homologue), cdc3 (profilin homologue), and cdc15 mutants exhibit no specific cdc12p staining during mitosis. cdc4 mutant cells exhibit a medial cortical cdc12p spot in place of a ring. mid1 mutant cells generally exhibit a cdc12p spot with a single cdc12p strand extending in a random direction. Based on these patterns, we present a model in which ring assembly originates from a single point on the cortex and in which a molecular pathway for the functions of cytokinesis proteins is suggested. Finally, we found that cdc12 and cdc3 mutants show a syntheticlethal genetic interaction, and a proline-rich domain of cdc12p binds directly to profilin cdc3p in vitro, suggesting that one function of cdc12p in ring assembly is to bind profilin.     

Yeast Srp1, a nuclear protein related toDrosophila and mouse pendulin,is required for normal migration,division, and integrity of nuclei during mitosis

This paper describes genes from yeast and mouse with significant sequence similarities to aDrosophila gene that encodes the blood cell tumor suppressor pendulin. The protein encoded by the yeast gene, Srp1p, and mouse pendulin share 42% and 51% amino acid identity withDrosophila pendulin, respectively. All three proteins consist of 10.5 degenerate tandem repeats of ～ 42 amino acids each. Similar repeats occur in a superfamily of proteins that includes theDrosophila Armadillo protein. All three proteins contain a consensus sequence for a bipartite nuclear localization signal (NLS) in the N-terminal domain, which is not part of the repeat structure. Confocal microscopic analysis of yeast cells stained with antibodies against Srp1p reveals that this protein is intranuclear throughout the cell cycle. Targeted gene disruption shows thatSRP1 is an essential gene. Despite their sequence similarities,Drosophila and mouse pendulin are unable to rescue the lethality of anSRP1 disruption. We demonstrate that yeast cells depleted of Srp1p arrest in mitosis with a G2 content of DNA. Arrested cells display abnormal structures and orientations of the mitotic spindles, aberrant segregation of the chromatin and the nuclei, and threads of chromatin emanating from the bulk of nuclear DNA. This phenotype suggests that Srplp is required for the normal function of microtubules and the spindle pole bodies, as well as for nuclear integrity. We suggest that Srp1p interacts with multiple components of the cell nucleus that are required for mitosis and discuss its functional similarities to, and differences fromDrosophila pendulin.     

A Conserved Functional Domain of Drosophila Coracle Is Required for Localization at the Septate Junction and Has Membrane-organizing Activity

The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members.     

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.