BIOLOGICAL OVERVIEW

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. Kelch functions to cross-link actin fibers. Biochemical studies using purified, recombinant Kelch protein show that full-length Kelch bundles actin filaments, and kelch repeat 5 contains the actin binding site. Kelch is tyrosine phosphorylated in a Src64-dependent pathway at tyrosine residue 627 (see Src oncogene at 64B). A Kelch mutant with tyrosine 627 changed to alanine (KelY627A) rescues the actin disorganization phenotype of kelch mutant ring canals, but fails to produce wild-type ring canals. Phosphorylation of Kelch is critical for the proper morphogenesis of actin during ring canal growth, and presence of the nonphosphorylatable KelY627A protein phenocopies Src64 ring canals. KelY627A protein in ring canals also dramatically reduces 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 Kelch-dependent actin cross-linking (Kelso, 2002).

In Drosophila, 15 syncytial nurse cells and 1 oocyte are enveloped by a monolayer of somatic follicle cells and constitutes an egg chamber, the structural and functional unit of the Drosophila ovary. A ring canal is a gateway through which mRNAs, proteins, and nutrients flow from nurse cells into the oocyte during the entire course of oogenesis. Ring canals are derived from arrested mitotic cleavage furrows that are modified by the addition of several proteins. These include abundant F-actin, at least one protein that is recognized by antiphosphotyrosine antibodies (PY protein), a mucin-like glycoprotein, the Hts ring canal protein (HtsRC), ABP280/filamin, Tec29 and Src64 tyrosine kinases, and Kelch (Xue, 1993; Robinson, 1997a; Kelso, 2002 and references therein).

As nurse cell cytoplasm transport proceeds, the diameter of ring canals grows from <1 µm to 10-12 µm. This represents the addition of over one inch of filamentous actin during a period in which the filament density remains constant. Near the end of oogenesis, the ring canal actin transforms from a single continuous bundle into several interwoven actin cables. Ring canal expansion probably involves the nucleation of new actin filaments and an increase in actin filament length, coupled with filament reorganization that requires the establishment of reversible actin cross-links (Kelso, 2002).

Kelch protein is required for ring canal morphogenesis (Xue, 1993; Tilney, 1996; Robinson and Cooley, 1997a). Ring canal actin in kelch mutant egg chambers is severely disorganized and partially occludes the lumen. This leads to a defect in cytoplasm transport and the production of small, sterile eggs (Xue, 1993). Kelch is a multidomain protein and a member of a superfamily of proteins defined, in part, by the presence of six 50-amino acid kelch repeats (KREPs). Based on sequence similarity to galactose oxidase, the KREP domain is predicted to fold into a six-bladed ß-propeller (Bork, 1994; Adams, 2000). In Limulus the KREP domain is present in at least three scruin proteins, each of which contains two KREP domains (Way, 1995). The KREP domains of alpha-scruin each form an F-actin binding domain that allows alpha-scruin to act as an actin filament-cross-linking protein (Tilney, 1975; Bullitt, 1988; Sanders, 1996; Sun, 1997). Another KREP protein, Mayven, is found in human brain extracts and tightly colocalizes with F-actin in cultured human U373-MG astrocytoma/glioblastoma cells (Soltysik-Espanola, 1999). The second conserved domain in Kelch is the BTB/POZ (broad complex, tramtrack, and bric-á-brac; also known as the poxvirus and zinc finger domain) dimerization domain. The molecular makeup of the Kelch protein and the morphology of the kelch mutant ring canals suggest that Kelch could organize actin filaments by acting as a dimeric cross-linking protein (Robinson, 1997a; Kelso, 2002 and references therein).

A signaling cascade that leads to malformed ring canals involves the Src family kinases (SFKs) Src64 and Tec29 (Btk family kinase at 29A). SFKs are associated with the phosphorylation of several important proteins involved in regulating F-actin-rich structures, including cell-substrate adhesions, cell-cell adhesions, and actin regulatory proteins such as p190 RhoGAP, cortactin, and ABP280/filamin. Src mutations in mice result in osteoclasts deficient in the formation of ruffled borders and defective in forming the peripheral actin ring. Mutations in Drosophila Src64 or tec29 lead to small ring canals that lack most phosphotyrosine staining, and egg chambers that have incomplete nurse cell cytoplasm transport (Kelso, 2002 and references therein).

Using a series of two-dimensional (2D) gel electrophoresis experiments, it has been determined that Kelch is phosphorylated in an SFK-dependent manner. Site-directed mutagenesis has been used to map the phosphorylated tyrosine residue. Thin section electron microscopy has revealed striking differences in actin organization and filament number in lines expressing wild-type Kelch when compared with src64^Delta17 and the nonphosphorylatable form of Kelch. This shows that phosphorylation of Kelch is necessary for normal filament organization. Binding studies show that the phosphorylated form of Kelch does not interact with actin. Therefore, Src64-mediated phosphorylation probably dissociates Kelch cross-links in ring canals. The nonphosphorylatable mutant also causes a reduction in actin monomer turnover kinetics. This suggests that reversible cross-links are required to allow dynamic actin monomer turnover and maintain overall ring canal morphology. These observations suggest that a major cytoskeletal target of Src64 signaling at the ring canal is the actin-cross-linking protein Kelch (Kelso, 2002).

The dynamics of actin filaments in ring canals have been elegantly described at the ultrastructural level (Riparbelli, 1995; Tilney, 1996). Ring canals are built at the positions of arrested cleavage furrows that form during the mitotic divisions of germline cells. The mechanism of cleavage furrow arrest is likely to be conserved among animal species because incomplete cytokinesis occurs during the proliferation of germline cells in many animals. In Drosophila, once egg chambers are fully assembled, ring canal growth happens in two phases. Initially, the thickness of the actin rim increases to ~0.3 µm as the diameter of the ring grows slowly to 2 µm. Subsequently, the thickness of the actin rim and the density of actin filaments remain constant while the rate of ring canal expansion increases. The net increase of actin within ring canals overall is 134-fold (Tilney, 1996). During the rapid phase of ring canal growth, actin filaments must be polymerized, probably at the plasma membrane, to expand the ring canal rim, and disassembled at the cytoplasmic face to maintain the lumen. This analysis of the Kelch protein has shown that precise regulation of actin filament cross-linking by phosphorylation is critical during rapid ring canal growth (Kelso, 2002).

The behavior of ring canals that contain KelY627A provides significant insight into Kelch function. The absence of Kelch phosphorylation leads to ring canals that accumulate more actin filaments than normal, possibly due to a slowing in the rate of actin depolymerization relative to the rate of polymerization. After about stage 8 of oogenesis, the failure to resolve the continuous sheet of actin filaments into discreet cables may be another consequence of inhibiting depolymerization. The presence of more 'permanent' Kelch cross-links may reduce the accessibility of the filament network to depolymerizing factors. In vitro experiments have demonstrated that actin-cross-linking proteins alone are capable of inhibiting the rate of pyrenyl F-actin depolymerization. Another possible explanation for these phenotypes is that because Kelch cross-links are no longer easily reversible, filament reorientation or sliding is restricted during ring canal growth (Kelso, 2002).

Fluorescence recovery after photobleaching (FRAP) experiments provide two additional insights into the actin dynamics at the ring canal.(1) Ring canal actin is highly dynamic. The rate of actin monomer turnover found in wild-type ring canals is comparable to the kinetics of actin turnover found in the leading edge of motile goldfish epithelial keratocytes. This would be consistent with a population of actin that is constantly undergoing a rapid cycle of polymerization and depolymerization. (2) The presence of nonregulated Kelch clearly results in a dramatic reduction in the dynamics of actin. This supports the model that mutant Kelch protein reduces accessibility to other actin-binding proteins, in this case proteins involved with polymerization or depolymerization. It is proposed that this effect could be due to bound Kelch acting as a stabilizing protein much in the same way that tropomyosin protects F-actin from actin depolymerizing factor/cofilin (Kelso, 2002 and references therein).

Studies involving the actin polymerization factor Arp2/3 have demonstrated that ring canal stability and growth is dependent on the presence of a functional Arp2/3 complex (Hudson, 2002). The effects of mutations in Arp2/3 complex subunits are progressively more severe as egg chambers develop, and by stage 6, ring canals begin to collapse. In kelch null mutants, the actin filaments are initially well organized, begin to show signs of disorganization around stage 4, and are completely disorganized starting at stage 6 (Tilney, 1996; Robinson, 1997a). Interestingly, thin section electron micrographs of kel^DE1;P[kelY627A]/+ show signs of actin filament disruption beginning at stage 6. The coincidence of kelch and Arp2/3 complex mutant phenotypes with the onset of rapid ring canal expansion and the presence of highly dynamic actin, suggest a model where ring canal growth is powered by de novo actin polymerization accompanied by regulated cross-links. Therefore, ring canal growth may be mechanistically similar to the movement of plasma membranes at the leading edge of motile cells. Future work on ring canal actin organization should include platinum replica electron microscopy to understand the overall organization of the ring canal actin filament network. This will allow direct comparison to the actin filament networks of lamellipodia in Xenopus laevis keratocytes and fibroblasts (Kelso, 2002 and references therein).

Intriguingly, the accumulation of actin during earlier stages of oogenesis is apparently independent of both Kelch and the Arp2/3 complex. Characterization of other mutants affecting ring canals has revealed genes required for initial stages of ring canal assembly. These include the cheerio gene that encodes the actin filament-cross-linking protein ABP280/filamin. In cheerio mutants, ring canal actin is absent. In addition, HtsRC is required for the early accumulation of actin filaments; however, it has not been determined whether HtsRC interacts directly with F-actin or affects actin polymerization. Therefore, additional research is needed to elucidate the mechanism of early ring canal biogenesis (Kelso, 2002 and references therein).

The regulation of Kelch-actin cross-links could be accomplished by Src64 directly phosphorylating Kelch. Alternatively, Src64 may activate another protein tyrosine kinase, such as Tec29, which in turn phosphorylates Kelch. However, the shared phenotype seen by electron microscopy of the src64 and P[kelY627A] ring canals is strongly suggestive of Kelch being the major downstream component of a Src64 cascade. Analysis of Kelch phosphorylation in tec29 mutants is difficult because available tec29 alleles are lethal. SFKs have been shown to signal rearrangements in the actin cytoskeleton in other contexts. In Drosophila, embryos mutant for src64 or tec29 fail to complete epidermal closure at the end of gastrulation. This is, in part, because the leading edge cells contain reduced quantities of F-actin, and the cells only partially elongate and fail to migrate completely. SFKs are also known to interact directly with cytoskeletal proteins, as in the case of c-Src and cortactin. Phosphorylation of cortactin by c-Src tyrosine kinase decreases its ability to cross-link F-actin in vitro. These examples suggest that there could be a critical role played by tyrosine phosphatases to ensure that F-actin does not become disorganized due to excessive phosphorylation of cross-linking proteins. There are several candidate phosphatases in Drosophila; however, their roles in ring canal development have not been studied (Kelso, 2002 and references therein).

It should be noted that not all Kelch family members are actin-binding proteins (for review see Adams, 2000). For example, nuclear restricted protein/brain (NRP/B) is a novel nuclear matrix protein that contains a highly conserved KREP domain. NRP/B is specifically expressed in primary neurons and participates in the regulation of neuronal process formation. A direct interaction with actin by the ectoderm neural cortex-1 protein has been demonstrated by coimmunoprecipitation; however, it does not exclusively colocalize with F-actin in Daoy cells, and it is perinuclear in neuronal cell lines (Kelso, 2002 and references therein).

A Kelch family member that interacts with actin is called Mayven. Mayven localizes to the leading edge of the lamellipodia in U373-MG astrocytoma/glioblastoma cells (Soltysik-Espanola, 1999). Mayven is also localized with the focal adhesion kinase (Soltysik-Espanola, 1999), suggesting it could play a role in actin reorganization at focal adhesion plaques. A role for phosphorylation in the regulation of Mayven has not been reported (Kelso, 2002).

In Limulus, it has been postulated that the Kelch homolog alpha-scruin acts as a protein that allows F-actin to rapidly twist and slide during acrosome extension or 'true discharge' (Sherman, 1999). Biochemical studies performed on alpha-scruin (Sun, 1997) have shown that the cysteine corresponding to Drosophila Kelch residue 628 lies within the alpha-scruin actin binding domain. Thus, both Kelch and alpha-scruin contain an actin binding site within KREP number 5. However, alpha-scruin does not have a tyrosine comparable to Kelch residue 627 in the primary sequence; therefore, regulation of alpha-scruin cross-linking is likely to be different from that for Kelch. alpha-Scruin regulation may target scruin-scruin interactions rather than scruin-actin interactions (Kelso, 2002).

GENE STRUCTURE

cDNA clone length - 5619

Bases in 5' UTR - 278

Exons - 6

Bases in 3' UTR - 907

PROTEIN STRUCTURE

Amino Acids - 689 (ORF1); 1477 (ORF2)

Structural Domains

The Drosophila kelch gene produces a single transcript separated into two open reading frames (ORFs) by a UGA stop codon. Only ORF1 and full length (ORF1 plus ORF2) kelch proteins are made (Xue, 1993; Robinson, 1997b). The ORF1 product is a member of a family of kelch-related proteins that includes several Pox virus ORFs, mammalian calicin, and Caenorhabditis elegans spe26. Currently, the protein databases contain four kelch family proteins from C. elegans and at least five mammalian kelch family proteins. Interestingly, the Drosophila kelch ORF1 contains ~110 amino acids (the NTR) at the amino terminus not found in other kelch-related proteins. The Drosophila kelch ORF2 domain encodes a protein with no significant homology to known proteins and so far is specific to Drosophila. Although the two Kelch protein (ORF1 and full length) motifs are conserved in several Drosophila species, the ORF1 protein is sufficient for kelch function (Robinson, 1997a; Robinson, 1997b).

Kelch ORF1 contains two conserved domains found in other kelch proteins as well as in nonkelch proteins. The first of these, the BTB or POZ domain, is a 120-amino acid motif that is found immediately after the amino-terminal region (NTR) in Kelch. This domain is also found in several zinc finger-containing transcription factors and it has been shown to mediate dimerization in vitro. A second domain consists of six 50-amino acid repeats known as kelch repeats (Xue, 1993). Kelch repeats are found in several nonkelch proteins including a recently characterized protein in Physarum polycephalum called actin-fragmin kinase. The kelch repeat sequence is predicted to fold into a superbarrel or ß-flower structure (Bork, 1994), similar to the repeat sequences in a family of bacterial, fungal, and influenza virus enzymes such as neuraminidase, galactose oxidase, and the sialidases (Robinson, 1997a).

date revised: 15 September 2003

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