kelch: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References
Gene name - kelch

Synonyms -

Cytological map position - 36D3

Function - actin binding protein

Keywords - oogenesis, ring canal, cytoskeleton

Symbol - kel

FlyBase ID: FBgn0001301

Genetic map position - 2L

Classification - kelch domain protein

Cellular location - cytoplasmic

NCBI link: Entrez Gene
kel orthologs: Biolitmine
Recent literature
Hudson, A. M., Mannix, K. M. and Cooley, L. (2015). Actin cytoskeletal organization in Drosophila germline ring canals depends on Kelch function in a Cullin-RING E3 ligase. Genetics [Epub ahead of print]. PubMed ID: 26384358
The Drosophila Kelch protein is required to organize the ovarian ring canal cytoskeleton. Kelch binds and crosslinks F-actin in vitro, and it also functions with Cullin 3 (Cul3) as a component of a ubiquitin E3 ligase. How these two activities contribute to cytoskeletal remodeling in vivo is not known. This study used targeted mutagenesis to investigate the mechanism of Kelch function. A model was tested in which Cul3-dependent degradation of Kelch is required for its function, but no evidence was found to support this hypothesis. However, mutant Kelch deficient in its ability to interact with Cul3 failed to rescue the kelch cytoskeletal defects, suggesting that ubiquitin ligase activity is the principal activity required in vivo. It was also determined that the proteasome is required with Kelch to promote the ordered growth of the ring canal cytoskeleton. These results indicate that Kelch organizes the cytoskeleton in vivo by targeting a protein substrate for degradation by the proteasome.
Hudson, A. M., Mannix, K. M., Gerdes, J. A., Kottemann, M. C. and Cooley, L. (2019). Targeted substrate degradation by Kelch controls the actin cytoskeleton during ring canal expansion. Development 146(1). PubMed ID: 30559276
During Drosophila oogenesis, specialized actin-based structures called ring canals form and expand to accommodate growth of the oocyte. Previous work demonstrated that Kelch and Cullin 3 function together in a Cullin 3-RING ubiquitin ligase complex (CRL3(Kelch)) to organize the ring canal cytoskeleton, presumably by targeting a substrate for proteolysis. This study used tandem affinity purification followed by mass spectrometry to identify HtsRC as the CRL3(Kelch) ring canal substrate. CRISPR-mediated mutagenesis of HtsRC revealed its requirement in the recruitment of the ring canal F-actin cytoskeleton. Genetic evidence is presented consistent with HtsRC being the CRL3(Kelch) substrate; biochemical evidence indicates that HtsRC is ubiquitylated and degraded by the proteasome. Finally, a short sequence motif was identified in HtsRC that is necessary for Kelch binding. These findings uncover an unusual mechanism during development wherein a specialized cytoskeletal structure is regulated and remodeled by the ubiquitin-proteasome system.
Hudson, A. M., Szabo, N. L., Loughran, G., Wills, N. M., Atkins, J. F. and Cooley, L. (2021). Tissue-specific dynamic codon redefinition in Drosophila. Proc Natl Acad Sci U S A 118(5). PubMed ID: 33500350
Translational stop codon readthrough occurs in organisms ranging from viruses to mammals and is especially prevalent in decoding Drosophila and viral mRNAs. Recoding of UGA, UAG, or UAA to specify an amino acid allows a proportion of the protein encoded by a single gene to be C-terminally extended. The extended product from Drosophila kelch mRNA is 160 kDa, whereas unextended Kelch protein, a subunit of a Cullin3-RING ubiquitin ligase, is 76 kDa. Previously tissue-specific regulation of readthrough of the first kelch stop codon was reported. This study characterizes major efficiency differences in a variety of cell types. Immunoblotting revealed low levels of readthrough in malpighian tubules, ovary, and testis but abundant readthrough product in lysates of larval and adult central nervous system (CNS) tissue. Reporters of readthrough demonstrated greater than 30% readthrough in adult brains, and imaging in larval and adult brains showed that readthrough occurred in neurons but not glia. The extent of readthrough stimulatory sequences flanking the readthrough stop codon was assessed in transgenic Drosophila and in human tissue culture cells where inefficient readthrough occurs. A 99-nucleotide sequence with potential to form an mRNA stem-loop 3' of the readthrough stop codon stimulated readthrough efficiency. However, even with just six nucleotides of kelch mRNA sequence 3' of the stop codon, readthrough efficiency only dropped to 6% in adult neurons. Finally, it was shown that high-efficiency readthrough in the Drosophila CNS is common; for many neuronal proteins, C-terminal extended forms of individual proteins are likely relatively abundant.
Sun, H., Shah, A. S., Bonfini, A., Buchon, N. S. and Baskin, J. M. (2023). Wnt/beta-catenin signaling within multiple cell types dependent upon kramer regulates Drosophila intestinal stem cell proliferation. bioRxiv. PubMed ID: 36865263
The gut epithelium is subject to constant renewal, a process reliant upon intestinal stem cell (ISC) proliferation that is driven by Wnt/beta-catenin signaling. Despite the importance of Wnt signaling within ISCs, the relevance of Wnt signaling within other gut cell types and the underlying mechanisms that modulate Wnt signaling in these contexts remain incompletely understood. Using challenge of the Drosophila midgut with a non-lethal enteric pathogen, \the cellular determinants of ISC proliferation, harnessing kramer, a recently identified regulator of Wnt signaling pathways, as a mechanistic tool. Wnt signaling within Prospero-positive cells supports ISC proliferation, and kramer regulates Wnt signaling in this context by antagonizing kelch, a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination. This work establishes kramer as a physiological regulator of Wnt/beta-catenin signaling in vivo and suggests enteroendocrine cells as a new cell type that regulates ISC proliferation via Wnt/β-catenin signaling.

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 src64Delta17 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 kelDE1;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).

Wnt/β-catenin signaling within multiple cell types dependent upon kramer regulates Drosophila intestinal stem cell proliferation

The gut epithelium is subject to constant renewal, a process reliant upon intestinal stem cell (ISC) proliferation that is driven by Wnt/β-catenin signaling. Despite the importance of Wnt signaling within ISCs, the relevance of Wnt signaling within other gut cell types and the underlying mechanisms that modulate Wnt signaling in these contexts remain incompletely understood. Using challenge of the Drosophila midgut with a non-lethal enteric pathogen, this study examined the cellular determinants of ISC proliferation, harnessing Kramer, a recently identified regulator of Wnt signaling pathways, as a mechanistic tool. Wnt signaling within Prospero-positive cells supports ISC proliferation and kramer regulates Wnt signaling in this context by antagonizing Kelch, a Cullin-3 E3 ligase adaptor that mediates Dishevelled polyubiquitination. This work establishes Kramer as a physiological regulator of Wnt/β-catenin signaling in vivo and suggests enteroendocrine cells as a new cell type that regulates ISC proliferation via Wnt/β-catenin signaling (Sun, 2023).

The adult Drosophila melanogaster intestine is a powerful model to study stem cell proliferation.1-5 The fly gut has many important physiological functions, most notably nutrient absorption, acting as a physical barrier and providing immunity to enteric pathogens and chemical insults.1,2 A conserved hallmark of the gut is the dynamic nature of its architecture.3-5 The Drosophila midgut is the largest and central portion of the intestines, and it is analogous to the mammalian small intestine in function and, to some extent, cellular architecture and composition (Sun, 2023).

The signature feature of the midgut is its epithelium, a single cell layer acting as a barrier to separate the lumen from internal tissues. In Drosophila, the midgut epithelium comprises four principal cell types. Intestinal stem cells (ISCs) can self-renew and also give rise to progenitor cells termed enteroblasts (EBs), which in turn can fully differentiate into enterocytes (ECs) that comprise the bulk of the epithelium. The specification of the fourth cell type, enteroendocrine cells (EEs), has not been fully elucidated, despite the importance of these cells in mediating important paracrine signaling events. EBs have been proposed to differentiate into EEs; however, recent studies demonstrated that ISCs, when Prospero-positive, divide into a distinct progenitor type termed pre-EEs, which subsequently differentiate into EEs. Disruption of ISC function can lead to either excessive proliferation or precocious differentiation, often resulting in disease. Therefore, a detailed understanding of the pathways and mechanisms regulating ISC proliferation is an important long-term goal with therapeutic implications (Sun, 2023 and references therein).

Numerous studies have shown that Wnt/β-catenin signaling, a morphogen signaling pathway that is highly conserved in animals, promotes ISC proliferation and differentiation under both physiological conditions and upon challenges such as enteric infection or chemical insults, both of which can damage the gut epithelium. More broadly, Wnt/β-catenin signaling, also known as canonical Wnt signaling, controls diverse cellular processes during animal development and homeostasis, including stem cell maintenance, cell fate specification, neural patterning, spindle orientation, cell migration, cell polarity, and gap junction communication. Dysregulation of canonical Wnt signaling caused by mutations of core components of this pathway is frequently linked to birth defects and many types of cancer. During tissue development and homeostasis, canonical Wnt signaling is thought to be the main pathway for regulating ISC proliferation and self-renewal, which drives massive renewal processes of intestinal epithelial cells (Sun, 2023).

Despite the fundamental importance of Wnt signaling in regulating ISC proliferation and subsequent tissue renewal in the gut epithelium, understanding of how Wnt signaling in different cell types within the gut contributes to these effects on ISCs is still rudimentary. Furthermore, recent studies have elucidated roles for β-catenin-independent, or non-canonical Wnt signaling pathways, which share some upstream components in the Wnt-receiving cell but do not activate β-catenin-dependent gene expression, in regulating ISC proliferation in the Drosophila midgut. Thus, knowledge of which cell types exhibit canonical and non-canonical Wnt signaling that collectively contribute to ISC proliferation and thus tissue maintenance in the midgut are major fundamental and unanswered questions (Sun, 2023).

A key shared player in all Wnt signaling pathways is Dishevelled (Dsh/DVL), which is recruited to the plasma membrane upon activation of Wnt receptors and co-receptors from the Frizzled and LRP families. Such recruitment catalyzes the disassembly of a multiprotein complex that facilitates proteasomal degradation of β-catenin, enabling its accumulation and subsequent translocation to the nucleus to activate TCF/LEF-dependent gene expression in the canonical pathway. Dsh recruitment to the plasma membrane also activates planar cell polarity and other non-canonical Wnt pathways, including the Wnt/Ca2+ pathway, by activation of Frizzled receptors. Thus, regulation of Dsh levels, which occurs via the ubiquitin-proteasome system and involves the action of several distinct E3 ubiquitin ligases, is a key control point in all Wnt signaling pathways (Sun, 2023).

Notably, a mammalian multi-subunit phosphoinositide-binding protein, pleckstrin homology domain-containing family A number 4 (PLEKHA4), promotes both Wnt/β-catenin and non-canonical Wnt signaling in human cell lines by antagonizing DVL polyubiquitination by the Cullin-3 (CUL3)-Kelch-like protein 12 (KLHL12) E3 ubiquitin ligase. This study found as well that Wnt/β-catenin signaling and subsequent cell proliferation in mouse models of melanoma was dependent upon PLEKHA4 expression (Shah, 2021). In Drosophila, knockout of the closest fly ortholog of PLEKHA4, kramer (kmr), impairs planar cell polarity in the adult wing, larval wing imaginal disc, and pupal wing disc epithelium. The absence of any discernable defects in canonical Wnt signaling in kmr knockout flies led the authors to question whether kmr indeed controlled Wnt/β-catenin signaling in this organism. It is proposed that the extent to which PLEKHA4/kmr loss affected canonical or non-canonical Wnt pathways might depend on cellular and tissue contexts, where expression of other factors downstream of DVL/Dsh might govern how tuning of DVL/Dsh levels would differentially affect outcomes from these pathways (Sun, 2023).

To test this prediction, this study investigated the role of kmr in controlling ISC proliferation in the Drosophila midgut, a physiological process dependent upon canonical Wnt signaling and recently linked to non-canonical Wnt signaling pathways as well. The experimental model used in this study involved challenge of adult flies with E. carotovora carotovora 15 (Ecc15), a gram-negative bacterium that produces non-lethal infection, to damage the midgut epithelium and induce repair pathways dependent upon ISC proliferation. Global knockout and cell type-specific knockdown of kmr was performed and effects on tissue pathophysiology were compared to those induced by knockdown of other established components of Wnt signaling pathways. As such, this study accomplished several goals. First, roles for kmr were established in controlling canonical Wnt signaling in Drosophila. Second, kmr was used as a tool to elucidate roles for canonical and non-canonical Wnt signaling within different cell types in the Drosophila midgut. These studies reveal not only that kmr-dependent canonical Wnt signaling controls ISC proliferation in the midgut but that such signaling occurs in several cell types, including Prospero-positive cells, suggesting that EEs, which these studies support derive from pre-EE progenitors, may play an unexpectedly important role in these processes (Sun, 2023).

Proliferation and differentiation of intestinal stem cells in the adult Drosophila midgut is essential to maintain the balance of tissue homeostasis and prevent excessive proliferation in this tissue. Wnt/β-catenin signaling plays central roles in tissue maintenance during development, including in the midgut. However, how canonical Wnt signaling is activated within intestinal stem cell progenitors and by fully differentiated progeny cells is still not well understood. This study discovered a new player, kramer (kmr), that regulates canonical Wnt signaling in the Drosophila midgut using a challenge with the non-lethal pathogen Erwinia carotovora carotovora (Ecc15) to induce massive ISC proliferation required to rebuild the damaged gut epithelium (Sun, 2023).

Using this system, kmr was established as a positive regulator of ISC proliferation and Wnt/β-catenin signaling. Inducible, cell type-specific kmr knockdown was used as a tool to interrogate the requirements for Wnt signaling within each cell type in the gut for controlling ISC proliferation. In addition to known effects of Wnt signaling within intestinal stem cells (ISCs), their immediate downstream progenitors termed enteroblasts (EBs), and fully differentiated enterocytes (ECs) in governing this process, this study unexpectedly points to roles for Wnt signaling within enteroendocrine cells (EEs) as a mechanism controlling stem cell proliferation in the Drosophila midgut (Sun, 2023).

It was first shown that interruption of kmr function decreases the expression of a canonical Wnt signaling target gene, fz3, in the posterior midgut, consistent with studies demonstrating fz3RFP expression in both ISCs and ECs showing that knockout of Wnt signaling components led to loss of fz3 expression in midgut region R5. Though ECs are the primary cell type in which Wnt signaling is activated, studies using kmr knockdown in multiple cell types suggest that Wnt signaling is also active in ISCs/EBs and EEs in posterior end of the midgut. These studies also revealed that loss of kmr in multiple cell types results in fewer EE cells and decreased ISC proliferation in the posterior midgut, indicating a role for kmr in EE differentiation (Sun, 2023).

Further, kmr knockdown in all cell types caused a decrease in proliferating, phospho-H3 positive (pH3+) cells, though staining with Armadillo/Prospero antibodies, which enables identification of ISCs/EBs and EEs, suggests that kmr knockdown in EE cells specifically downregulates stem cell proliferation. This distinction is important because pH3+ cells include all dividing progenitor cells, including ISCs/EBs and pre-EEs, and the data showed that kmr knockdown in all cell types significantly decreased dividing stem cells. Notably, kmr knockdown in ISCs/EBs and in ECs did not result in a defect in number of Arm+/Pro- cells (i.e., ISCs/EBs); However, kmr knockdown in Pro+ cells led to a reduction in number of ISCs/EBs, suggesting that kmr expression in EE cells regulates the proliferation of neighboring ISCs in a non-autonomous manner. These data also provide support to a model wherein EEs derive not from EBs but instead from a distinct set of progenitors termed pre-EEs (Sun, 2023).

This study also sheds light on the mechanisms by which kmr expression in distinct midgut cell types might regulate the physiological response to Ecc15 infection. Prior work on the mammalian ortholog of kmr, PLEKHA4, revealed that it promotes Wnt signaling pathways via physical interaction with KLHL12, a CUL3 E3 ubiquitin ligase substrate-specific adaptor and negative regulator of DVL. By binding to KLHL12 and sequestering it in plasma membrane-associated clusters, PLEKHA4 prevents DVL polyubiquitination by CUL3-KLHL12, ultimately causing elevated DVL levels and enhanced Wnt signaling in Wnt-receiving cells (Sun, 2023).

Because of the pleiotropic roles for DVL in both canonical Wnt/β-catenin and non-canonical β-catenin-independent pathways, this study found that PLEKHA4 knockdown affected both of these pathways. Previous studies on kmr in Drosophila, focusing on hair patterning, identified defects in planar cell polarity (PCP), a Frizzled- and Dsh-dependent pathway in flies. However, the present study identifies for the first time a role for kmr in promoting canonical Wnt/β-catenin signaling in vivo. By analyzing knockdown of the two fly KLHL12 orthologs, kelch and diablo, this study found that kmr acts in opposition to kelch, much as PLEKHA4 does with KLHL12 in mammalian cells, supporting the established mechanism of action. Finally, Wnt/β-catenin and PCP signaling were independently blocked and ISC proliferation following Ecc15 challenge was assessed; only blockade of Wnt/β-catenin signaling was found to exactly phenocopy loss of kmr (Sun, 2023).

In conclusion, this study reveals that the Dsh regulator kmr is a new, important regulator of Wnt/β-catenin signaling and ISC proliferation in vivo. As well, kmr was harnessed as a tool to systematically investigate the role of Wnt signaling in each of the major cell types in the Drosophila midgut in controlling ISC proliferation and differentiation during epithelial repair following challenge with an enteric pathogen. These studies suggest that Wnt signaling within enteroendocrine cells can control this process, adding a new layer of regulation to this important physiological process. Future studies will be necessary to identify downstream mechanisms by which EEs may non-autonomously regulate ISC proliferation in the midgut, as well as the existence of similar pathways in mammalian systems (Sun, 2023).


cDNA clone length - 5619

Bases in 5' UTR - 278

Exons - 6

Bases in 3' UTR - 907


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).

kelch: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 25 September 2023

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