InteractiveFly: GeneBrief

sol narae: Biological Overview | References

Wnt/ Wingless (Wg) is essential for embryonic development and adult homeostasis in all metazoans, but the mechanisms by which secreted Wnt/Wg is processed remain largely unknown. A Drosophila Sol narae (Sona) is a member of A Disintegrin And Metalloprotease with ThromboSpondin motif (ADAMTS) family, and positively regulates Wg signaling by promoting Wg secretion. This study reports that Sona and Wg are secreted by both conventional Golgi and exosomal transports, and Sona cleaves extracellular Wg at the two specific sites, leading to the generation of N-terminal domain (NTD) and C-terminal domain (CTD) fragments. The cleaved forms of extracellular Wg were detected in the extracellular region of fly wing discs, and its level was substantially reduced in sona mutants. Transient overexpression of Wg-CTD increased wing size while prolonged overexpression caused lethality and developmental defects. In contrast, Wg-NTD did not induce any phenotype. Moreover, the wing defects and lethality induced by sona RNAi were considerably rescued by Wg-CTD, indicating that a main function of extracellular Sona is the generation of Wg-CTD. Wg-CTD stabilized cytoplasmic Armadillo (Arm) and had genetic interactions with components of canonical Wg signaling. Wg-CTD also induced Wg downstream targets such as Distal-less (Dll) and Vestigial (Vg). Most importantly, Cyclin D (Cyc D) was induced by Wg-CTD but not by full-length Wg. Because Sona also induces Cyc D in a cell non-autonomous manner, Wg-CTD generated by Sona in the extracellular region activates a subset of Wg signaling whose major function is the regulation of cell proliferation (Won, 2019).

Cellular communication via components in the extracellular matrix (ECM) is essential for cell survival and proliferation as well as differentiation. Extracellular proteases play important roles in regulating activity, localization and stability of the ECM proteins. Despite the importance of these proteases, their specific functions are still largely unexplored. ADAMTS family contains extracellular proteases that are present only in metazoans. Six and nineteen members have so far been identified in flies and mammals, respectively. Mammalian ADAMTSs are involved in cell proliferation, angiogenesis and organogenesis, so their malfunctions result in various diseases such as cancer, arthritis, and arteriosclerosis. An ADAMTS Sol narae (Sona) is essential for fly development. Loss of sona decreases the level of extracellular Wg, and sona exhibits positive genetic interaction with wntless (wls) that encodes a cargo protein for Wg. Therefore, intracellular Sona seems to cooperate with Wls in Wg secretion. A new function of extracellular Sona in cell survival and cell proliferation has been reported (Tsogtbaatar, 2019). sona has genetic interactions with cell death-related genes such as Death-associated inhibitor of apoptosis (Diap1) and reaper. Interestingly, Sona upregulates Cyclin D (Cyc D) in a cell non-autonomous manner, and increases tissue size. Cyc D is a G1 Cyclin to initiate the cell cycle by responding to the mitogen signals Therefore, it is possible that extracellular Sona generates a yet unidentified signaling molecule that induces Cyc D in the signal-receiving cells (Won, 2019).

Wnt family is essential for animal development, and has been extensively studied since a mutant of fly Wg, the homolog of vertebrate Wnt1, was described a century ago. Wnt is secreted by both conventional Golgi-mediated transport and exosomal secretion pathway. Interaction between Wnt and Frizzled (Fz) receptors initiates a cascade of intracellular responses in the responding cells that lead to downstream gene expression. In flies, Wg is involved in cell proliferation, differentiation, and survival by inducing Wg effector components including Vestigial (Vg), Distal-less (Dll) and Senseless (Sens). In mammals, Wnt signaling promotes cell proliferation by transcriptional activation of multiple target genes such as c-Myc and Cyc D and its malfunction leads to various diseases such as cancer, neurodegenerative diseases, inflammatory disease, and diabetes (Won, 2019).

The role of extracellular Sona was explored in this study; Sona was found to generate NTD and CTD fragments of Wg by cleaving extracellular Wg. The Wg-CTD fragment was similar to full-length Wg in activating canonical Wg signaling but was dissimilar to full-length Wg in Cyc D induction, lack of Sens induction, and protein instability. Thus, one of the main functions of Sona is to generate Wg-CTD that carries out subsets of Wg signaling (Won, 2019).

This paper reports that Sona cleaves extracellular Wg into Wg-NTD and Wg-CTD, and the Wg-CTD is a new form of active Wg. Because Wg-CTD substantially rescued the sona loss-of-function phenotypes such as lethality and wing defects, generation of Wg-CTD seems to be one of Sona’s major functions. Wnt modifications such as lipidation and glycosylation have been extensively studied, but Wnt cleavage has not been addressed except for the Xenopus Tiki protease. Tiki reduces Wnt secretion by cleaving the amino-terminal region of intracellular Wnt that is required for the lipidation of Wnt. While Tiki aims to decrease the amount of secreted Wnt, Sona aims to generate a new active form of Wg from an already active WgFL (Won, 2019).

Genetic interaction between wg-CTD and other Wg signaling components indicates that Wg-CTD activates Wg signaling similar to WgFL. However, there are several differences between these two forms of Wg. First, Wg-CTD but not WgFL increased the level of Cyc D. Overexpressed Cyc D-Cdk4 in flies accelerates cell division of undifferentiated cells such as wing disc cells. Sona also induces Cyc D and promotes cell proliferation in a cell non-autonomous manner. Therefore, Wg-CTD generated by extracellular Sona seems to induce Cyc D in the neighboring cells for cell proliferation. Second, both forms of Wg-CTD, Wg-CTDL1 and Wg-CTDL2, are less stable than WgFL. Instability of Wg-CTD may be an essential feature because mitogens and their downstream components are often removed by degradation to prevent excessive cell proliferation. Presence of Wg-CTDL2-like structures in wing discs, however, implies that these Wg-CTDL2-like structures may be stabilized in vivo by ECM components to achieve spatiotemporal regulation of the mitogenic activity. Third, Wg-CTD is not able to induce Sens. Sens expression in the DV midline is required for differentiation of wing margin bristles, unlike Vg that is essential for cell proliferation and cell survival (Won, 2019).

The difference between the two Wg forms in Sens induction may be due to their differential affinity to Fz receptors, based on the report that NTD and CTD of vertebrate Wnts able to interact Fz receptors independently from each other with different affinity. It has been proposed that Wnt is generated during evolution via the fortuitous fusion of two ancestral proteins analogous to its NTD, homologous to a class of lipid-interacting proteins, and CTD, homologous to a group of cytokines involved in cell signaling. This explains why NTD mutants are unable to be secreted, while CTD mutants are secreted but inactive. Given the evolutionary conservation of the components of Wnt signaling, ADAMTSs may also be involved in the generation of functional Wnt-CTD in mammals. Further study on the relationship between Wnts and ADAMTSs will expand understanding of Wnt signaling and Wnt-related diseases (Won, 2019).

An ADAMTS Sol narae is required for cell survival in Drosophila

Cell survival is essential for all living organisms to cope against multiple environmental insults. Intercellular signaling between dying and surviving cells plays an important role to ensure compensatory proliferation, preventing tissue loss after environmental stresses. This study shows that Sol narae (Sona), a Disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) in Drosophila is required for cell survival. sona exhibited a positive genetic interaction with Death-associated inhibitor of apoptosis 1 (Diap1), and a negative genetic interaction with reaper (rpr). Transcription patterns of sona, Diap1, and rpr genes in the pouch region of wing discs were coordinately changed after irradiation. Interestingly, there was a negative correlation in the expression levels of Sona and DIAP1, and both cell types, one with high Sona level and the other with high Diap1 level, were resistant to irradiation-induced cell death. The sona-expressing cells rarely entered into cell cycle themselves but promoted the nearby cells to proliferate in irradiation conditions. These sona-expressing cells are able to upregulate Cyclin D (Cyc D) and increase tissue size. Furthermore, transient Sona overexpression increased survival rate and promoted development of flies in irradiation conditions. It is proposed that the two types of radiation-resistant cells, one with high Sona level and the other with high Diap1 level, communicate with dying cells and between each other for cell survival and proliferation in response to irradiation (Tsogtbaatar, 2019).

This study has shown that Sona functions in two different ways. First, Sona is required cell autonomously for cell survival, and the level of sona transcription correlates with the degree of cell survival under the irradiated condition. Cells expressing a high level of either sona or Diap1 are resistant to cell death, so there are at least two different types of surviving cells in wing discs. Second, Sona induces cell proliferation cell non-autonomously. Transient expression of sona upregulated Cyc D in neighboring cells but not in sona-expressing cells, suggesting that radiation-resistant sona-expressing cells secrete signaling molecule(s) that upregulates Cyc D in neighboring cells. In conjunction with genetic interactions between sona, Diap1 and rpr, cells expressing sona may dynamically interplay with other cells that express Diap1 and rpr for damage control (Tsogtbaatar, 2019).

Sona is expressed in the patchy pattern, and sona clones with different sizes were generated in the wing pouch region. It is speculated that the large sona clones are generated where sona is not expressed. Similar result was reported in the loss-of-function clone analysis of the vestigial (vg) gene that is essential for survival and proliferation of wing disc cells. vg RNAi clones are generated where Vg is not expressed or expressed at a low level in wing discs. That is, the loss-of-function vg clones are rarely formed where Vg is required for cell survival and proliferation. Indeed, Vg drives cell cycle progression by inducing dE2F1 gene whose product is essential for G1 to S transition in cell cycle. Formation of clones with different sizes may be a general phenomenon for the genes that are expressed unevenly and are required for cell proliferation or cell survival. Varied levels of sona and vg expression in different parts of wing discs may be required to create regional differences in growth rate in order to form wings with proper size and shape (Tsogtbaatar, 2019).

What are the distinct functions of sona-expressing cells? In normal conditions, sona is expressed in a widely varing level in a patchy pattern, and acts as a signal to promote cell proliferation for wing development. In irradiated conditions, the transcriptional level of sona dramatically increased among sona-expressing cells that may be required to cope against extensive cell death. This irradiation generated two major cell groups in the wing pouch. The first group of cells are determined to grow and proliferate or, alternatively, to die upon a high level of irradiation. The second group of cells are resistant to irradiation and send out signals to regulate the first group of cells. The first group of cells should express proteins for cell growth, cell proliferation, cell survival and cell death including Diap1 and Rpr. The second group of cells should have capacity to sense the changes in environment and send out signals to regulate the first group of cells. In this study, Sona was identified as one of the proteins expressed in the second group of cells in a patchy pattern. Interestingly, a phosphorylated form of ribosomal protein S6 (pS6) that should be regulated in the first group of cells is also present in a patchy pattern. It is speculated that pS6 and sona may express in the first and the second groups of cells, respectively (Tsogtbaatar, 2019).

This study showed that Sona induces Cyc D in nearby cells, which is consistent with the finding that overexpressed Sona increases tissue size. Cyc D is known as a target protein of Wnt signaling in mammals, and acts in a G1 phase of cell cycle that responds to mitogens. In flies, overexpression of Cyc D/Cdk4 accelerates cell division in proliferating wing disc cells while increasing the cell size in post-mitotic cells by endoreplication. Because Cyc D is required for cell division of surviving cells, it should be upregulated in Diap1-expressing cells. Indeed, Diap1 is induced by Yorkie and STAT in Hippo pathway and JAK-STAT pathway, respectively, and these two pathways are essential for cell proliferation in a cell autonomous manner. An important question is the identity of the direct signal(s) that upregulate Cyc D. Secreted Sona may act as a direct signal or, as a metalloprotease, may modulate the activity of other signaling molecules such as Wg. It has been reported that irradiation activates a damage-activated enhancer BRV118 of the wg gene. Therefore, irradiation changes the transcription pattern of both sona and wg, and may coordinate the functions of Sona and Wg for induction of Cyc D, although Sona may activate yet another signaling molecules (Tsogtbaatar, 2019).

An interesting new mechanism to repair tissue damage has been recently discovered, in which cells of the presumptive hinge region in the wing disc migrate into the pouch region after irradiation. These hinge cells also migrate into the pouch region when the pouch cells are killed by hid expression. These hinge cells are resistant to cell death due to the activation of Wg signaling and STAT signaling that suppress the transcription of rpr52. Resistance to irradiation and lack of rpr expression are also characteristics of sona-expressing cells, suggesting that the sona-expressing cells in the wing pouch may be functionally related to the radiation-resistant hinge cells. Because intense irradiation physically induces DNA breakage even in these radiation-resistant cells, the mechanism by which these sona-expressing cells cope against DNA damage is an important question to be explored (Tsogtbaatar, 2019).

Several mammalian metalloproteases in ECM are also shown to prevent apoptosis. Overexpression of MMP-15 (matrix metalloprotease-15) prevents apoptosis of Hela and human adenocarcinoma. Furthermore, ADAM-12 increases apoptosis of stromal cell but decreases that of tumor cells. In ADAMTS family, ADAMTS is shown to be required for cell survival. ADAMTS is mutated in belted (bt) mice that show cell death of melanoblast. Sona is also involved in cell survival by increasing resistance against irradiation and by promoting cell proliferation of neighboring cells. Identification of Sona substrate and its function will greatly help understand the role of Sona in cell survival, which is currently ongoing in our laboratory (Tsogtbaatar, 2019).

Anastral spindle 3/Rotatin stabilizes Sol narae and promotes cell survival in Drosophila melanogaster

Apoptosis and compensatory proliferation, two intertwined cellular processes essential for both development and adult homeostasis, are often initiated by the mis-regulation of centrosomal proteins, damaged DNA, and defects in mitosis. Fly Anastral spindle 3 (Ana3) is a member of the pericentriolar matrix proteins and known as a key component of centriolar cohesion and basal body formation. This study reports that ana3^m19 is a suppressor of lethality induced by the overexpression of Sol narae (Sona), a metalloprotease in a disintegrin and metalloprotease with thrombospondin motif (ADAMTS) family. ana3^m19 has a nonsense mutation that truncates the highly conserved carboxyl terminal region containing multiple Armadillo repeats. Lethality induced by Sona overexpression was completely rescued by knockdown of Ana3, and the small and malformed wing and hinge phenotype induced by the knockdown of Ana3 was also normalized by Sona overexpression, establishing a mutually positive genetic interaction between ana3 and sona. p35 inhibited apoptosis and rescued the small wing and hinge phenotype induced by knockdown of ana3. Furthermore, overexpression of Ana3 increased the survival rate of irradiated flies and reduced the number of dying cells, demonstrating that Ana3 actively promotes cell survival. Knockdown of Ana3 decreased the levels of both intra- and extracellular Sona in wing discs, while overexpression of Ana3 in S2 cells dramatically increased the levels of both cytoplasmic and exosomal Sona due to the stabilization of Sona in the lysosomal degradation pathway. It is proposed that one of the main functions of Ana3 is to stabilize Sona for cell survival and proliferation (Cho, 2021).

The ability to resist and recover from external stresses is important for all living organisms that face stresses such as heat, reactive oxygen species, and irradiation during development and in the adult stage. Damaged cells need to be removed by apoptosis and replaced with newly formed cells by compensatory proliferation. The wing imaginal disc of Drosophila melanogaster is the primordium of the adult wing, and shows a very low level of cell death during normal larval development. In contrast, it shows extensive cell death by environmental stresses, and yet can develop into a normal wing even after 40% to 60% cell death (Cho, 2021).

The centrosome consists of a pair of centrioles and pericentriolar materials (PCMs). DNA damage and mitotic defects cause the overduplication of centrosomes and the formation of multipolar spindles, leading to mitotic failure and cell death. Defects in PCMs interrupt spindle assembly and activate the spindle assembly checkpoint. Fly Anastral spindle 3 (Ana3) is a PCM responsible for the cohesion of centrioles, prevention of premature centriolar segregation, and formation of basal bodies (Stevens, 2009). Ana3 and its mammalian homolog Rotatin (RTTN) contain multiple Armadillo repeats known to interact with Wnt signaling components and potentiate the Wnt pathway (Song, 2003). Wnt has critical roles in growth, development, adult homeostasis, and regeneration. Ana3 and RTTN are also important for the formation of cilia and basal bodies (Kia, 2012; Stevens, 2009). Loss of RTTN causes polymicrogyria (PMG), situs inversus, isomerism, and heterotaxia in humans (Cho, 2021).

From a previous genetic screen, 28 mutants were found to be as responsible for the suppression of lethality caused by the overexpression of Sol narae (Sona) (Kim, 2020). The present study identified one of suppressors as ana3^m19. Sona is a member of a disintegrin and metalloprotease with thrombospondin motif (ADAMTS) family (Kim, 2016). Most ADAMTSs are secreted proteases that cleave components in the extracellular matrix, and their malfunctions result in multiple diseases including cancer. Sona is positively involved in Wingless (Wg) signaling, and secreted by both the exosomal secretion pathway and Golgi transport (Kim, 2016; Won, 2019). Sona cleaves the linker region of extracellular Wg and generates a new functional form of Wg that is specialized in cell proliferation (Cho, 2021).

Sona is important for cell survival, with the level of Sona correlated with the extent of cell survival (Tsogtbaatar, 2019). Cells expressing a high level of sona are cell autonomously resistant to γ-ray irradiation, while Sona secreted from these cells induces Cyclin D (Cyc D) in the neighboring cells for cell survival and proliferation in a non-cell autonomous manner. Interestingly, Wg-CTD but not full-length Wg induces Cyc D, which demonstrates that Sona is involved in intercellular communication to support the normal development of damaged tissues by regulating Wg signaling. Consistent with this, sona suppressors such as wntless, arrow, pou domain motif 3, and archipelago are related to Wg signaling (Cho, 2021).

This paper reports that Ana3 is also important for cell survival. Furthermore, overexpression of Ana3 increased the survival rate of irradiated flies, and the amount of Ana3 correlated with the extent of organism survival under irradiation. The level of Ana3 in wing discs was significantly increased by 1 h after irradiation, indicating that Ana3 may be one of the proteins that respond to irradiation at the front line. Ana3 expressed in S2 cells increased the level of both intracellular and secreted Sona by negatively regulating the lysosomal degradation pathway, which is consistent with the finding of ana3^m19 as a sona suppressor. These data demonstrate a new role of Ana3 in the stabilization of Sona (Cho, 2021).

This paper reports that the ana3^m19 mutant is a suppressor of Sona-induced lethality. Fly ana3 has a positive genetic interaction with sona that encodes a metalloprotease involved in Wnt signaling, establishing a potential link between Ana3/RTTN and Wnt signaling. Ana3/RTTN is a peripheral member of the centrosome complex whose malfunction leads to embryonic lethality in both ana3 mutant flies and RTTN knockout mice. Both Ana3 and Sona are involved in cell survival and resistance to irradiation. Consistent with their positive genetic interaction and functional similarity, this study found that Ana3 stabilizes Sona and increases the level of Sona in both wing discs and S2 cells. The truncated region in ana3^m19 protein is the most conserved region in Ana3/RTTN homologs, suggesting that this region plays a key role in stabilizing Sona. Some PMG mutations have also been identified in the Armadillo repeats in this carboxyl region of RTTN protein (Cho, 2021).

Both lethality and the small wing phenotype induced by Sona overexpression were completely rescued by knockdown of Ana3, suggesting that one of the main functions of Ana3 is to stabilize Sona. It is worth noting that a degradation of Sona occurs in the lysosome but not in the proteasome complex, as well as that another sona suppressor Arr also stabilizes Sona (Han, 2020). Since the original genetic screen was aimed at identifying suppressors that reduce Sona activity, it makes sense that both ana3 and arr mutants are identified as sona suppressors. Interestingly, Ana3 dramatically increased the level of exosomal Sona but not soluble Sona. This suggests that Ana3 stabilizes Sona in the exosomal secretion pathway that is interconnected with the lysosomal degradation pathway and the endosomal pathway but not in Golgi transport (Cho, 2021).

The loss of ana3 induced cell death, which is a common phenotype of centrosome components. Interestingly, overexpression of Ana3 enhanced the survival rate of irradiated flies, with wing discs showing the increased level of Ana3 1 h after irradiating the larvae, indicating that signals initiated by irradiation increase the level of Ana3 to prevent cell death. Since Ana3 stabilizes Sona, and knockdown of ana3 completely rescues the lethality caused by overexpressed Sona, the ability of Ana3 in promoting cell survival may stem from stabilized Sona. Previous work has shown that Sona-expressing cells are resistant to irradiation in a cell autonomous manner, and Sona secreted from these cells enables neighboring cells to survive and proliferate in a non-cell autonomous manner (Tsogtbaatar, 2019). Thus, it is possible that the increased level of Ana3 by irradiation contributes to increasing the level of Sona, which in turn functions to promote cell survival in both cell-autonomous and non-cell autonomous manners (Cho, 2021).

Extracellular Sona cleaves Wg and generates Wg-CTD that increases the level of Cyc D for initiating cell cycles (Won, 2019). Cyc D1 in mammalian cells promotes cell proliferation in response to mitogens, but overexpression of Cyc D1 leads to centrosome amplification, deregulation of the mitotic spindle, and chromosome abnormalities. Cyc D1 is oncogenic in many human cancer cells because it contributes to malignant transformation, with centrosome amplification by ras oncogene depending on Cyc D1. The link between fly Cyc D, Sona, and Wg-CTD, as well as the association of many components in Wnt signaling such as Disheveled, Armadillo/β-catenin, Axin, and Arrow/LRP6 with centrosomes, suggests that Sona may participate in the regulation of centrosomal duplication for the initiation of cell cycles (Cho, 2021).

Wg secreted by conventional Golgi transport diffuses and forms Wg gradient whereas Wg tethered to extracellular vesicles do not diffuse

Wingless (Wg)/Wnt family proteins are essential for animal development and adult homeostasis. Drosophila Wg secreted from the dorsal-ventral (DV) midline in wing discs forms a concentration gradient that is shaped by diffusion rate and stability of Wg. To understand how the gradient of extracellular Wg is generated, the secretion route of NRT-Wg, an artificial membrane-tethered form of Wg that is supposedly not secreted but still supports fly development, was compared to that of wild-type Wg. Wild-type Wg is secreted by both conventional Golgi transport and via extracellular vesicles (EVs), and NRT-Wg can be also secreted via EVs. Furthermore, wild-type Wg secreted by Golgi transport diffused and formed Wg gradient but Wg-containing EVs did not diffuse at all. In case of Wg stability, Sol narae (Sona), a metalloprotease that cleaves Wg, contributes to generate a steep Wg gradient. Interestingly, Wg was also produced in the presumptive wing blade region, which indicates that NRT-Wg on EVs expressed in the blade allows the blade cells to proliferate and differentiate without Wg diffused from the DV midline. It is proposed that EV-associated Wg induces Wg signaling in autocrine and juxtaposed manners whereas Wg secreted by Golgi transport forms gradient and acts in the long-range signaling, and different organs differentially utilize these two types of Wg signaling for their own development (Won, 2020).

Dynein Heavy Chain 64C Differentially Regulates Cell Survival and Proliferation of Wingless-Producing Cells in Drosophila melanogaster

Dynein is a multi-subunit motor protein that moves toward the minus-end of microtubules, and plays important roles in fly development. This study identified Dhc64C^m115, a new mutant allele of the fly Dynein heavy chain 64C (Dhc64C) gene whose heterozygotes survive against lethality induced by overexpression of Sol narae (Sona). Sona is a secreted metalloprotease that positively regulates Wingless (Wg) signaling, and promotes cell survival and proliferation. Knockdown of Dhc64C in fly wings induced extensive cell death accompanied by widespread and disorganized expression of Wg. The disrupted pattern of the Wg protein was due to cell death of the Wg-producing cells at the DV midline and overproliferation of the Wg-producing cells at the hinge in disorganized ways. Coexpression of Dhc64C RNAi and p35 resulted in no cell death and normal pattern of Wg, demonstrating that cell death is responsible for all phenotypes induced by Dhc64C RNAi expression. The effect of Dhc64C on Wg-producing cells was unique among components of Dynein and other microtubule motors. It is proposed that Dhc64C differentially regulates survival of Wg-producing cells, which is essential for maintaining normal expression pattern of Wg for wing development (Kim, 2021).

Exosomal arrow (Arr)/lipoprotein receptor protein 6 (LRP6) in Drosophila melanogaster increases the extracellular level of Sol narae (Sona) in a Wnt-independent manner

Wg/Wnt as a signaling protein binds Frizzled (Fz) and Arrow (Arr), two Wg co-receptors essential for Wg signaling for cell proliferation, differentiation, and cell survival. Arr has a long extracellular region, a single transmembrane domain and an intracellular region. This study reports that a new arr^m7 mutant is identified in a genetic screen as a suppressor of lethality induced by overexpression of Sol narae (Sona), a secreted metalloprotease in ADAMTS family involved in Wg signaling. arr^m7 allele has a premature stop codon, which encodes Arr^m7 protein missing the intracellular region. arr^m7 clones show cell death phenotype and overexpression of Arr^m7 protein also induces cell death. Levels of extracellular Sona were decreased in both arr^m7 and arr² null clones, demonstrating that Arr increases the level of extracellular Sona. Indeed, Arr but not Arr^m7, increased levels of Sona in cytoplasm and exosome fraction by inhibiting the lysosomal degradation pathway. Interestingly, Arr itself was identified in the exosome fraction, demonstrating that Arr is secreted to extracellular space. When Sona-expressing S2 cells were treated with exosomal Arr, the extracellular level of active Sona was increased. These results show that exosomal Arr dictates Sona-expressing cells to increase the level of extracellular Sona. This new function of Arr occurred in the absence of Wg because S2 cells do not express Wg. It is proposed that Arr plays two distinct roles, one as an exosomal protein to increase the level of extracellular Sona in a Wnt-independent manner and the other as a Wg co-receptor in a Wnt-dependent manner (Han, 2020).

This study reporta that the loss of arr decreases the level of extracellular Sona in flies, and Arr increases the extracellular level of Sona by stabilizing intracellular Sona in S2 cells through inhibition of lysosomal pathway. Interestingly, Arr was present in the fraction containing exosomes, and the exosomal Arr also increases the level of extracellular Sona by unknown mechanisms. It is proposed that exosomal Arr inhibits lysosomal degradation pathway and increases the level of exosomal Sona. When Arr is absent, Sona enters the lysosomal pathway to be degraded (Han, 2020).

The m7 suppressor with both arr^m7 and pdm3^m7 was identified in the screen using ethyl methanesulfonate (EMS) as a mutagen<. It is worth noting that arr^m7 has a G to A transition, whereas pdm3^m7 has an insertion of a defective hobo element in the untranslated region of the pdm3 gene. The point mutation in the arr gene seemed to occur before the insertion of the hobo element in the pdm3 gene, because other suppressors did not have hobo elements in their pdm3 genes. Therefore, both inhibition of wg transcription by pdm3^m7 and decrease in the level of extracellular Sona by arr^m7 may have been crucial for the m7 suppressor to be identified in the screen (Han, 2020).

Arr^m7 protein does not have the intracellular domain but still has the LDLR domain involved in dimerization, so dimers formed between Arr^m7 and wild-type Arr in arr^m7 heterozygotes become inactive. Besides this Arr-Arr^m7 dimer, Arr-Arr and Arr^m7-Arr^m7 dimers are also formed in arr^m7 heterozygotes, and the amount of Arr-Arr dimers may be enough to support normal development of arr^m7 heterozygotes. This explanation is in line with the finding that en > arr RNAi flies develop normally despite the low level of Arr protein in these flies. Although arr RNAi expression induced no visible phenotypes in these experiments, it suppressed phenotypes induced by overexpression of Arr or Sona (Han, 2020).

All biochemical experiments in this report were carried out with S2 cells that do not express Wg, indicating that stabilization of Sona by Arr occurs in the absence of Wg. Similar conclusion was drawn in case of LRP6 and gap junction protein connexin 43 (Cx43) in cardiac gap junction assembly. Transcription of Cx43 gene is induced by Wnt signaling independent of LRP6, but instead LRP6 post-transcriptionally promotes traffic of Cx43 from endoplasmic reticulum to Golgi apparatus. Reduction of LRP6 leads to retention of Cx43 in ER, which leads to the lysosomal degradation of immature Cx43. Such relationship between LRP6 and Cx43 is remarkably similar to that of Arr and Sona. It has been also reported that LRP6 deficiency results in lethal dilated cardiomyopathy and cardiac dysfunction by activation of dynamin-related protein 1 signaling. Therefore, Arr/LRP6 seems to play new post-transcriptional roles independent of Wnt signaling (Han, 2020).

This study shows that coexpression of Arr and Sona in S2 cells stabilizes Sona and increases the level of exosomal Sona by inhibiting the lysosomal degradation pathway. In addition, Arr is present in exosomes and these exosomal Arr also stabilizes Sona. It is actually common to find Wg signaling components in EV populations such as Wnts, β-catenin/Arm, Wls, and Fzd-10. LRP6 is also found in the exosome fraction by proteome profiling, although further confirmation is required. It seems that both Arr/LRP6 and Fz are secreted via EVs, and interaction between the Wnt co-receptors and Wnt in EVs may promote Wnt signaling but may also perform extracellular EV-specific functions independent of Wnt signaling as reported in this study. Further understanding mechanisms of secreted exosomal Arr in stabilization of Sona will greatly help to reveal Wnt-independent functions of Arr (Han, 2020).

Sol narae (Sona) is a Drosophila ADAMTS involved in Wg signaling

ADAMTS (a disintegrin and metalloproteases with thrombospondin motif) family consists of secreted proteases, and is shown to cleave extracellular matrix proteins. Their malfunctions result in cancers and disorders in connective tissues. This paper reports that a Drosophila ADAMTS named Sol narae (Sona; CG9850) promotes Wnt/Wingless (Wg) signaling. sona loss-of-function mutants are lethal and rare escapers had malformed appendages, indicating that sona is essential for fly development and survival. sona exhibited positive genetic interaction with wntless (wls) that encodes a cargo protein for Wg. Loss of sona decreased the level of extracellular Wg, and also reduced the expression level of Wg effector proteins such as Senseless (Sens), Distalless (Dll) and Vestigial (Vg). Sona and Wg colocalized in Golgi and endosomal vesicles, and were in the same protein complex. Furthermore, co-expression of Wg and Sona generated ectopic wing margin bristles. This study suggests that Sona is involved in Wg signaling by regulating the level of extracellular Wg (Kim, 2016).

Search PubMed for articles about Drosophila Sol narae

Cho, D. G., Lee, S. S. and Cho, K. O. (2021). Anastral spindle 3/Rotatin stabilizes Sol narae and promotes cell survival in Drosophila melanogaster. Mol Cells 44(1): 13-25. PubMed ID: 33510049

Han, J. H., Kim, Y. and Cho, K. O. (2020). Exosomal arrow (Arr)/lipoprotein receptor protein 6 (LRP6) in Drosophila melanogaster increases the extracellular level of Sol narae (Sona) in a Wnt-independent manner. Cell Death Dis 11(11): 944. PubMed ID: 33139721

Kia, S. K., Verbeek, E., Engelen, E., Schot, R., Poot, R. A., de Coo, I. F., Lequin, M. H., Poulton, C. J., Pourfarzad, F., Grosveld, F. G., Brehm, A., de Wit, M. C., Oegema, R., Dobyns, W. B., Verheijen, F. W. and Mancini, G. M. (2012). RTTN mutations link primary cilia function to organization of the human cerebral cortex. Am J Hum Genet 91(3): 533-540. PubMed ID: 22939636

Kim, G. W., Won, J. H., Lee, O. K., Lee, S. S., Han, J. H., Tsogtbaatar, O., Nam, S., Kim, Y. and Cho, K. O. (2016). Sol narae (Sona) is a Drosophila ADAMTS involved in Wg signaling. Sci Rep 6: 31863. PubMed ID: 27535473

Kim, J. Y., Tsogtbaatar, O. and Cho, K. O. (2021). Dynein Heavy Chain 64C Differentially Regulates Cell Survival and Proliferation of Wingless-Producing Cells in Drosophila melanogaster. J Dev Biol 9(4). PubMed ID: 34698231

Song, D. H., Dominguez, I., Mizuno, J., Kaut, M., Mohr, S. C. and Seldin, D. C. (2003). CK2 phosphorylation of the armadillo repeat region of beta-catenin potentiates Wnt signaling. J Biol Chem 278(26): 24018-24025. PubMed ID: 12700239

Stevens, N. R., Dobbelaere, J., Wainman, A., Gergely, F. and Raff, J. W. (2009). Ana3 is a conserved protein required for the structural integrity of centrioles and basal bodies. J Cell Biol 187(3): 355-363. PubMed ID: 19948479

Tsogtbaatar, O., Won, J. H., Kim, G. W., Han, J. H., Bae, Y. K. and Cho, K. O. (2019). An ADAMTS Sol narae is required for cell survival in Drosophila. Sci Rep 9(1): 1270. PubMed ID: 30718556

Won, J. H., Kim, G. W., Kim, J. Y., Cho, D. G., Kwon, B., Bae, Y. K. and Cho, K. O. (2019). ADAMTS Sol narae cleaves extracellular Wingless to generate a novel active form that regulates cell proliferation in Drosophila. Cell Death Dis 10(8): 564. PubMed ID: 31332194

Won, J. H. and Cho, K. O. (2020). Wg secreted by conventional Golgi transport diffuses and forms Wg gradient whereas Wg tethered to extracellular vesicles do not diffuse. Cell Death Differ. PubMed ID: 33028960

date revised: 19 February 2022

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.