Abstracts
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. Here we report 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.
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Introduction
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 proteins1,2,3. Despite the importance of these proteases, their specific functions are still largely unexplored. ADAMTS family contains extracellular proteases that are present only in metazoans4,5. Six and nineteen members have so far been identified in flies and mammals, respectively6,7. Mammalian ADAMTSs are involved in cell proliferation, angiogenesis and organogenesis, so their malfunctions result in various diseases such as cancer, arthritis, and arteriosclerosis7,8,9. An ADAMTS Sol narae (Sona) is essential for fly development10. Loss of sona decreases the level of extracellular Wg, and sona exhibits positive genetic interaction with wntless (wls) that encodes a cargo protein for Wg10,11,12. Therefore, intracellular Sona seems to cooperate with Wls in Wg secretion10. We recently reported a new function of extracellular Sona in cell survival and cell proliferation13. 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 signals14. Therefore, it is possible that extracellular Sona generates a yet unidentified signaling molecule that induces Cyc D in the signal-receiving cells.
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 ago15,16,17. Wnt is secreted by both conventional Golgi-mediated transport and exosomal secretion pathway18,19,20. Interaction between Wnt and Frizzled (Fz) receptors initiates a cascade of intracellular responses in the responding cells that lead to downstream gene expression21,22. 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)23,24,25,26. In mammals, Wnt signaling promotes cell proliferation by transcriptional activation of multiple target genes such as c-Myc and Cyc D27,28,29 and its malfunction leads to various diseases such as cancer, neurodegenerative diseases, inflammatory disease, and diabetes30,31,32.
We asked the role of extracellular Sona in this study and found that Sona generates 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.
Results
Sona and Wg are secreted by both conventional Golgi and exosomal transports
Sona is an ADAMTS protease secreted as an active form to the extracellular region10, and has biochemical and genetic interactions with Wg that is secreted by both conventional Golgi and exosomal transports18,19,20. As a first step toward understanding the role of extracellular Sona, we examined by which pathway Sona is secreted. To this end, we obtained conditioned media from the culture of S2 sona-HA cell line, and precleared it to obtain the initial supernatant fraction, SN0. Centrifugation of SN0 at 100,000×g yielded two fractions: a supernatant fraction (SNΔ) that contains soluble proteins secreted by Golgi transport, and a pellet fraction (P100) that contains exosomes19,33. The cell extract (CX) contained both full-length Sona-HA (red arrow) and the active form of Sona-HA lacking its pro-domain (black arrow), while the SNΔ and P100 fractions contained only the active form of Sona-HA (Fig. 1a). Our data demonstrate that Sona is also secreted by both Golgi and exosomal transports (Fig. 1b). To prove further that Sona is secreted by exosomal pathway, we examined the P100 fraction of Sona-HA in detail. Purity of the P100 fraction was verified by the presence of the exosomal markers Syntaxin 1A (Syx1A) and Alix as well as the absence of the ER marker Calnexin33,34 (Fig. 1c). Particulate structures with 70–250 nm diameter were detected in the P100 fraction of S2 sona-HA or S2 GFP-wg by Nanoparticle tracking analysis (NTA), and Sona and Wg were present in the fraction with 1.09–1.11 g / mL density in sucrose step gradient33 (Fig. 1d and Supplemental Fig. S1A-B). Furthermore, Sona-HA was also present on the outer surface of vesicles (Fig. 1e and Supplemental Fig. S1C, D). To confirm the presence of Sona on exosomes in vivo, we examined whether Sona-HA and the exosomal marker CD63-GFP colocalize in wing discs. Unlike the lysosomal markers (Supplemental Fig. S2), half of CD63-GFP-positive (+) vesicles contained Sona-HA detected by both anti-HA and Sona-Pro antibodies (52.3 ± 10.3%, n = 65, Supplemental Fig. S1E)10, and about half of these CD63+ Sona+ vesicles contained endogenous Wg (21.5 ± 2.1%, n = 65, Supplemental Fig. S1F). These results demonstrate that Sona is present on exosomes.
The active form of extracellular Sona is essential for cleavage of the Wg linker region
Coimmunoprecipitation of Sona and Wg10 suggested that Sona may cleave Wg. To test this, we examined whether any small Wg fragments are generated in the presence of Sona. In fact, a 65 kDa fragment smaller than 83 kDa full-length GFP-Wg (GFP-WgFL) was detected by the 4D4 Wg antibody in both SNΔ and P100 fractions only when Sona was coexpressed with GFP-Wg (black arrows in Fig. 1f’, f”). Such Sona-dependent changes were not detected in CX (Fig. 1f and Supplemental Fig. S3A, B). Interestingly, anti-GFP antibody detected both 65 kDa and an additional 60 kDa fragment (black arrowhead in Fig. 1h), indicating that these two fragments have the N-terminal region where GFP is inserted (Fig. 1k). Since these 65 and 60 kDa fragments were not produced by the protease-dead SonaE475A or in the presence of a zinc chelator, EDTA, metalloprotease activity of Sona is essential for Wg cleavage (Fig. 1g, h and Supplemental Fig. S3A-C). We located the two cleavage sites designated as L1 and L2 in the Wg linker based on two features. First, the 65 kDa but not the 60 kDa fragment has the 4D4 epitope (Fig. 1g, h). Second, the 4D4 epitope is located between amino acids 229 and 360 of Wg35,36. Cleavage at L1 and L2 should produce fragments named NTDL1, CTDL1, NTDL2, and CTDL2 (Fig. 1k). NTDL1 and NTDL2 are 60 kDa and 65 kDa fragments, respectively. Meanwhile, a 23 kDa fragment was detected with the 4D4 antibody when untagged Wg or GFP-Wg was coexpressed with Sona (red arrowheads in Fig. 1i’ and Supplemental Fig. S3A’), or detected with the HA antibody when Sona and Wg-3XHA with three HA tags at the C-terminus37 were co-expressed (red arrowhead in Fig. 1j). Therefore, this 23 kDa fragment is CTDL1 because it contains both the C-terminal part of Wg and the 4D4 epitope (Fig. 1k). However, CTDL1 fragments generated from the untagged Wg or GFP-Wg were detectable only after long exposure, and the intensity of CTDL1 fragment was much lower than that of NTDL2 (Fig. 1i-i’ and Supplemental Fig. S2A’). Moreover, the CTDL2 fragment was never detected when Wg-3XHA was coexpressed with Sona (Fig. 1j). The low levels of CTDL1 and the absence of the CTDL2 may be due to protein instability, which will be addressed in Fig. 2.
The linker of Wg is necessary and sufficient for Wg cleavage by Sona
To examine whether the linker is the only region required for Wg cleavage, we generated two constructs, GFP-NTD and linker-GFP that encode 53 kDa GFP-NTD without the linker region and 48 kDa Linker-GFP with the linker region fused to GFP, respectively (Fig. 2a, d). When GFP-NTD and Sona-HA were coexpressed, Sona-dependent cleavage was not detected (Fig. 2c). In contrast, we found all cleavage products such as a 42 kDa L1 cleavage product (black arrows), a 34 kDa L2 cleavage product (red arrowheads), and a 12 kDa L2 cleavage product (black arrowheads) when Linker-GFP and Sona-HA were coexpressed (Fig. 2f, h). Furthermore, Wg cleavage by Sona occurred when Sona and Wg were prepared from different cells or different secretion pathways (Supplemental Fig. S3D-F). Therefore, the linker region is necessary and sufficient for Wg cleavage by Sona regardless of origin.
The CTD domain of Wg-CTD fragments is responsible for protein instability
We previously mentioned that both CTDL1 and CTDL2 fragments might be unstable (Fig. 1i). However, the 42 kDa L1 Linker-GFP fragment that is equivalent to the 23 kDa Wg-CTDL1 fragment was readily detected (Compare Fig. 1i to Fig. 2h). This raised a possibility that the CTD domain itself is responsible for instability of Wg-CTD fragments. To test this, we generated linker-CTD that encodes 29 kDa Linker-CTD and compared its Sona-dependent cleavage products to those of Linker-GFP (Fig. 2i). When Linker-CTD and Sona were coexpressed, a 12 kDa 4D4-positive L2 cleavage product was detected (black arrowheads) but the expected 23 kDa CTDL1 was not detected even with multiple attempts (Fig. 2k). Moreover, the 34 kDa L2 linker-GFP fragment was detected (red arrowhead in Fig. 2f), which is equivalent to the 17 kDa Wg-CTDL2 fragment. As shown before, this 17 kDa Wg-CTDL2 fragment was never detected throughout this study (Fig. 1j, k). Therefore, both Wg-CTD fragments are less stable than their equivalents due to the CTD itself.
Wg is directly cleaved by Sona
To test the enzyme-substrate relationship between Sona and Wg, we asked whether Sona cleaves Wg in vitro. Since the NTD of Wg is too hydrophobic to be expressed in E. coli, we generated GST-linker-CTD (Fig. 2l). We hypothesized that Sona may cleave GST-linker-CTD because the linker is sufficient for cleavage by Sona (Fig. 2l). When GST-linker-CTD purified from E. coli was incubated with active Sona purified from the SNΔ fraction of S2 sona-HA (Supplemental Fig. S4A), the 4D4 antibody detected a Sona-dependent 18 kDa fragment (red arrowhead in Fig. 2m and Supplemental Fig. S4B; black arrow in Fig. 2n and Supplemental Fig. S4C). This 18 kDa fragment is the L1 cleavage product that is equivalent to the 23 kDa Wg-CTDL1 fragment, but is smaller because the CTD domain in the GST-linker-CTD is not glycosylated (Supplemental Fig. S4D). This suggests that Wg is a substrate of Sona.
If Sona directly cleaves Wg, the amount of cleaved Wg would positively correlate with that of Sona. To test this, the fixed amount of SN0 containing Wg was incubated with the increasing amounts of SN0 containing Sona-HA. As expected, the amount of Wg-CTDL1 fragment positively correlated with that of Sona (Fig. 2o). When the fixed amount of the P100 containg GFP-Wg was incubated with the increasing amounts of the P100 containing Sona-HA, the amount of Wg-NTDL1 and Wg-NTDL2 fragments also proportionally increased (Fig. 2p). This result is consistent with direct cleavage of Wg by Sona.
Sona is required for cleavage of extracellular Wg in vivo
We next asked whether cleavage of extracelluar Wg also occurs in vivo. Wg is highly expressed along the DV midline of wing discs38,39,40. To detect cleaved Wg forms, we examined extracellular Wg-HA pattern in the DV midline of wg[KO; Wg-HA]41. Assuming Sona cleaves Wg at L1 and L2 cleavage sites in vivo, we expected to detect extracellular structures including Wg-HAFL and four additional Wg fragments except NTDL1 in wg[KO; Wg-HA] wing discs using anti-HA and 4D4 antibodies (Fig. 3a). We found not only yellow structures (HA+ 4D4+) that contain full-length Wg but also the green (HA+ 4D4–) and the red structures (HA– 4D4+) that represent cleaved Wg fragments (Fig. 3a, e). These cleaved structures were also detected in wg > GFP-wg discs (Supplemental Fig. S5). Thus, cleaved Wg fragments were present in the extracellular region.
If Sona plays a major role in the cleavage of WgFL, sona mutants should have more WgFL than wild-type. To test this, we compared the percentage of yellow structures (HA+ 4D4+) in sona18/ sona47 wg[KO; Wg-HA] discs with that in control wg[KO; Wg-HA] discs (Fig. 3d–h). While 74.1% (648/875) were yellow in sona18/sona47 discs, only 32.0% (460/1436) were yellow in wild-type discs (Fig. 3h). This suggests that Wg cleavage occurred at a lesser extent in the sona discs. This supports that Sona is a major player in cleavage of Wg in vivo.
Prolonged overexpression of Wg-CTD induces morphological defects and lethality
We reasoned that at least one of the cleaved forms of Wg should be active because Sona positively regulates Wg signaling10. To test which Wg fragment is active, we performed luciferase assay in S2R+ cells that expressed Wg-NTD or Wg-CTD. Neither Wg-NTD nor Wg-CTD, however, showed any Wg activity (Supplemental Fig. S6A). This was unexpected because artificially engineered Wnt7a-CTD is reported to be active in TopFlash reporter assay42. It is possible that S2 R+ cells lack some essential components that are required for Wg-CTD activity43, or Wg-CTD is too unstable in S2 R+ cell culture (Supplemental Fig. S6). Instead of finding the better condition for luciferase assay, we decided to test the activity of Wg fragments in vivo with UAS-GFP-wg-NTD and UAS-wg-mycCTD transgenic flies (Fig. 2a and Supplemental Fig. S7A).
Overexpression of GFP-Wg-NTD with engrailed (en)-Gal4 or nubbin (nub)-Gal4 produced no phenotypes (Fig. 4b and Supplemental Fig. S7B). In contrast, en > wg-mycCTD and nub > wg-mycCTD wings were small and deformed (Fig. 4a, d, k, l), and the posterior region of en > wg-mycCTD wing discs were smaller than that of control discs (Supplemental Fig. S7K, L). Furthermore, cubitus interruptus (ci) > wg-mycCTD eyes were small and rough (Supplemental Fig. S7N, O). Expression of Wg-mycCTD by actin-Gal4 or tubulin-Gal4 induced early larval lethality (n > 40) but that of GFP-wg-NTD produced no lethality. Expression of the untagged Wg-NTD and Wg-CTD also generated phenotypes similar to the tagged counterparts (Fig. 4c, e and Supplemental Fig. S7A, E, F, J, M). Taken together, Wg-CTD but not NTD is an active Wg form in vivo.
Transient overexpression of Wg-CTD stimulates cell proliferation
Wg stimulates cell proliferation as a mitogen44,45, and moderate Wg overexpression increases the number of phosphohistone 3 (PH3)-positive cells44,46. To check whether Wg-CTD also induces cell proliferation, we transiently expressed Wg-CTD using Gal80ts in order to avoid cell death or cell cycle arrest by prolonged Wg signaling47,48,49. Indeed, expression of Wg-mycCTD or untagged Wg-CTD for ten hours increased wing size by 17.6 and 31.3%, respectively (n = 31, Fig. 4f, g). Likewise, the number of PH3-positive cells was increased along the ptc region compared to control ptc > GFP discs (Fig. 4h–j). Therefore, Wg-CTD is able to promote cell proliferation.
Wg-CTD rescues the loss of sona phenotypes
Wg-CTD would rescue the loss-of-function phenotypes of extracellular Sona if generation of Wg-CTD is a main function of extracellular Sona. To test this, sona RNAi-111-4 (sona RNAi, hereafter) was coexpressed with Wg-mycCTD and the wing phenotype and lethality were compared to those of sole sona RNAi expression10. All nub > sona RNAi wings were small, wrinkled or both10 (n = 95, Fig. 4k, m) but 68% of nub > sona RNAi, wg-mycCTD wings were normal (n = 109, Fig. 4n, s). Notched wing phenotype was observed in 55% of wg > sona RNAi (n = 71) but only in 6% of wg > wg-mycCTD, sona RNAi (n = 68) flies (Fig. 4o–r). Furthermore, notched wing phenotype of ptc > wg-mycCTD was rescued in ptc > sona RNAi, wg-mycCTD flies (Supplemental Fig. S8E-G). Therefore, Wg-CTD expression rescued loss of sona phenotypes.
Wg-CTD activates canonical Wg signaling
Cytoplasmic Arm becomes stabilized by activation of canonical Wg signaling50. Because Wg-CTD expression rescued the lethality and wing defects induced by arm RNAi, Sgg or dTCFDN expression (Fig. 5a–p), we tested whether Wg-CTD stabilizes the cytoplasmic Arm. In fact, ci > GFP, wg-mycCTD wing discs had the increased level of Arm in the anterior region (Fig. 5q, r). More Arm was also present in the CX of nub > wg-mycCTD compared to control nub-Gal4 wing discs (Fig. 5s).
We next examined whether Wg-CTD increases levels of Wg effector proteins, Vg and Dll that are induced by canonical Wg signaling51,52,53,54. Transient expression of Wg-CTD by en-Gal4 increased the level of Vg and Dll in the posterior region of wing discs (Fig. 6a–d), and Wg-CTD-expressing flp-out clones had higher level of Dll (Fig. 6e–g). The untagged Wg-CTD expression in en > wg-CTD discs also increased the level of Dll (Supplemental Fig. S9B). GFP-Wg-NTD and untagged Wg-NTD failed to change the level of Wg effector proteins (Fig. 6h, i and Supplemental Fig. S9A). Therefore, Wg-CTD is a new form of active Wg that induces canonical Wg signaling.
Wg-CTD upregulates the level of Cyc D for cell proliferation
Overexpressed WgFL partially rescued the lethal phenotype of wg mutants but Wg-CTD did not (Supplemental Fig. 9C-E). This demonstrates that Wg-CTD can carry out only subsets of Wg signaling and so called ‘Wg signaling’ is induced by combined activity of both WgFL and cleaved Wg-CTD. An important question is then whether Wg-CTD has any unique functions unshared by WgFL. We hypothesized that Wg-CTD may be more specialized for cell proliferation than WgFL because Sona increases the level of Cyc D13. Indeed, Cyc D was upregulated by prolonged expression of Wg-CTD in the anterior region of ci > wg-mycCTD wing discs and by transient expression of Wg-CTD in the posterior region of en > wg-mycCTD, Gal80ts discs cultured for 12 h at 30 °C (Fig. 7a, b; Supplemental Fig. S9F, G). GFP-WgFL or GFP-Wg-NTD expression, however, did not change the level of Cyc D (Fig. 7c, d). Taken together, Wg-CTD is able to induce Cyc D. Wg signaling plays an important role in neuronal differentiation by inducing sens in the DV margin of wing discs55,56,57. Transient expression of GFP-Wg increased the level of Sens in ptc > GFP-wg, Gal80ts discs, and induced ectopic sensory bristles in nub > GFP-wg, Gal80ts wings, which are consistent with previous reports58,59 (Fig. 7e–h). In contrast, transient expression of Wg-CTD did not induce ectopic Sens in ptc > wg-mycCTD Gal80ts and en > wg-mycCTD Gal80ts wing discs (Fig. 7i and Supplemental Fig. S9I). Wings of nub > wg-mycCTD, Gal80ts, nub > wg-mycCTD, and nub > wg-NTD flies also had no ectopic bristles (Fig. 7j and Supplemental Fig. S9K-L). Thus, Wg-CTD is not able to induce Sens unlike WgFL.
Discussion
We report here that Sona cleaves extracellular Wg into Wg-NTD and Wg-CTD, and the Wg-CTD is a new form of active Wg (Supplemental Fig. S10). Because Wg-CTD substantially rescued the sona loss-of-function phenotypes such as lethality and wing defects (Fig. 4), 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 Wnt60. 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.
Genetic interaction between wg-CTD and other Wg signaling components indicates that Wg-CTD activates Wg signaling similar to WgFL (Fig. 5). However, there are several differences between these two forms of Wg. First, Wg-CTD but not WgFL increased the level of Cyc D (Fig. 7). Overexpressed Cyc D-Cdk4 in flies accelerates cell division of undifferentiated cells such as wing disc cells61. Sona also induces Cyc D and promotes cell proliferation in a cell non-autonomous manner13. Therefore, Wg-CTD generated by extracellular Sona seems to induce Cyc D in the neighboring cells for cell proliferation. Second, both 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 proliferation62 (Fig. 4). Presence of Wg-CTDL2-like structures in wing discs (Fig. 3), however, implies that these Wg-CTDL2-like structures may be stabilized in vivo by ECM components to achieve spatiotemporal regulation of the mitogenic activity63,64. Third, Wg-CTD is not able to induce Sens (Fig. 7 and Supplemental Fig. S8). Sens expression in the DV midline is required for differentiation of wing margin bristles55,56,57, unlike Vg that is essential for cell proliferation and cell survival23,65.
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 affinity66. 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 signaling42,66,67. This explains why NTD mutants are unable to be secreted68, while CTD mutants are secreted but inactive69. Given the evolutionary conservation of the components of Wnt signaling, ADAMTSs may also be involved in the generation of functional Wnt-CTD in mammals70,71. We expect that further study on the relationship between Wnts and ADAMTSs will expand our understanding on Wnt signaling and Wnt-related diseases.
Materials and methods
Drosophila strains, transgenic lines and generation of ectopic clones
sona mutants, sona RNAi lines, UAS-sona, and UAS-sona-HA are described elsewhere10. The UAS-wg-mycCTD, UAS-GFP-wg-NTD, UAS-wg-CTD and UAS-wg-NTD flies were generated for this study. UAS-CD63-GFP72, UAS-GFP-wg73, wg[KO; Wg-HA]41, UAS-GFP-lamp74, wgGal475 and ci-Gal476 were kindly provided by other labs that produced them. All other lines were obtained from the Bloomington stock center.
DNA constructs
The pAc-GFP-wg and pAc-wg-3XHA were constructed by recombining the pAc5.1 vector with GFP-wg or wg-3XHA obtained from MK33-GFP-wg (a gift from J.P. Vincent, unpublished) or UAS-wg-3XHA37. To generate the GFP-wg-NTD (GFP-NTD) and wg-mycCTD constructs, a myc tag was inserted in the DNA corresponding to the region between Arg367 and Tyr368 in GFP-Wg. DNA fragments representing GFP-NTD (1–245) and mycCTD (1–22, 245–468) were then amplified by PCR and inserted into pUAST vectors by recombination cloning methods.
Cell lines, cell culture, and exosome preparation
Drosophila S2 tub-wg, S2R+, and S2 cell lines were obtained from DGRC. S2 GFP-wg, wg-3XHA, and sona-HA stable cell lines were generated by selection under 2.5 μg/ml hygromycin B (Invitrogen) as follows. Drosophila S2 cell were grown in M3 media (Sigma-Aldrich) supplemented with 10% IMS (Sigma-Aldrich) at 25 °C. Stable cell lines were grown with hygromycin in 10% IMS M3 media, and S2 tub-wg cells were cultured in 10% FBS M3 media. Transfections were carried out with Effectene (Qiagen) or Cellfectin (Invitrogen) according to the manufacturers’ instructions. For exosome preparation, 7–40 ml of conditioned media obtained from cultures (1.25 × 106 cells / ml) were used as described19. The size and number of the exosomes in the P100 fraction were measured by Nanosight NC300 (Malvern Instruments).
Immunocytochemistry and Western analysis
Fly larvae were cultured at 25 °C unless stated otherwise. Wing discs from the late third instar larvae were used for intracellular staining and extracellular staining24. For immunocytochemistry, we used Sona-Pro, 1:300–500; Golgi (Calbiochem, mouse), 1:200; GFP (Abd serotec, sheep), 1:100; Senseless (a gift from H. Bellen, guinea pig), 1:1000; HRS (a gift from H. Bellen, guinea pig), 1:1000; WgN (sc-28646 Santa Cruz, rabbit), 1:100; Wg (DSHB, mouse), 1:1000; HA (Roche, rat), 1:300; HA (Santa Cruz, rabbit), 1:300; Vg (gift from Sean B. Carroll, rabbit), 1:100; Wg (DSHB, mouse), 1:100; Dll (Santa Cruz, goat), 1:100. For the extracellular staining of proteins, we used 10 times more antibodies than for the intracellular staining. Fluorescent images were captured using a Zeiss LSM laser scanning confocal microscope and processed with Adobe Photoshop.
Western analysis was carried out as described10. For western analysis, we used Sona-Pro (our lab, rabbit), 1:5000; HA (Santa Cruz, rabbit), 1: 250; GFP (Abcam, rabbit), 1:10,000; Wg (DSHB, mouse), 1:500–1000; Syntaxin 1A (DSHB, mouse), 1:25; Alix (gift from T. Aigaki, mouse), 1:500; Actin (DSHB, mouse), 1:500; Calnexin (gift from N.J. Colley, rabbit), 1:2000.
Electron microscopy
For immunogold labeling, P100 fraction from S2 sona-HA cells were plated on grids, blocked with 5% BSA in PBS and incubated with anti-HA antibody (1:5). Then, samples were washed with 0.1% BSA in PBS and incubated in secondary anti-rabbit antibody conjugated with 15 nm gold particles (AURION). After 8 times wash with PBS for 5 min each, samples were incubated in 1% glutaraldehyde for 5 min. Then, samples were washed with H2O for 8 times before staining with Phosphotungstic acid (PTA). Sample grids were air-dried completely and visualized using a transmission electron microscope (Talos F200X).
In vitro GST-Wg cleavage assay
For purification of GST-Wg, pGEX-4T-1-WgCterm was expressed in BL21 E. coli strain. Then, we purified the GST-Wg protein by standard column-based protocols (GST-column, 1st SP Sepharose column, 2nd SP Sepharose column). For purification of active Sona from S2 cell culture, CX and SNΔ fractions were prepared and lysis buffer without EDTA and Protein inhibitor cocktail (PIC) were added to these fractions. Active Sona was obtained by mixing with HA-conjugated bead and precipitating the beads. GST-Wg and active Sona were mixed and incubated at 25 °C overnight.
Sucrose step gradient
Exosome pellets were resuspended in 0.25 M sucrose and loaded on top of a sucrose step gradient before being centrifuged at 100,000xg in a Beckman SW41Ti rotor for 3 h as described77. Ten to twelve fractions of 1 mL each were then manually collected from the bottom of the gradient.
Wg reporter assay
The Wg reporter assay was carried out by conventional methods. WISIR vector that contains both firefly luciferase under the control of a Wg-responsive promoter and Renilla luciferase under the control of a Copia promoter was transfected into S2R+ cells. After one day of culture, cells were splitted to a 48 well plate and incubated for 3~4 h until the experimental treatment started. After 24 h of treatment, cells were lysed by following the manufacturer’s instructions of the Dual-Luciferase Repoter Assay System (Promega). Each condition was tested in triplicate.
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Acknowledgements
We thank K.W. Choi and K.H. Kang for their critical reading of this manuscript. We also thank Munashingha P. R. for preparation of GST-Wg protein. We are indebted to J. P. Vincent, K. Basler, S. Eaton, S. Hayashi, S. M. Cohen, L. S. Shashidhara, R. Holmgren, and H. Kramer for fly lines, to K. Basler, J. P. Vincent, and S.T. Hong for DNA constructs, and to H. Bellen, N.J. Colley, T. Aigaki and Sean B. Carroll for antibodies. We thank Bloomington Stock Center, Drosophila Genetic Resource Center, and Developmental Studies Hybridoma Bank for fly strains and antibodies. This research was supported by grants from the National Research Foundation of Korea (NRF-2017R1A2B4009254 and NRF-2019R1H1A2039726) and National Research Council of Science and Technology (DRC-14-KRISS).
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Won, JH., Kim, GW., Kim, JY. et al. ADAMTS Sol narae cleaves extracellular Wingless to generate a novel active form that regulates cell proliferation in Drosophila. Cell Death Dis 10, 564 (2019). https://doi.org/10.1038/s41419-019-1794-8
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DOI: https://doi.org/10.1038/s41419-019-1794-8
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