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, we compared the secretion route of NRT-Wg, an artificial membrane-tethered form of Wg that is supposedly not secreted but still supports fly development, to that of wild-type Wg. We found that 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. We propose 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.

Role of Armadillo repeat 2 and kinesin-II motor subunit Klp64D for wingless signaling in Drosophila.

Armadillo (Arm) is crucial for transducing Wingless (Wg) signaling. Previously, we have shown that Klp64D, a motor subunit of Drosophila kinesin-II, interacts with Arm for Wg signaling. Molecular basis for this interaction has remained unknown. Here we identify a critical Arm repeat (AR) required for binding Klp64D and Wg signaling. Arm/[Formula: see text]-catenin family proteins contain a conserved domain of 12 Arm repeats (ARs). Five of these ARs can interact with Klp64D, but only the second AR (AR2) binds to the cargo/tail domain of Klp64D. Overexpression of AR2 in wing imaginal disc is sufficient to cause notched wing margin. This phenotype by AR2 is enhanced or suppressed by reducing or increasing Klp64D expression, respectively. AR2 overexpression inhibits Wg signaling activity in TopFlash assay, consistent with its dominant-negative effects on Klp64D-dependent Wg signaling. Overexpression of the Klp64D cargo domain also results in dominant-negative wing notching. Genetic rescue data indicate that both AR2 and Klp64D cargo regions are required for the function of Arm and Klp64D, respectively. AR2 overexpression leads to an accumulation of Arm with GM130 Golgi marker in Klp64D knockdown. This study suggests that Wg signaling for wing development is regulated by specific interaction between AR2 and the cargo domain of Klp64D.

ADAMTS Sol narae cleaves extracellular Wingless to generate a novel active form that regulates cell proliferation in Drosophila.

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.

Physicochemical Modification Effect on Homogeneously Aligned Liquid Crystals Based on the Nickel Oxide Thin Film.

We demonstrated homogeneous liquid crystal (LC) alignment on Nickel Oxide (NiO) films subjected to ion beam (IB) irradiation. Uniform LC alignment was achieved at high IB intensity values of 1200 and 1800 eV. To determine the mechanism of LC alignment following IB irradiation, physicochemical analysis was performed using atomic force microscopy and X-ray photoelectron spectroscopy. IB irradiation with high intensity increases uniformity of the surface, and IB irradiation induces the formation of oxygen vacancies and increases the NiO (Ni2+) phase components. Hence, both of the smooth surface and the strong van der Waals interactions between the NiO film and the LC molecules provides the LC molecules with a stable anchor on the surface, leading to uniform LC alignment on NiO films after IB irradiation. IB-irradiated NiO exhibited a high transparency of 85% in the visible light range as compared with the 83% average transmittance of conventional polyimide, which makes the IB-irradiated NiO alignment layer attractive in display devices.

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. We report here that a Drosophila ADAMTS named Sol narae (Sona) 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.

Cell type-specific responses to wingless, hedgehog and decapentaplegic are essential for patterning early eye-antenna disc in Drosophila.

The Drosophila eye-antenna imaginal disc (ead) is a flattened sac of two-layered epithelia, from which most head structures are derived. Secreted morphogens like Wingless (Wg), Hedgehog (Hh), and Decapentaplegic (Dpp) are important for early patterning of ead, but the underlying mechanisms are still largely unknown. To understand how these morphogens function in the ead of early larval stages, we used wg-LacZ and dpp-Gal4 markers for the examination of wild-type and mutant eads. We found that the ead immediately after hatching was crescent-shaped with the Bolwig's nerve at the ventral edge, suggesting that it consists of dorsal domain. In a subsequent step, transcriptional induction of dpp in the cells along the Bolwig's nerve was followed by rapid growth of the ventral domain. Both Wg and Hh were required for the formation of the ventral domain. Wg was crucial for the growth of the entire ead, but Hh was essential for cell division only in the dorsal domain. In the ventral domain, Hh regulated dpp transcription. Based on these data, we propose that signaling among distinct groups of cells expressing Wg, Dpp, or Hh in the ead of the first-instar larvae are critical for coordinated growth and patterning of ead.

Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake.

Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH-optimum and Mg(2+)-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.