Uas mcherry nls

srpHemo-GAL4 (mac>) was provided by K. Brückner (UCSF, USA) [9 (link)]. Oregon R (control), P{CaryP}attP2 (control), P{CaryP}attP40 (control), kay²(Dfos²), (UAS-Fra)2 (Dfos), UAS-Rho1.N19 Rho1DN), UAS-fbz (DfosDN), UAS-kayak RNAi (Dfos RNAi) TRiP HMS00254 and TRiP JF02804, UAS-dia RNAi TRiP HM05027, UAS-LamC RNAi TRiP JF01406 and TRiP HMS00308, e22c-GAL4 (ecto>), Resille::GFP, UAS-GFP::nls, UAS-dia::EGFP, UAS-diaRBD::EGFP, UAS-mCherry::nls, UAS-CD8::GFP lines were obtained from the Bloomington Stock Center (Indiana, USA). kay¹ (Dfos¹) line was provided by O. Schuldiner (WIS, Israel). UAS-dia::deltaDad::EGFP (diaCA) and srpHemo-GAL4 UAS-CLIP::GFP (mac>CLIP::GFP) lines were provided by B. Stramer (KCL, UK). UAS-cher::FLAG (cher) line was provided by M. Uhlirova (CECAD, Germany). w[1118] (control), UAS-cher RNAi KK107451, UAS-TM4SF RNAi KK102206, UAS-Lam RNAi¹ GD45636, UAS-Lam RNAi² KK107419 lines were obtained from the Vienna Drosophila Resource Center (Austria).

Fly stocks used for this study are as follows: wild-type Drosophila melanogaster Canton-S (CS), ap::GFP [Bloomington stock center (BS), #38423], Pdf-GAL4 (BS, #6900), nSyb-GAL4 (BS, #51941), c929 (BS, #25373), Mai179 (obtained from Dr. Orie T. Shafer, University of Michigan), Pdf-GAL80 (obtained from Leslie C. Griffith, Brandeis University), UAS-ap RNAi (NIG-fly, 8376R-1), UAS-ap^ΔHD (see next section), UAS-ap^ΔLIM (see next section), UAS-Chi RNAi (VDRC, 43934), UAS-Chi^ΔLID (obtained from Dr. Veronica Rodriguez, Tata Institute of Fundamental Research), UAS-Chi^ΔDD (obtained from Dr. Veronica Rodriguez), UAS-dORK1ΔNC (BS, #6587), UAS-mCherry.NLS (BS, #38424), UAS-IVS-mCD8::RFP (BS, #32218), and UAS-GFP RNAi (NIG-fly, GFP-IR-2). Flies were raised on glucose-yeast-cornmeal medium at 25.0 ± 0.5 °C in a 12-h light:12-h dark (LD) cycle. All lines except for UAS-mCherry.NLS and UAS-IVS-mCD8::RFP were outcrossed for at least six generations to white flies with the CS genetic background.

Flies were maintained on standard yeast medium at 25 °C unless otherwise noted. Flies bearing the following mutations and transgenes were obtained from the Bloomington Drosophila Stock Center (Indiana University, IN, USA): w¹¹¹⁸, repo-GAL4 (BL7415), UAS-mCherry-NLS (BL38424), tubulin-GAL80^ts (BL7019), UAS-mCD8::GFP (BL32186), NRE-EGFP1 (referred to as NRE-GFP, BL30728), UAS-Notch^intra (BL52008), an Eaat1-GAL4^{34 (link)} (BL8849), elav-GAL4 (BL458). spen^k07612 and spen⁰³³⁵⁰ P element insertions (Drosophila Genomics Resource Center [DGRC] #102574 and BL11295) were previously characterized as homozygous lethal spen loss-of-function mutations¹⁷ and were used here as heterozygotes. UAS-PLIN1::GFP^{40 (link)} was obtained from RP Kuhnlein (University of Graz, Austria). The EP line spen-GS2279 (Kyoto DGRC Stock Center) was used to overexpress spen, and is referred to as UAS-spen. The UAS-spen^RNAi line was a gift from KM Cadigan^{50 (link)} and was previously characterized in studies of Drosophila retina development^{14 (link)}. spen mutants and transgenic flies were outcrossed to a w¹¹¹⁸ control stock. We used the temperature-sensitive TARGET system^{45 (link)} to restrict spen RNAi expression to adult glial cells. Briefly, flies carrying repo-GAL4, tubulin-GAL80^ts, and UAS-spen^RNAi were raised at 18 °C to inhibit GAL4 activity and switched to 29 °C as adults to induce expression of spen^RNAi.

The following stocks were used: yw for wild-type control, ap-GAL4 (Bloomington 50156), UAS-mCD8::GFP (Bloomington 5130), UAS-myr::mRFP (Bloomington 7118), UAS-mCherry.NLS (Bloomington 38424), sas15 (null mutant)(Bloomington 2098), Sage-GAL4 (a gift from Deborah J. Andrew), Ptp10DEP1172 (Bloomington 11332), UAS-dArc1 (Bloomington 37532), UAS-Numb (a gift from Yuh Nung Jan), UAS-SasFL and UAS-V5-SasFL (Lee et al., 2013 (link)), Arc1esm18 (Bloomington 37530). Crosses and embryo collections were performed at room temperature. For overexpression experiments, embryos were shifted to 29 °C for at least 120 min prior to fixation and staining and 3rd instar larvae were shifted to 29 °C for overnight for further analysis. For the EV targeting experiments, imaginal discs from 3rd instar larvae were harvested at room temperature and incubated in 200 µl of S2 supernatant overnight at 29 °C before fixation and staining. There are 10,000–50,000 cells in a 3rd instar imaginal disc. Given the results from the NTA analysis, we can conclude that ~140,000 EVs are present in 200 µl of supernatant from V5-Sas^FL-expressing S2 cells. We used 5 wing discs per incubation, so the ratio of EVs to cells is ~0.5 to~2. The relative V5 signal intensities on the imaginal discs were measured by densitometry analysis using ImageJ software.