Dextran alexa568

Live imaging of larval salivary glands was performed as previously reported (Tran et al., 2015 (link)). In short, naturally secreting isolated glands were placed in a glass-bottom imaging dish, covered with a Isopore 0.1 µm PC membrane (Merck) and 50 µl Schneider's Drosophila medium. For long-term imaging, the imaging chamber was humidified with a wet tissue paper and sealed with parafilm to prevent evaporation. Dissected glands were imaged with a Zeiss CellObserver Z.1 with a Yokogawa CSU-X1 spinning disc scanning unit and an Axiocam MRm CCD camera (6.45 µm×6.45 µm). For additional Dextran imaging, dissected glands were incubated for 1 h with 200 µM Dextran-Alexa568 (molecular mass 10,000, Invitrogen) in Schneider's Drosophila medium, washed three times with medium and then placed on an imaging chamber as described above.

CHO WT and CHO ΔXylT cells were seeded on coverslips for pulse-chase experiments. In brief, CHO cells were first incubated with dextran-Alexa568 (10,000 MW, 0.4 mg/mL) (Invitrogen) for 4 h, washed and incubated in dextran-free CHO medium for 18 h. Cells were then pulsed with dextran-Alexa488 (10,000 MW, 0.4 mg/mL) for 10 min, washed and incubated in CHO medium for 30 min. CHO WT and CHO ΔXylT cells were fixed with 4% PFA, and stained with phalloidin-iFluor 647 Reagent (1:1000) (Abcam) and DAPI. Images of 20 random FOVs were acquired on Zeiss Apotome.2 microscope with 63 × oil immersion objective using AxioVision 4.9.1 software (Zeiss). Spatial resolution of images was 9.7674 pixels per micron, pixel size: 0.1024 × 0.1024 micron². Endo-lysosomal fusion was scored by quantifying co-localization between the two labeled dextrans using ImageJ version 1.52e and JACoP plugin for pixel intensity spatial correlation analysis (37 (link)). Pearson’s correlation coefficient and Manders split coefficients (M1 and M2, thresholds set manually for both channels) were calculated.

Embryos were dechorionated in 50% bleach solution for 2 min and then washed with water. Embryos were then relocated to a slide with a gas-permeable membrane in Halocarbon 27 oil (Sigma-Aldrich), covered with a coverslip and imaged. Live imaging of embryos was performed on a CSU10b Yokogawa spinning-disk confocal from Zeiss and Solamere Technologies Group with a 63×/1.4 NA objective. For slow movies, images were acquired in 30 s intervals with 27-30 z-slices at a 0.5 µm interval. Fast movies involved a single z-slice and 0.5-1 s intervals. For drug injection, after dechorionation as above, embryos were dehydrated for 15 min, covered with Halocarbon 700 oil and then injected with either colchicine (Sigma-Aldrich, C3915, 1 mg/ml in H₂O) or dextran Alexa568 (Thermo Fisher Scientific, D22912). Embryos were imaged immediately after the injection.