Lsm 880

The acceptor photobleaching module of Zeiss LSM880 software was employed for FRET analysis. The cells were co-stained with anti-CP110 and anti-LC3 antibodies followed by Alexa Fluor 488-conjugated anti-rabbit and 555-conjugated anti-mouse secondary antibodies, respectively. The 488-nm laser line was used to excite endogenous CP110 and the 561-nm laser line was used to excite and bleach endogenous LC3. A region containing centrosomes co-stained with CP110 and LC3 was selected for bleaching. The LC3 dots were bleached using 50 iterations of 561 nm laser line at 60% intensity. Images were taken each 6-s intervals for 5 cycles. FRET efficiency (E) was calculated as follows: E = (Dpost − Dpre)/Dpost for all Dpost > Dpre. Dpre and Dpost are donor fluorescence intensity before and after photobleaching, respectively. The fluorescence intensities were collected from the Mean ROI module of Zeiss LSM880.

Worms were anesthetized using 100 mM of sodium azide (NaN₃) and mounted on 5% agarose on glass slides. All images (except Figure 8 and Figure 7—figure supplement 1) were acquired using a Zeiss confocal microscope (LSM880). Several z-stack images (each ~0.4 μm thick) were acquired with the ZEN software. Representative images are shown following orthogonal projection of 2–10 z-stacks. Images shown Figure 8 and Figure 7—figure supplement 1 were taken using an automated fluorescence microscope (Zeiss, AXIO Imager Z1 Stand). Acquisition of several z-stack images (each ~1 μm thick) was performed with the Micro-Manager software (Version 3.1). Representative images are shown following max-projection of 2–10 z-stacks using the maximum intensity projection type. Image reconstruction was performed using ImageJ software (Schneider et al., 2012 (link)).
For quantification of UNC-7::GFP puncta shown in Figure 7C, images were acquired and z-stack were generated as described above. Manual counting of the UNC-7::GFP puncta was performed using the cell counter plug-in of the ImageJ software.
For the quantification of eat-4 and cho-1 expression in AIM for the analysis shown in Figure 5F, images were acquired using a Zeiss confocal microscope (LSM880) and the fluorescence intensity mean was obtained with the ZEN software tool.

Alizarin-Red-S vital dye, MS-222 anesthesia, and mounting for microscopy were performed as previously described (Aman et al., 2021 (link)). Images in Figure 1—figure supplement 1B, Figure 2C and D, Figure 4C, Figure 5E, and Figure 8A, D and E were acquired on a Zeiss LSM880 in fast Airyscan mode. Images in Figure 2—figure supplement 1A and Figure 4A were acquired on a Zeiss LSM880 in conventional confocal mode. Images in Figure 1A, Figure 1—figure supplement 1C, Figure 3C and E, Figure 5D, Figure 6E, and Figure 6—figure supplement 1A and B were acquired on a Zeiss Observer equipped with Yokogawa CSU-X1 spinning disc. Images in Figure 8—figure supplement 2 were acquired on a Zeiss SteREO Discovery V12 stereomicroscope. Orthogonal views were produced using FIJI (Schindelin et al., 2012 (link)). Brightness and contrast were adjusted in Adobe Photoshop, and nonlinear gamma adjustments were applied to images when necessary to highlight relevant cell types. Photoconversion of nuclear EOS was done using a 405 nm laser on a Zeiss LSM800.

Control and KO cells were fixed with 4% formaldehyde for 10 min, washed with PBS twice and incubated with 20 µg/mL biotinylated lectin for 1 h on ice, followed by staining with 1 µg/mL Cy5-Streptavidin. The levels of lectin binding were visualized using a confocal microscope (LSM 880; Zeiss, Oberkochen, Germany), or analyzed by a flow cytometer (Cytoflex LX; Beckman Coulter, Brea, CA, USA). Control and KO cells were infected with IAV for 60 min on ice, which allowed attachment but prevented internalization, then fixed. The cells were incubated with the corresponding primary antibody and fluorescent secondary antibody for 2 h and 1 h, respectively, and then the nuclei were stained with DAPI for 15 min at room temperature. A confocal microscope (LSM 880; Zeiss, Oberkochen, Germany) was used to obtain images.

MEF cells were plated at a density of 2.5 × 10⁴ cells cm⁻³. Three days after seeding, cells were treated with 5 µM liposomes, 5 µM solubilized lipids, or 20 µM exosomes diluted in DMEM medium with HEPES (pH7.4) and incubated at 37 °C for 30 to 60 min. BSA-solubilized lipids were made up of 10 mM lipid in 12% w/v BSA with 150 mM NaCl and solubilized by vortexing^{58 (link)}. In total, 100 nM Lysotracker-Red/Deep Red (Invitrogen) or 5 mg mL⁻¹ transferrin-Alexa594/Alexa488 (Invitrogen) were mixed with the lipid uptake solution if needed. For endocytosis inhibition, cells were pretreated with 100 µM of Dyngo-2a (Abcam) or nystatin (Sigma) for 15 min at 37 °C before adding in the liposome mixture. Cells were rinsed with PBS to wash off unbound reagents and fixed with 4% PFA. Mounted slides were stored at 4 °C and confocal microscopy images were taken with LSM 880 (Zeiss) within 5 days post-fixation. For pixel intensity measurement, images quantification was conducted as described in the cell culture and immunocytochemistry section. For lysosomal colocalization, z-stack confocal images were acquired under the optimal acquisition setting of LSM 880 (Zeiss). Built-in Coloc function in the Imaris software (Bitplane) was used for volume rendering, spot detection, and overlapping percentage count.