― 253 ―transducer (Validyne, DP15), a pressure amplifier (Validyne, PA501), and a digital multimeter.FRAP measurementsFluorescence recovery after photobleaching (FRAP) was measured using confocal laser scanning microscopy (FV3000). A region of interest (ROI) was photobleached with full laser power, and fluorescence recovery was recorded over time. The fluorescence intensities were normalized to the pre-bleach values, and recovery curves were fitted to a single exponential function.MIC assaysMinimum inhibitory concentrations (MICs) were determined using the broth micro-dilution method according to the Japanese Society of Chemotherapy. E. coli was cultured in Mueller-Hinton medium (Becton Dickinson) at 37°C for 18 hours to determine the MIC.Microscopic imaging of bacteriaTo observe the localization of Nur peptides and ribosomes, E. coli strains ATCC25922 and CJW7020 (expressing L1-msfGFP) were cultured to log-phase in LB. After adjusting the cultures to an OD600 of 0.1, cells were incubated with or without Nur peptides at 37°C. Observations were performed using a confocal laser microscopy (Olympus, FV3000) equipped with a ×100 objective lens (NA 1.4). To assess antibiotic-induced changes in ribosome localization, E. coli strain QC101 (expressing L9-mCherry) was grown to log phase in LB medium. After adjusted to OD600 to 0.1, cells were washed three times in EZ medium and diluted 1:50 in fresh EZ. The bacterial suspension was treated with Nur peptides at final concentrations of 200 μg/ml (much higher than MIC) for 45 minutes at room temperature. Cells were imaged using FV3000.Results and DiscussionFirst, we aimed to establish an in vitro fluorescence microscopy-based assay to observe lipid transfer mediated by the Atg2-Atg18 complex. GUVs containing the fluorescent lipid NBD-PE and another set of GUVs containing PI3P, which binds Atg18, were prepared. We brought these two types of GUVs into contact using micropipette manipulation and confirmed that, at this stage, there was no transfer of NBD-PE between the GUVs. We then introduced the Atg2-Atg18 complex into this system; however, NBD-PE transfer between GUVs still did not occur. Next, we examined SUVs containing fluorescent lipids. Initially, the Atg2-Atg18 complex was bound to PI3P-containing GUVs, after which SUVs containing NBD-PE were added to the system. Following incubation for a defined period, Proteinase K was added to degrade the Atg2-Atg18 complex, and the GUVs were observed under fluorescence microscopy. As a result, we detected the transfer of NBD-PE fluorescence onto the GUVs. Subsequently, we designed a mutant Atg2 based on the AlphaFold-predicted structure (Figure 1), introducing mutations that narrowed the central cavity, and purified this mutant Atg2. When this Atg2 mutant–Atg18 complex was added to the previously established SUV-GUV system, a significant reduction in NBD-PE transfer to the GUVs was observed. Together, these results strongly suggest that the Atg2-
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