HOPS function is required for SE recovery. (A) Schematic diagram of the method for monitoring SEs fusion with vacuoles (Vac). (B and C) Tracing of SEs by LLSM during recovery of vps11-1 mutant cells (from Video 5). vps11-1 cells expressing mGFP-tagged Ivy1 were grown at 24°C in a synthetic medium, incubated at 37°C for 1 h, and then tracked and analyzed by LLSM at 24°C. Vacuoles were stained with CMAC. The 3D stacks were cropped by Imaris after deconvolution, and the different channels were split by ImageJ. Scale bar, 2 µm. Imaris-defined xy, xz, and yz planes of the 32-min time point are shown. The analyzed dot is indicated in B by white arrows. Ivy1 fluorescent intensity was analyzed by vantage time plots in Imaris, and the plot statistic values were measured by surpass objects with spots (C). (D) 3D view of Ivy1 after dots dispersion. The image was extracted from Video 5, and all views are shown. (E) Quantification of Ivy1 dots in Video 6. The numbers of Ivy1 dots were counted manually and analyzed by ImageJ. Error bars represent SD of three independent experiments.