Fig. 4. M-C₆-NBD-PC is trafficked to the vacuole in an energy-requiring, vesicle-mediated process. (A) The wild-type diploid strain CRY3 was grown to early log phase in SDC at 30°C. Cells were then labeled with DAPI for 10 minutes prior to being placed on ice and chilled. After approximately 10 minutes on ice, cells were labeled with 10 µM DMSO-solubilized lipid for 90 minutes. Cells were then harvested and washed three times with either ice-cold SCNaN₃+F or ice-cold SDC. Aliquots from each batch of cells were then incubated in their respective media at 30°C for 1 hour. Cells were subjected to a final wash with ice-cold SCNaN₃+F prior to analysis and imaging by fluorescence microscopy. (B) The end4 mutant strain and its isogenic parent strain were grown to early log phase in SDC at 30°C. The isogenic parent and end4 strains were labeled with DMSO-solubilized M-C₆-NBD-PC (1.0 µM and 0.5 µM, respectively) on ice for 60 minutes, followed by washing twice in ice-cold SDC. Cells were warmed to 30°C and labeled with FM4-64 (40 µM) for 15 minutes, washed twice in SDC (30°C) and resuspended in SDC (30°C) for 60 minutes. Cells were centrifuged, then resuspended in ice-cold SCNaN₃ prior to imaging by fluorescence and differential interference contrast (DIC) microscopy.