Fig. 8. Cells from shi^ts animals reversibly arrest mBSA uptake at the cell surface. (A) Protocol designed to test the reversibility of the temperature-sensitive shibire inhibition of endocytosis and the nature of fluid-phase endosomes formed at the restrictive temperature (31°C). (B) Cells derived from the indicated alleles were incubated as in panel A, fixed and imaged on a wide-field microscope. Cy3-mBSA (red in merge) and Cy5-mBSA (blue in merge) colocalize (arrows) in a population of F-Dex (FDx)-containing endosomes (green in merge) that were formed at 31°C, confirming that F-Dex-containing endosomes formed in shi^ts cells at 31°C are capable of mixing with surface-arrested Cy3mBSA when the cells are returned to permissive temperatures. (C) Protocols designed to test the kinetics of sequestration of B-Cy3mBSA when cells from shi^ts1 animals were pre-incubated at 21°C (left panel) or 32°C (right panel) and then rapidly transferred to ice. The percentage of cellular B-Cy3mBSA accessible to Cy5-SA in the depicted cell is indicated at the top right in each image. Note that after transfer from 32°C to ice, much of the B-Cy3mBSA fluorescence remains in peripheral endosomes (arrowheads) inaccessible to Cy-SA. (D) Histogram showing the relative accessibility of B-Cy3mBSA incubated with shi^ts1 cells at the indicated temperatures (white bars) and then transferred to ice (0°C, blue bars), relative to cells directly labeled on ice (arrow). Data shown are mean±s.e.m. obtained from one experiment with duplicate dishes with at least 30 cells in each dish. Similar results were obtained in two independent experiments with cells from of shi^ts1and shi^ts2 animals. Bar, 5 µm.