FIGURE 5.

Re-recruitment of adaptors after 20 °C block occurs initially on recycling endosomes in CD8-M6PR-expressing cells. HeLaM cells expressing CD8-M6PR for 24 h were untreated (37 °C) or temperature-blocked (20 °C) for 4 h before return to 37 °C for the indicated times. Cells were stained for CD8 and GGA1 (A and C), GGA1 and TfR (B and E), or GGA1 and GM130 (D). CD8-M6PR (C) staining was also imaged by wide field microscopy and used to generate isosurfaces into which GGA1 recruitment was quantified and normalized to cells kept at 20 °C for 4 h. GGA1 recruitment to CD8-M6PR was statistically significantly decreased during 20 °C block and returned to control levels only 15 min into recovery. GGA1 intensity within CD8-defined isosurfaces was analyzed using analysis of variance, and asterisks indicate p < 0.01 compared with steady state (n > 5 cell analyzed). D and E, confocal images were acquired and quantified using M1 and M2 coefficient, as described under “Experimental Procedures.” Bar graphs represent mean Mander's coefficients of GGA1 intensity that is also GM130-positive (D) or GGA1 intensity that is also TfR-positive (E). Error bars indicate S.E. Asterisks indicate p < 0.01 when evaluated with a one-way analysis of variance and a Dunnett post-test. All comparisons were made to staining in HeLaM maintained at 37 °C.