Figure 8.

GRASP55 is required for Golgi fragmentation and mitotic entry in a phosphorylation-dependent manner. (A) As reported previously (Sütterlin et al., 2002) NRK cells were arrested in S phase with thymidine. The cells were washed to remove thymidine, and then they were injected with either C100, [201-304], or BSA as a control. Between 5.5 and 9.5 h post S-phase release, cells were fixed and stained with phospho-histone H3 and Hoechst 33342 to determine the number of mitotic cells. For each time point 200 cells were counted. The data shows an average of four independent experiments. The maximum mitotic index was observed at 7.5 h postthymidine release. (B) Wild-type [201-304], [201-304]EET, [201-304]TTE, [201-304]EEE, KHM buffer, or His-tagged GFP were tested in an in vitro assay reconstituting Golgi fragmentation in NRK cells by mitotic extract. The percentage of cells with fragmented Golgi membranes was determined by immunofluorescence microscopy using anti-giantin antibody. (C) Wild-type [201-304], [201-304]EET, [201-304]TTE or [201-304]EEE were injected into post-S phase-arrested NRK cells as described previously (Sütterlin et al., 2002). Cells were fixed at 7.5 h and processed to determine mitotic index compared with control or noninjected cells. The data represents and average of the total experiments (control, n = 14; C100 injected, n = 4; each [201-304] peptide, n = 4).