Fig. 4

HURP p725 is required for the formation of Golgi ribbon. A Differential localization of HURP p725 and np725. HeLa cells were examined for the subcellular localization of HURP p725 and np725 by immunofluorescence. EGFP-TRIP11 was used to label GA. Three independent experiments were performed. B Colocalization of HURP p725 with cis-Golgi marker. Immunofluorescence was conducted in HeLa cells to visualize HURP p725, trans-Golgi (GRASP55), cis-Golgi (GRASP65) and the ER-Golgi intermediate compartment (ERGIC3). Three independent experiments were performed. C HURP was localized to GA area and displayed morphological correlation with GA. Immunofluorescence was conducted in HeLa cells to visualize HURP p725 and GA (GRASP65) (I). The cells with HURP p725 in crescent ribbon-like bundle (ribbon), compact or fragment were selectively examined, and % of those selected cells having GA in crescent ribbon, compact or fragment form was counted and plotted (II). By contrast, the cells with GA in crescent ribbon (ribbon), compact or fragment were selectively examined, and % of those selected cells having HURP p725 in crescent ribbon-like bundle (ribbon), compact or fragment form was counted and plotted (III). To analyze the structure of GA or HURP p725, 200 cells for each independent experiment were examined, and 3 independent experiments were performed. D HURP p725 displayed extended fiber-like distribution and np725 was localized to spindle in mitotic cells. HeLa cells were applied to immunofluorescence to visualize the distribution of HURP, HURP p725, np725 and spindle. Mitotic cells were selectively examined. Three independent experiments were performed. E The cells losing HURP p725 or containing longer HURP p725 fiber had more fragmented or elongated GA. The HeLa cell doublets undergoing cytokinesis were selectively examined, including cells with symmetric or asymmetric segregation of HURP p725. HURP p725 and GA (GRASP65) were detected by immunofluorescence (I). The % of cells with or without HURP p725 containing GA fragments distributed in 1, 2–5 or greater than 5 clusters were counted and plotted (II). The length of HURP p725 (III) or GA (IV) in cell doublets with symmetric or asymmetric segregation of HURP p725 was measured. The length of HURP p725 or GA in the cell doublet containing symmetric HURP p725 was the average of the two connected cells. Length of GA or HURP p725 was measured only in the cells with HURP p725 when HURP p725 was asymmetrically segregated. To analyze the structure of GA or HURP p725, 200 cells for each independent experiment were examined, and 3 independent experiments were performed. As to the measurement of the length of HURP p725 or GA, 20 cells were examined with the image analysis software MetaVue version 7.8.0.0 (Molecular Devices) for each independent experiment and 3 independent experiments were performed. F Knockdown of HURP fragmented GA, and that was not rescued by HURP 725A. HeLa cells harboring shLuc or shHURP plus each one of pcDNA3.1 empty vector, HA-HURP WT, 725E or 725A were analyzed for the GA structure by immunofluorescence (I, II), or knockdown efficiency by Western blot (III). The % of cells with various forms of GA was counted and plotted. To analyze the structure of GA stained by GRAST65 antibodies, 200 cells either with shLuc, or shHURP plus HA-HURP WT, 725E or 725A, for each independent experiment were examined, and 3 independent experiments were performed. G HURP 122K lost the Golgi ribbon constructing activity. HeLa cells harboring shLuc, shHURP plus EGFP, or shHURP plus each one of EGFP-HURP versions were analyzed for the GA structure by immunofluorescence (I, II), or knockdown efficiency by Western blot (III). The % of cells with various forms of GA was counted and plotted. To analyze the structure of GA stained by GRAST65 antibodies, 200 cells either with shLuc, or shHURP plus HA-HURP WT, 122K or 122F, for each independent experiment were examined, and 3 independent experiments were performed. H Knockdown of Ajuba fragmented GA. HeLa cells harboring shLuc or shAjuba were analyzed for the GA structure by immunofluorescence (I, II), or knockdown efficiency by Western blot (III). To analyze the structure of GA stained by GRAST65 antibodies, 200 cells either with shLuc or shAjuba for each independent experiment were examined, and 3 independent experiments were performed. The % of cells with various forms of GA was counted and plotted. *, **, and *** stand for statistical significance by Student’s t-test with p < 0.05, 0.01 and 0.001 respectively. Scale bar: 10 μm