1. Increased levels of MISP at the cell cortex promote spindle orientation defects.
Increased levels of MISP at the cell cortex promote spindle orientation defects. (A,B) HeLa cells were transfected with Flag or Flag–MISP for 48 h and plated onto fibronectin-coated coverslips. (A) Quantification showing the spindle angle of transfected cells. Data represent mean±s.d. from three independent experiments, n>65 cells. Cells are shown in a maximum z-projection, stained for α-tubulin (green) and pericentrin (PCNT, magenta), and co-stained with Hoechst (DNA, blue). The side view shows a z-slice of the pericentrin signal. Scale bars: 5 µm. (B) Quantification showing the normalized pole-to-cortex difference in distance for the cells. Data represent mean±s.d. from three independent experiments, n>68 cells. (C–E) HeLa cells were transfected as indicated and cells from a maximum projection of three planes stained for MISP (magenta) and ezrin (green), co-stained with Hoechst (DNA, blue) are presented. The spindle angle (D) or spindle position (E) of the indicated treatments was quantified. Data represent mean±s.d. from three independent experiments, n>65 cells. (F,G) Caco-2 BBe cells were transfected with indicated siRNAs and cystogenesis allowed to proceed for 4 days. (F) Cells were stained with antibodies against MISP (magenta) and ezrin (green), and co-stained with Hoechst (blue, DNA). Maximum projections of three confocal z-stacks through the middle region of the cysts are shown. Scale bars: 5 µm. The percentage of cells with single (normal) lumen or multiple lumens was quantified. Data represent mean±s.d. from three independent experiments, n>115 cells per experiment. (G) Quantification showing the spindle angle formed from the middle of the spindle and the mid point of the cyst. Data represent mean±s.d. from three independent experiments, n>61 cells. **P<0.01, ***P<0.001, ****P< (unpaired Student's t-test).
Yvonne T. Kschonsak, and Ingrid Hoffmann J Cell Sci 2018;131:jcs214544
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