Figure 7. MISP is involved in spindle orientation and positioning. (A) Representative examples of mitotic UPCI:SCC114 cells transfected for 48 h with a siRNA against luciferase (left) or MISP (right) on an L-shaped fibronectin micropattern, stained with antibodies against pericentrin (red) and α-tubulin (green); DNA (Hoechst 33342) is shown in blue. Luciferase-siRNA-transfected cells orient their mitotic spindles along the hypotenuse of the L-shaped micropattern. In contrast, in MISP-1-siRNA-transfected cells the spindle orientation deviates from this orientation. Scale bar, 5 µm. (B) Frequency of angular deviations of spindle orientation away from the hypotenuse of L-shaped micropatterns for cells treated with siRNAs to luciferase or MISP. The graph represents the sum of all mitotic cells evaluated in three independent experiments. (C) Representative images (upper panel) of UPCI:SCC114 cells transfected with indicated siRNAs for 48 h and stained with antibodies against pan-cadherin to mark cell boundaries and α-tubulin reveal that mitotic spindles in MISP-depleted cells are located outside the cell center. White lines depict cell boundaries. Scale bar, 10 µm. A graph depicting the distribution of mitotic spindle positions relative to the cell center after transfection of the indicated siRNAs is shown in the lower panel. The graph represents the sum of mitotic cells evaluated in three independent experiments. (D) Representative images (upper panel) of GFP-MISP-U2OS cells transfected with indicated siRNAs stained with an antibody to α-tubulin and Hoechst 33342. The mitotic spindle is displaced from the cell center upon depletion of MISP by siRNAs MISP-1 and MISP-2, both of which deplete endogenous as well as overexpressed GFP-MISP. In cells transfected with MISP-3-siRNA targeting only endogenous MISP mitotic spindles are positioned at the cell center. Expression of GFP-MISP was induced 24 h after siRNA transfection for further 24 h. White lines depict cell boundaries. Scale bar, 10 µm. Determination of spindle positions (lower panel) shows that mitotic spindles in cells transfected with siRNAs MISP-1 and MISP-2 are displaced from the cell center, whereas overexpression of GFP-MISP rescues spindle positioning to the cell center. The graph shows the sum of mitotic cells evaluated in three independent experiments.