Figure 5.

MLK3 overexpression disrupts microtubule organization. HEK293 cells containing a stable, inducible, wild-type MLK3 expression plasmid grown on coverslips were incubated with tetracycline (1 μg/ml; Ac and d, Bc and d; Induced) or vehicle (ethanol) alone (0.1%; Aa and b, Ba and b; Uninduced) for 23 h. Cells were then fixed in methanol and stained for α-tubulin. (A) metaphase cells. To detect maximal immunofluorescence signal in the astral area, images were collected using conventional microscopy (Aa and c). For clarity, confocal images (antibody and d) were also collected. (B) interphase cells. Shown are representative single fields of both uninduced (Ba and b) and induced (Bc and d) cells from which confocal optical slices were captured at two different depths. Ba and c are images focused close to the topmost cellular peripheries, whereas Bb and d are images focused 2–3 μM more internally, toward the cell centers. Scale bars, 10 μM, applies to image pairs Aa and c, Ab and d and to all images of a given field in B.