The lateral LGN complex controls the stereotypical dynamics of spindle movements. (a) Still image from a time-lapse movie showing a metaphase wild-type cell expressing Centrin2-GFP only in the same field as a mutant cell expressing Centrin2-GFP + high levels of Gαi-G203A-ires-H2B-GFP (see Materials and methods). (b) Time-course of the spindle αZ variations during metaphase indicates systematic random movements upon Gαi-G203A expression (left graph) compared with quick planar orientation of Centrin2-GFP–expressing control cells (right graph) from the same field. (c) Time-lapse analysis of spindle movements relative to the apico–basal axis in a control Centrin2-GFP (top) and a Gαi-G203A-ires-H2B-GFP + Centrin2-GFP–expressing cell (bottom), taken from the same field in the same embryo. Apical is at the bottom and Centrin2-GFP–expressing centrosomes are pseudo-colored in red and green. In the control cell, most of the Z rotation (blue arrows) occurs within the first minutes of metaphase and is directed toward planar orientation. In the Gαi-G203A–expressing cell, Z rotation occurs throughout metaphase and is randomly oriented, and anaphase occurs with a random oblique axis. (d–f) Spindle Z-rotation is decreased by reduction of NuMA (d) or LGN (e) levels with RNAi and by expression of a dominant-negative form of LGN (f). (g) Global spindle dynamics during metaphase increases when Gαi-G203A increases, and decreases in the absence of cortical LGN and NuMA. Each pair of bars compares the average dynamics of mutant cells to Centrin2-GFP–expressing cells in the same field, in the same embryo (see Materials and methods). Numbers in the bars indicate the number of cells analyzed for each genotype. Spindle dynamics was not changed by expression of H2B-GFP or a control RNAi hairpin directed against luciferase. Error bar = SEM, *, P < 0.01; unpaired student t test. Bar, 5 µm.
