Figure 4. The number of microtubule bundles changes as mitosis proceeds.

(A) Cells were fixed and stained with antibodies (anti-α-tubulin clone DM1A, green) to detect microtubules and DAPI to label DNA (magenta). Cells with spindles perpendicular to the coverslip were imaged using confocal microscopy and deconvolved using Autoquant software (top panels), and 3D reconstructions were rendered using ChimeraX software (bottom panels, not to scale). Individual z planes are shown for slices approximately 25%, 50%, and 75% through the spindle for three representative cells. Numbers (upper left) indicate the number of distinct microtubule bundles in that position of the spindle.

(B) The number of microtubule bundles throughout the spindle length in metaphase spindles, imaged as in (A). Some spindles (top) had a fairly consistent number of microtubule bundles throughout the spindle, while other spindles (bottom) had a peak in the number of bundles toward the midpoint. Each line represents one spindle, pooled from three experimental replicates.

(C) The maximum number of microtubule bundles from confocal images of metaphase cells (calculated from 4 experimental replicates encompassing 21 cells). Each point represents one cell.

(D) Line scans show the relative DNA and tubulin fluorescence intensity from sum intensity projections of spindles lying in the plane of the coverslip, imaged as in (A). Metaphase spindles were grouped based on the shapes of tubulin curves (no shoulders, left; unclear shoulders, center; two clear shoulders denoted by asterisks, right); three individual examples are shown in each panel (also see Figures S4G and S4H). Each line represents one spindle; a total of 15 representative spindles were selected from 25 analyzed images from two experimental replicates.

(E) Quantification of DNA (top) or tubulin (bottom) from line scans obtained as in (D). Metaphase was categorized as early or late based on the presence (late) or absence (early) of shoulders (stages where no clear classification could be assigned were excluded). Each point represents the area under the curve for one spindle line scan, and lines indicate the mean ± SD. Values were calculated from 52 spindles, pooled from 4 experimental replicates.

See also Figures S4 and S6, Videos S1 and S2, and Table S2.