Figure 2.

Spindle Actin Is Branched and Precedes Kinetochore MTs

(A) Confocal live-cell imaging of RPE-1 cells, stably expressing shuttling actin-chromobody-TagGFP2 (actin, green), stained with SiR-tubulin (MTs, magenta). Spindle actin is assembled at centrosomes and microtubules during formation of the mitotic spindle (arrowheads). Scale bar, 5 μm; time stamp, min:s.

(B) Quantification of spindle actin duration in NIH 3T3 and RPE-1 cells. Data are shown as mean + SD of two independent experiments together with individual data points; n > 5 events per cell line.

(C) Confocal live-cell imaging of an individual centrosome (asterisk, 00:00) in NIH 3T3 cells during prometaphase, stably expressing shuttling actin-chromobody (actin, green) and mCherry-β-tubulin (MTs, red). Individual actin filaments aligning with forming spindle MTs are indicated by arrowheads. Scale bar, 1.5 μm; time stamp, min:s.

(D) Quantification of spindle actin branch angle in NIH 3T3 cells, as indicated in (C) (00:01) by dashed, violet lines. Data are shown as a scatter box plot; n = 11 actin filament branches.

(E) Confocal live-cell imaging of NIH 3T3 cells stably expressing shuttling actin-chromobody (actin, green) and mCherry-β-tubulin (MTs, red) during prometaphase. Kymographs corresponding to the violet arrows are shown on the right and illustrate actin assembly before MT polymerization on individual tracks at the equatorial plane. Scale bar, 5 μm; time stamp, min:s.