Figure 3.

The Arp2/3 Complex Nucleates Spindle Actin, and Spindle Actin Is Required for Mitotic Spindle Formation

(A) Centrosomal Arp2/3 complex localization in inter- and prometaphase. NIH 3T3 cells were stained with anti-Arp2 (green) and Centrin 1 (red). Arrowheads indicate centrosomes, and the individual centrosome in interphase is shown as a magnification. For prometaphase, a maximum intensity projection was calculated to show both centrosomes in a single image. Scale bar, 10 μm or 1 μm (magnification).

(B) Chromosome congression in NIH 3T3 cells stably expressing mCherry-β-tubulin (MTs, red). Cells were stained with SiR-DNA (magenta) and treated with indicated inhibitors. See also Videos S6, S7, and S8. Scale bar, 5 μm; time stamp, min:s.

(C) Quantification of integrated sAC-TagGFP2 fluorescence intensities at centrosomes with indicated treatments during prometaphase. Data are normalized to DMSO and presented as box plots; n = 6 (CK-689, CK-666, SMIFH2) or 7 (DMSO, CK-869) events from two independent experiments.

(D) Live-cell imaging of NIH 3T3 cells labeled as in (B) treated with CK-666 during mitotic progression. Note the lack of centrosome-mediated MT assembly, whereas chromatin-mediated MT nucleation is not affected (white arrowheads). Scale bar, 5 μm; time stamp, min:s.