Figure 8.

TPX2/Aurora A complex recruits WDR62 to GDP-MTs in vitro. (A) Time-lapse images showing progressive accumulation of 3 nM TagBFP-TPX2 (green) on GDP-MT over time (compare the TPX2 intensity between two vertical dashed lines at indicated time points), leading to biased deposition of TPX2 on older lattice segment near the seed and no binding to newly generated segment of GDP-MT (arrows). The distance between two vertical dashed lines is 2 µm, and the position of the right dashed line is MT plus end at 0 min. (B) Images and corresponding kymographs showing MT binding of 3 nM, 7.5 nM, and 15 nM TagBFP-TPX2 (green). Vertical arrows in images and horizontal arrows in kymographs indicate the absence of TPX2 (3 nM and 7.5 nM) from newly generated segment of GDP-MT. (C) Plots of average intensity of TagBFP-TPX2 at indicated concentrations on a 2-µm-long segment of GDP-MT as in A (between two vertical dashed lines) against time. The values were normalized to the maximum intensity of 15 nM TPX2. n = 10–20 MTs from two experiments. (D) Plots of intensities of TagBFP-TPX2 (green) and MT (red) against distance (denoted by the horizontal dashed line at 1 min in A), showing stronger TPX2 intensity on older GDP lattice. (E) Images and corresponding kymographs of GFP–Aurora A (AurA) alone (green) or together with TagBFP-TPX2 (blue) at indicated concentrations. In the presence of TPX2, Aurora A was recruited to MTs and colocalized with TPX2. The "x" in left panels indicates that TPX2 was not included in the assay. Vertical arrows in images and horizontal arrows in kymographs indicate the absence of TPX2 and Aurora A (3 nM and 7.5 nM) from newly generated segment of GDP-MT. (F) Quantification of intensities of 15 nM GFP–Aurora A on GMPCPP seeds and GDP MTs in the absence or presence of 15 nM TagBFP-TPX2 after 5 min of GDP-MT growth from seed. n = 21 or 22 MTs from two experiments. (G) Plots of average TagBFP-TPX2 or GFP–Aurora A intensity on a 2-µm-long segment of GDP-MT at indicated concentrations against time as in C. The values were normalized to the maximum intensity in the condition of TPX2 and Aurora A both at 15 nM. n = 10–20 MTs from two experiments. (H) Images of MTs grown in the presence of 30 nM WDR62-GFP (green) together with 15 nM TagBFP–Aurora A (blue) or 15 nM TagBFP-TPX2 (blue). Note that Aurora A can weakly localize to seeds in the presence of WDR62 (compare E and H). (I) Quantification of intensities of 30 nM WDR62-GFP on the GMPCPP seeds and GDP MTs in the absence or presence of 15 nM TagBFP–Aurora A or 15 nM TagBFP-TPX2 after 5 min of GDP-MT growth from seed. n = 20–30 MTs from two experiments. (J) Images and corresponding kymographs of MTs grown in the presence of WDR62-GFP (green) and TPX2/TagBFP–Aurora A complex (blue) at indicated concentrations. Vertical arrows in images and horizontal arrows in kymographs indicate the absence of WDR62 from newly generated segment of GDP-MT at all TPX2/Aurora A concentrations. (K) Plots of average WDR62-GFP or TPX2/TagBFP–Aurora A intensity on a 2-µm-long segment of GDP-MT at indicated concentrations against time as in C. The values were normalized to the maximum intensity in the condition of 30 nM WDR62 with 15 nM TPX2/Aurora A. n = 10–16 MTs from two experiments. (L) Quantification of intensities of 30 nM WDR62-GFP on GMPCPP seeds and GDP MTs in the absence or presence of TPX2/TagBFP–Aurora A at indicated concentrations after 5 min of GDP-MT growth from seed. The values were normalized to the intensity of WDR62 alone on GMPCPP seeds. n = 17–30 MTs from two experiments. Horizontal scale bar, 2 µm; vertical scale bar, 1 min. Data represent mean ± SD. ***, P < 0.001; two-tailed t test.