Figure 6.

CENP-OPQUR and NDC80 Complexes Bind Microtubules Cooperatively

In (A)–(C) and (E), Taxol-stabilized, rhodamine-labeled microtubules were tethered to glass coverslips and incubated in the presence of fluorescent recombinant proteins. The scale bar represents 1 μm.

(A) Alexa488-labeled CENP-OP (green channel) was unable to bind microtubules (red channel) in isolation and bound microtubules only in presence of Alexa647-labeled CENP-QU subcomplex (blue channel).

(B) NDC80-GFP complex (green channel) and Alexa-647-labeled CENP-OPQU (blue channel) interact with an overlapping surface to microtubules, as shown by reciprocal concentration-dependent competition.

(C) CENP-QU made deficient in microtubule binding by FAM labeling is translocated to microtubules through the interaction of CENP-OP with rKT21. Microtubules were incubated in the presence of CENP-QU N-terminally labeled with Fluorescein (green) and/or rKT21 in which CENP-A^MN was fused to BFP (blue) and CENP-LN was labeled with Alexa-647 (purple).

(D) PrDos (Ishida and Kinoshita, 2007) disorder prediction of the CENP-Q (black) and NDC80/HEC1 (red) N-terminal tails. Dotted line indicates the disorder threshold; false positive rate 5%.

(E) CENP-OPQUR and rKT21 bind microtubules cooperatively. Microtubules (red channel) were incubated with the indicated concentrations of Alexa-647-labeled CENP-OPQUR (purple), rKT21 containing Alexa-488-labeled KMN (green), or combinations thereof. At the bottom, the same experiment carried out in presence of CENP-OPQ^68-CUR shows that microtubule binding by the N-terminal region of CENP-Q is required for augmentation of microtubule binding affinity. Experiments in (A)–(C) and (E) are representative of at least 3 repeats. See also Figure S4 and Figure S7.