Fig. 2: Phosphorylation of PRC1 by CDK1 reduces PRC1 binding to antiparallel microtubule overlaps, indirectly affecting phosphorylation by PLK1 in overlaps.

A Schematic of the antiparallel microtubule pair assay (left) and an example time sequence of TIRF microscopy images of the formation of an antiparallel microtubule (red) overlap in the presence of PRC1 at the indicated concentrations. B TIRF microscopy images of unphosphorylated (top) or fully CDK1-phosphorylated (bottom) PRC1-Alexa546 binding to antiparallel overlaps of Alexa647-microtubules elongating from surface immobilized microtubule ‘seeds’. Alexa647-tubulin concentration is 18 µM, PRC1 concentrations and scale bar as indicated. Same data displaying also the microtubule channel in Suppl. Figure 2A. C Quantification of the PRC1-Alexa546 fluorescence intensity measured in antiparallel microtubule overlaps for unphosphorylated and fully CDK1-phosphorylated PRC1 as a function of the PRC1 concentration. A Hill function fit to the data of unphosphorylated PRC1 yields a dissociation constant of 10 ± 0.75 nM. A linear regression is shown for the data of phosphorylated PRC1. Error bars are standard deviation. n = 3, total length of 100 µm antiparallel overlaps for each tested concentration. D TIRF microscopy images of unphosphorylated (top) or fully PLK1-phosphorylated (bottom) PRC1-Alexa546 binding to antiparallel overlaps of Alexa647-microtubules elongating from surface-immobilized microtubule ‘seeds’. Alexa647-tubulin concentration is 18 µM, PRC1 concentrations and scale bar as indicated. Same data displaying also the microtubule channel are shown in Suppl. Figure 2B. E PRC1-Alexa546 fluorescence intensity in antiparallel microtubule overlaps for unphosphorylated and PLK1-phosphorylated PRC1 as a function of the PRC1 concentration. A Hill function fit to the data of unphosphorylated and PLK1-phosphorylated PRC1 yields dissociation constants of 9.3 ± 0.46 nM and 9.6 ± 0.68 nM. Error bars are standard deviation. n = 3 independent experiments, total length of 100 µm antiparallel overlaps for each tested concentration. Fluorescence intensity in (C) and (E) are converted to electron count. F Phos-tag gel of 800 nM PRC1-SNAP phosphorylated for 5 min by 100 nM PLK1 with or without stabilized microtubules at different (polymerized tubulin) concentrations, or unphosphorylated PRC1 as negative control. n = 5 independent experiments. Source data are provided as a Source Data file.