Fig. 3: Phosphorylation of PRC1 by CDK1 prevents stable minimal midzone organization in the presence of KIF4A.

A Schematic of the minimal midzone self-organization assay. Purified PRC1 and KIF4A organize microtubules into bundles with compacted antiparallel overlaps at the center. PRC1 crosslinks antiparallel microtubules and recruits KIF4A to the antiparallel overlap zone. In turn, KIF4A walks towards the microtubule plus-end pulling on PRC1, generating antiparallel microtubule sliding, which compacts the antiparallel overlap at the bundle center. Parallel microtubule minus segments grow outward, gnerating an organization similar to anaphase midzone bundles. B TIRF microscopy images taken ~20 min after self-organization of microtubule bundles in the presence of 20 nM unphosphorylated (top) or fully CDK1-phosphorylated (bottom) PRC1-Alexa546 (green), 5−50 nM KIF4A-mGFP (blue), and 18 µM Alexa647-tubulin (red). See Suppl. Figure 4 for same data displaying the individual channels separately. Scale bar as indicated. C Example TIRF microscopy images of a time series of (left) a bundle with a stable compacted antiparallel microtubule overlap (minimal midzone) in the presence of 20 nM unphosphorylated PRC1 and 30 nM KIF4A (left), a bundle that fails to form a compacted overlap in the presence of 20 nM CDK1-phosphorylated PRC1 and 10 nM KIF4A (middle), and an antiparallel bundle that first forms a compacted overlap that is then however unstable and bends in the presence of 20 nM CDK1-phosphorylated PRC1 and 30 nM KIF4A. The Alexa647-tubulin concentration was always 18 µM. Scale bar as indicated. D Phase spaces of antiparallel bundle self-organization for unphosphorylated and CDK1-phosphorylated PRC1. Each symbol represents a minimum of 3 individual experiments with 10–20 observed microtubule bundles per experiment, and a symbol with two colors represents a condition at the boundary between two regimes where the outcome of self-organization can lead to either one or the other type of organization. Symbols as in (C).