Differential regulation of single microtubules and bundles by a three-protein module

A remarkable feature of the microtubule cytoskeleton is the coexistence of subpopulations having different dynamic properties. A prominent example is the anaphase spindle, where stable antiparallel bundles exist alongside dynamic microtubules and provide spatial cues for cytokinesis. How are the dynamics of spatially proximal arrays differentially regulated? We reconstitute a minimal system of three midzone proteins: microtubule-crosslinker PRC1 and its interactors CLASP1 and Kif4A, proteins that promote and suppress microtubule elongation, respectively. We find that their collective activity promotes elongation of single microtubules while simultaneously stalling polymerization of crosslinked bundles. This differentiation arises from (1) strong rescue activity of CLASP1, which overcomes the weaker effects of Kif4A on single microtubules, and (2) lower microtubule- and PRC1-binding affinity of CLASP1, which permits the dominance of Kif4A at overlaps. In addition to canonical mechanisms where antagonistic regulators set microtubule length, our findings illuminate design principles by which collective regulator activity creates microenvironments of arrays with distinct dynamic properties.