A multivalent docking platform and Rcn1-mediated inhibition control the extent of calcineurin recruitment to the cell division site for the dephosphorylation of multiple cytokinetic proteins.

Cytokinesis requires coordinated signaling to ensure the accurate physical separation of daughter cells. Calcineurin (CN), a conserved Ca 2+ /calmodulin-dependent phosphatase, is required for cytokinesis in organisms ranging from yeast to humans, yet how CN is regulated at the division site and the substrates through which it promotes cell division remain poorly understood. Here we use the fission yeast Schizosaccharomyces pombe, which display striking cell division defects in the absence of CN, to define how CN is anchored at the cell division site. We show that CN recruitment to the cytokinetic ring (CR) requires its PxIxIT- and LxVP-binding surfaces and is mediated by multivalent interactions with the CR components paxillin-like Pxl1 and the F-BAR protein Cdc15. Disrupting these interactions nearly eliminates CN from the CR and causes gross cytokinetic defects similar to complete loss of CN function. Cell cycle stage-specific quantitative phosphoproteomics combined with proximity labeling-based proteomics were used to identify candidate CN substrates involved in cytokinesis. Validation of a cohort of these proteins localizing to the CR, including the F-BAR protein Rga7, the actin regulator Aim21 and three protein kinases, revealed that CN targets a broad network of structural and signaling components involved in cell division. We also identify the conserved CN inhibitor Rcn1 as a CN substrate and show that Rcn1 restricts CN accumulation at the CR to provide an additional layer of spatial regulation. Thus, spatial control of CN enables proper protein dephosphorylation for successful cytokinesis.