Cytokinesis requires supervillin‐mediated binding to myosin II and synergism with anillin (962.1)

Abstract

Cytokinesis requires coordination of cortical myosin II activation with central spindle alignment. Supervillin and anillin are two actin‐ and myosin II‐associated vertebrate proteins implicated in maintenance of the cytokinetic furrow in early cytokinesis, The nature of their cross‐talk is unknown. Point mutations within an evolutionarily conserved sequence in supervillin, residues 99 ‐ 153, eliminate binding to the nonmuscle myosin II heavy chain (MHC), confer dominant‐negative inhibition of cytokinesis, and abrogate the ability of full‐length supervillin to rescue RNAi‐mediated depletion of supervillin. While an observed ~2‐fold increase of anillin in supervillin‐knockdown cells does not eliminate cytokinetic failure, knockdown of both proteins together yields more than additive increases in numbers of multinucleated cells, as compared with depletion of either protein alone. Localizations of supervillin, anillin, MHC (total myosin), and phosphorylated regulatory myosin II light chain (pMRLC, activated myosin) in dividing cells suggest that supervillin and anillin synergize to confine MHC to the furrow. Anillin alone is sufficient to maintain normal levels of pMRLC at the furrow in late anaphase, but supervillin is required for normal pMRLC localization in early anaphase and around the cytokinetic bridge during daughter cell separation. Protein affinity isolations of GFP‐tagged supervillin and anillin indicate that these proteins do not significantly associate with each other although myosin II, actin and other cytoskeletal proteins co‐purify with each bait protein. None of these proteins co‐isolated with GFP alone. Stark differences between specifically co‐isolating kinases, phosphatases, and other signaling proteins reinforce the conclusion that supervillin and anillin regulate cytokinesis through separate pathways.Grant Funding Source: Supported by the National Institutes of Health and the University of Massachusetts Medical School.