Anillin Phosphorylation Controls Timely Membrane Association and Successful Cytokinesis

Abstract

During cytokinesis, a contractile ring generates the constricting force to divide a cell into two daughters. This ring is composed of filamentous actin and the motor protein myosin, along with additional structural and regulatory proteins, including anillin. Anillin is a required scaffold protein that links the actomyosin ring to membrane and its organizer, RhoA. However, the molecular basis for timely action of anillin at cytokinesis remains obscure. Here, we find that phosphorylation regulates efficient recruitment of human anillin to the equatorial membrane. Anillin is highly phosphorylated in mitosis, and is a substrate for mitotic kinases. We surveyed function of 46 residues on anillin previously found to be phosphorylated in human cells to identify those required for cytokinesis. Among these sites, we identified S635 as a key site mediating cytokinesis. Preventing S635 phosphorylation adjacent to the AH domain disrupts anillin concentration at the equatorial cortex at anaphase, whereas a phosphomimetic mutant, S635D, partially restores this localization. Time-lapse videomicroscopy reveals impaired recruitment of S635A anillin to equatorial membrane and a transient unstable furrow followed by ultimate failure in cytokinesis. A phosphospecific antibody confirms phosphorylation at S635 in late cytokinesis, although it does not detect phosphorylation in early cytokinesis, possibly due to adjacent Y634 phosphorylation. Together, these findings reveal that anillin recruitment to the equatorial cortex at anaphase onset is enhanced by phosphorylation and promotes successful cytokinesis.

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“…And Anillin, a phylogenetically conserved protein, is the essential component that plays the scaffold protein role and links the actomyosin ring to the membrane and RhoA, which is a pivotal effector directing the localization of the contractile actomyosin ring [ 23 ]. In other words, dysfunction of Anillin could impair the efficient integration of RhoA functions and leads to failure of cytokinesis [ 24 ].…”

Active Ran regulates anillin function during cytokinesis

“…This autoinhibition could ensure that anillin’s C-terminus has access to importin or microtubules only when RhoA-GTP levels increase during mitotic exit. A recent study showed that anillin is also regulated by phosphorylation, via a site that lies just N-terminal to the RBD ( Kim et al. , 2017 ).…”

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“…This was also underpinned by the evidence of phosphomimetic-mutant S635D, the negative charge of D at the 635 residues which partially recovered the localization. S635 phosphorylation helps ANLN improve the efficacy of the Rho integration with its upstream and downstream regulators, which contributes to the success of cytokinesis [58]. To date, the kinases that are responsible for S635 remain unidentified, so there is a need to determine which kinases are responsible for the phosphorylation at S635.…”

“…CDK1 was found to interact with ANLN [56,57], and it is indicated that ANLN mobility is directly or indirectly regulated by CDK1 via phosphorylation [58]. The ANLN-actin-binding protein has been identified as being involved in PI3K/PTEN signaling, which is critical in cell life/death control [59].…”