Summary Anillin is a scaffolding protein that organizes and stabilizes actomyosin contractile rings and was previously thought to function primarily in cytokinesis [1–10]. Using Xenopus laevis embryos as a model system to examine Anillin’s role in the intact vertebrate epithelium, we find that a population of Anillin surprisingly localizes to epithelial cell-cell junctions throughout the cell cycle, whereas it was previously thought to be nuclear during interphase [5, 11]. Further, we show that Anillin plays a critical role in regulating cell-cell junction integrity. Both tight junctions and adherens junctions are disrupted when Anillin is knocked down, leading to altered cell shape and increased intercellular spaces. Anillin interacts with Rho, F-actin, and Myosin II [3, 8, 9], all of which regulate cell-cell junction structure and function. When Anillin is knocked down, active Rho (Rho-GTP), F-actin, and Myosin II are misregulated at junctions. Indeed, increased dynamic “flares” of Rho-GTP are observed at cell-cell junctions, while overall junctional F-actin and Myosin II accumulation is reduced when Anillin is depleted. We propose that Anillin is required for proper Rho-GTP distribution at cell-cell junctions and for maintenance of a robust apical actomyosin belt, which is required for cell-cell junction integrity. These results reveal a novel role for Anillin in regulating epithelial cell-cell junctions.

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Anillin is a scaffolding protein that organizes and stabilizes actomyosin contractile rings and was previously thought to function primarily in cytokinesis. Using Xenopus laevis embryos as a model system to examine Anillin’s role in the intact vertebrate epithelium, we find that a population of Anillin surprisingly localizes to epithelial cell‐cell junctions throughout the cell cycle, whereas it was previously thought to be nuclear during interphase. Further, we show that Anillin plays a critical role in regulating cell‐cell junction integrity. Both tight junctions and adherens junctions are disrupted when Anillin is knocked down, leading to abnormal intercellular spaces and increased permeability to fluorescent dextran. Further, fluorescence recover after photobleaching (FRAP) studies show that Anillin perturbation affects the dynamics of the tight junction protein ZO‐1. Anillin interacts with RhoA, F‐actin, and Myosin‐2, all of which regulate cell‐cell junction structure and function. When Anillin is knocked down, active RhoA (RhoA‐GTP), F‐actin, and Myosin‐2 are misregulated at junctions. Indeed, while overall accumulation of RhoA‐GTP is reduced when Anillin is depleted, increased dynamic “flares” of RhoA‐GTP are observed at cell‐cell junctions. Junctional accumulation of F‐actin and Myosin‐2 are also reduced when Anillin is depleted. We propose that Anillin is required for proper RhoA‐GTP distribution at cell‐cell junctions in order to maintain a robust apical actomyosin belt, which is required for cell‐cell junction integrity. These results reveal a novel role for Anillin in regulating epithelial cell‐cell junctions. Grant Funding Source: Supported by R00GM089765, start‐up funds, NSF Predoc Fellowships & T32GM007315 to C.R. and E.B.

Anillin is a contractile ring protein that is required for cytokinesis in several model organisms. Anillin can link multiple components of the contractile ring including F‐actin, Myosin‐2, and the small GTPase RhoA. While Anillin is important for stable furrow positioning in cultured cells, little is known about Anillin's function in multicellular organisms or whether Anillin plays roles outside of cytokinesis. Here, we used Xenopus laevis embryos to examine Anillin's function in the intact epithelium. We find that a population of Anillin is localized at cell‐cell junctions throughout the cell cycle. Both tight junctions and adherens junctions are disrupted in Anillin knock down embryos. Additionally, fluorescent dextran can penetrate into intercellular spaces in Anillin KD embryos, indicating that the epithelial barrier function is compromised. Because Anillin is reported to interact with RhoA, we tested the effect of knocking down Anillin on RhoA activity at cell‐cell junctions. Anillin knock down results in increased spontaneous flares of active RhoA, which are prominent at cell‐cell junctions in both dividing cells and non‐dividing regions of the epithelium. We propose that Anillin is required to properly distribute tension and RhoA activity in the apical actomyosin belt in order to maintain cell‐cell junctions. Anillin's dual functions at the cytokinetic contractile ring and apical actomyosin belt may be particularly important during the process of cytokinesis in epithelial cells when the dividing cell must maintain and remodel cell‐cell junctions with neighboring cells as well as manage the contractile forces associated with cytokinesis. This work was supported R00 GM089765 to A.L.M., and NSF Predoctoral Fellowships to C.C.R. and E.B.