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, 16 (1), 193-201

Anillin Binds Nonmuscle Myosin II and Regulates the Contractile Ring

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Anillin Binds Nonmuscle Myosin II and Regulates the Contractile Ring

Aaron F Straight et al. Mol Biol Cell.

Abstract

We demonstrate that the contractile ring protein anillin interacts directly with nonmuscle myosin II and that this interaction is regulated by myosin light chain phosphorylation. We show that despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. We propose a role for anillin in spatially regulating the contractile activity of myosin II during cytokinesis.

Figures

Figure 8.
Figure 8.
Depletion of anillin by siRNA in HeLa cells. (A) Western blot with anti-anillin of whole cell lysates after treatment with no RNA, control siRNA, or siRNA directed against human anillin. (B) Frequency of multinucleation in control or siRNA-treated HeLa cells. (C) immunofluorescence staining of DNA (blue), anillin (green), and myosin II (red) in HeLa cells treated with control or anillin siRNA. (D) Single frames of YFP-Myosin fluorescence from Movie S4, control, and Movie S8, anillin siRNA. White arrow indicates position of contractile ring. Bars, 5 μm.
Figure 1.
Figure 1.
Characterization of X. laevis anillin. (A) Western blot of 20 μg of crude X. laevis egg extract with preimmune serum, immunized serum, and affinity-purified antibody. (B) Coimmunoprecipitation of anillin and actin from Xenopus egg extracts. Lane 1, interphase extract precipitated with rabbit IgG; lane 2, CSF extract precipitated with IgG; lane 3, interphase extract precipitated with anti-anillin; lane 4, CSF extract precipitated with anti-anillin. (C) immunofluorescence images XTC cells stained for DNA, anillin, actin, and myosin II.
Figure 2.
Figure 2.
Anillin affinity chromatography. (A) Eluates from anillin (full length [FL] or without pleckstrin homology domain [-PH]), Polo kinase (PLX1), or GST columns. Left, cytostatic factor arrested extracts. Right, interphase extracts. (B) Eluates from GST and fulllength anillin columns immunoblotted with antibodies to nonmuscle myosin II heavy chain.
Figure 3.
Figure 3.
Delineation of myosin II binding region in anillin. (A) Schematic of anillin truncation fragments with minimal myosin binding region, region homologous to actin binding region, and pleckstrin homology domain highlighted. (B) Eluates from affinity columns for each of the fragments of anillin and Polo kinase (PLX1). Myosin II is the prominent band at 200 kDa. (C) Homology alignment of Xenopus, human, and Drosophila anillin in the myosin binding region.
Figure 4.
Figure 4.
Anillin binds activated myosin II. Myosin II precipitation by anillin after myosin II phosphorylation by MLCK, Cdc2/CyclinB, Polo kinase, or no kinase.
Figure 5.
Figure 5.
Myosin II and anillin organization in arrested contractile rings. (A) DNA, tubulin, and anillin staining in a blebbistatin-arrested HeLa cell. (B) Image plane at the coverslip surface in a blebbistatin-arrested HeLa cell stained for anillin and myosin II. (C) Enlarged view of B. Bars, 5 μm.
Figure 6.
Figure 6.
Depletion of anillin by dsRNA in Drosophila cells. (A) Western blot of anillin depletion at varying dsRNA concentrations. (B) Quantitation of multinucleate phenotype in untreated or 75 nM anillin dsRNA-treated cells. (C) Image of control or anillin dsRNA-treated cells stained for DNA and cell volume. (D) Image of Drosophila cell untreated or anillin dsRNA treated during cytokinesis stained for DNA, anillin, and myosin II. Bar, 5 μm.
Figure 7.
Figure 7.
Depletion of myosin II by dsRNA in Drosophila cells. (A) Western blot of myosin II after control and 100 nM myosin II dsRNA treatment. (B) Quantitation of multinucleate phenotype in untreated or myosin II dsRNA-treated cells. (C) DNA staining of multinucleate cells in control or myosin II dsRNA-treated cells. (D) Image of control or myosin II dsRNA-treated cells during cytokinesis stained for DNA, anillin, and myosin II. Bar, 5 μm.

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