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. 2015 Nov 15;26(23):4187–4196. doi: 10.1091/mbc.E15-05-0258

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© 2015 Sumiyoshi et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 5: — Kinase activity of AIR-1 is required for the formation and/or maintenance of cytoplasmic MTs in meiotic embryos. (A) Ex time-series images of meiotic embryos expressing GFP::β-tubulin (black). (a) Control embryo, (b) air-1(RNAi) embryo, and (c) air-1(RNAi) embryo expressing a kinase-inactive form of AIR-1 (AIR-1^K73RT201A). Right, magnified views. Bar, 10 μm (images showing whole embryos), 5 μm (magnified views). Contrast of original images was inverted and enhanced to visualize cytoplasmic MTs. (B) Quantification of cytoplasmic MTs in meiotic embryos. From images as shown in A, the size of the area covered with cytoplasmic MTs was measured and normalized by the size of the embryo. Each dot represents a single embryo (control, n = 9; air-1(RNAi), n = 9; and air-1(RNAi)–expressing AIR-1^K73RT201A, n = 11). Pairs with significant difference, **p < 0.01). (C) Localization of AIR-1 in the cytoplasm of a meiotic embryo at late anaphase. Immunofluorescence images of a meiotic embryo stained with an anti–α-tubulin antibody (green) and an anti–AIR-1 antibody (red) along with DAPI (blue). Right, magnified views of the cytoplasmic region. Contrast was enhanced to visualize cytoplasmic MTs. Bar, 10 μm. (D) Localization of the kinase-active form of AIR-1 in the cytoplasm of a meiotic embryo at late anaphase. Immunofluorescence images of a meiotic embryo stained with an anti–phospho-AIR-1 antibody (green), which recognizes the kinase-active form of AIR-1, and an anti–AIR-1 antibody (red), along with DAPI (blue). Right, magnified views. Contrast was enhanced to visualize cytoplasmic MTs. Bar, 10 μm. (E) Movement of cytoplasmic MTs in meiotic embryos. Ex vivo time-series images of cytoplasmic MTs around a meiotic spindle visualized with GFP::β-tubulin. Green and red arrows indicate two ends of moving cytoplasmic MTs. (a) MT entering the meiotic spindle. (b) MT exiting the meiotic spindle. Frames were taken every 2 s. Indicated is the time (in seconds) elapsed from the start of the movie. Contrast was inverted and enhanced to visualize cytoplasmic MTs. sp, meiotic spindle. Bar, 5 μm. (F) Velocity of cytoplasmic MTs. Each dot represents the velocity of cytoplasmic MTs entering or exiting meiotic spindles. Data were collected from four meiotic embryos.