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. 2017 Sep 1;28(18):2400–2409. doi: 10.1091/mbc.E16-11-0756

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© 2017 Kanfer 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 1: — CENP-F tracks growing microtubules in vivo. (A) Top, domain organization of CENP-F. Bottom, position of the internal GFP tag (CENP-F^GFP). (B) Live images of U2OS cells coexpressing N-terminally GFP-tagged CENP-F (GFP-CENP-F, left) or internally GFP-tagged CENP-F (CENP-F^GFP, right) and a mitochondrial marker (mtBFP, red). Arrowheads show mitochondrial localization. Scale bars, 10 µm (C) Top, localization of CENP-F^GFP to the nuclear envelope (arrowheads) in early prophase. DNA is counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Bottom, localization of CENP-F^GFP to the kinetochores (arrowheads). Condensed chromatin is counterstained using an anti-phosphohistone H3 (H3P). Scale bars, 10 µm. (D) Cells overexpressing FLAG-Miro (magenta) and stained for endogenous CENP-F (left, green) or cotransfected with CENP-F^GFP (right, green) show recruitment of both endogenous and transfected CENP-F onto the mitochondrial network without any obvious localization elsewhere. Scale bars, 10 µm. (E) TIRF images of live U2OS cells coexpressing CENP-F^GFP (green) and RFP-EB1 (red). Arrowheads point at CENP-F foci found at extreme tips of growing microtubules. Scale bar, 5 µm. (F) Kymograph of the live images in E. Scale bars, 1 µm and 1 s. (G) Quantification of the distance traveled (top), lifetime (middle), and speed of comets labeled with EB1 (left) and CENP-F (right). Note that all comets bearing CENPF^GFP signal were also positive for EB1, whereas the reverse was not true.