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. 2023 Jul 5;11:981425. doi: 10.3389/fcell.2023.981425

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Copyright © 2023 Didaskalou, Efstathiou, Galtsidis, Kesisova, Halavatyi, Elmali, Tsolou, Girod and Koffa.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Aurora A-dependent HURP phosphorylation at the Ser627 alters HURP localization and recovery dynamics after photobleaching (A) Schematic representation of WT HURP. Four potential Aurora A phosphorylation sites (blue lines) are located in the C-terminus. Black arrow indicates the Serine residue that is mutated to Alanine to create the HURP S627A mutant. (B) Left: Representative images of HeLa Kyoto cells transfected with either eGFP-HURP WT or eGFP-HURP S627A mutant and arrested at metaphase using MG132. Immunofluorescence has been performed against α-tubulin. DNA was counterstained with Hoechst. Right: Intensity plot profiles along metaphase spindle long axis of HeLa Kyoto cells transfected with either eGFP-HURP WT or eGFP-HURP S627A mutant. Lines indicate the mean values whereas dots represent the $\pm$ S.D. (eGFP-HURP WT, n = 46 cells; eGFP-HURP S627A mutant, n = 69 cells). (C) Representative images of cells expressing eGFP-HURP WT (upper panel) or eGFP-HURP S627A (lower panel) at different timepoints post photobleaching. Scale bars denote 5 μm. (D) Left: Fluorescence recovery curves of eGFP-HURP. Bold-lines indicate the mean value and dashed-lines represent the ±S.D. Right: One-phase exponential fitting resulted in half-lives of $t_{1 / 2} = 22 \pm 7 \sec$ for HURP WT (n = 17 cells), and $t_{1 / 2} = 47 \pm 16 \sec$ for HURP S627A (n = 26 cells) (mean ± S. D.; ****p < 0.0001). (E) Left: eGFP-HURP fluorescence decay curves of the unbleached spindle half. Bold-lines indicate the mean value and dashed-lines represent the ± S.D. Right: One-phase decay fitting resulted in half-lives of $t_{1 / 2} = 26 \pm 9 \sec$ for HURP WT (n = 14 cells), and $t_{1 / 2} = 63 \pm 28 \sec$ for HURP S627A (n = 23 cells) (mean ± S. D.; ****p < 0.0001). (F) Co-immunoprecipitation assays using an antibody against GFP in HeLa Kyoto mitotic extracts. HeLa Kyoto cells were transfected with eGFP, eGFP- HURP WT, or eGFP-HURP S627A, and bound complexes were analyzed by Western blot using antibodies against Kif5B, TPX2, Importin β, Aurora A, and GFP. Input lanes represent 4% of the total amount of protein used for the immunoprecipitation assays.