Journal: Nucleic Acids Research

Article Title: KIF2C condensation concentrates PLK1 and phosphorylated BRCA2 on kinetochore microtubules in mitosis

doi: 10.1093/nar/gkaf476

Figure Lengend Snippet: BRCA2 pT207 binds to the N -terminal domains of KIF2C and KIF2A. ( A ) NMR analysis of the interaction between BRCA2 167-260 phosphorylated by PLK1 (F1 P ) and the N -terminal domain of KIF2C (KIF2C Nt). 2D NMR 1 H- 15 N SO-FAST HMQC spectra of F1 P , where each peak represents the chemical environment of a BRCA2 residue, were recorded before (black) and after (red) addition of KIF2C Nt (ratio 1:1). Superimposition of these spectra revealed that, upon binding, several peaks disappeared, while other peaks shifted (marked with arrows). The associated graph displays the peak intensity decrease upon addition of KIF2C Nt as a function of the BRCA2 residue number. ( B ) NMR analysis of the interaction between BRCA2 167-260 phosphorylated by PLK1 (F1 P ) and the N -terminal domain of KIF2A (KIF2A Nt). Here again, the 2D NMR 1 H- 15 N SO-FAST HMQC spectra of phosphorylated Avi-tagged BRCA2 167-260 recorded before (black) and after (magenta) addition of the N -terminal domain of KIF2A (KIF2A Nt; ratio 1:1) were superimposed. The peaks affected by the interaction are labeled. ( C ) NMR characterization of the KIF2C-bound state of phosphorylated BRCA2 48-218 . On the left, 2D NMR 1 H- 15 N SO-FAST HMQC spectra acquired on phosphorylated BRCA2 48-218 in the presence and absence of the N -terminal domain of KIF2C (ratio 1:1) were superimposed; several peaks, including those corresponding to S205, pT207, L209, and V11, disappeared after addition of KIF2C. On the right, 15 N CEST analysis performed on phosphorylated BRCA2 48-218 in the presence of the KIF2C N -terminal domain (ratio 1:0.15); the major and minor dips observed for residues S205, pT207, L209, and V211 indicate the 15 N chemical shifts in the free and bound states, respectively.

Article Snippet: After production and purification of the 6His-Avi-BRCA2 167-260 construct, half of the peptide sample was phosphorylated by adding PLK1 (provided by the recombinant protein platform of Institut Curie, Paris) at a molar ratio of about 200:1, as published [ , ].

Techniques: Residue, Binding Assay, Labeling

Journal: Nucleic Acids Research

Article Title: KIF2C condensation concentrates PLK1 and phosphorylated BRCA2 on kinetochore microtubules in mitosis

doi: 10.1093/nar/gkaf476

Figure Lengend Snippet: BRCA2 phosphorylated by the mitotic kinase PLK1 interacts with the microtubule depolymerases KIF2C and KIF2A. ( A ) NMR characterization of BRCA2 167-270 before (F1; black) and after phosphorylation by PLK1 (F1 P ; red). Superposition of the 2D NMR 1 H- 15 N SO-FAST HMQC spectra of F1 and F1 P , where each peak represents the chemical environment of a BRCA2 residue, identified phosphorylated residues; peaks corresponding to the four phosphorylated residues are labeled. ( B ) Volcano plots showing proteins from mitotic HeLa cell extracts that were identified by mass spectrometry as binding to (left) BRCA2 167-270 phosphorylated by PLK1 (F1 P ) versus non-phosphorylated BRCA2 167-270 (F1), and (right) BRCA2 167-270 phosphorylated by PLK1 (F1 P ) versus phosphorylated BRCA2 167-270 variant containing the T207A mutation (F1 T207A P ). ( C ) Domain organization of KIF2C and KIF2A, with residues of KIF2C mutated in this study indicated by colored circles: K52 and K54 in the N -terminal domain, G495 in the catalytic site, and S715 involved in KIF2C dimerization. ( D ) AlphaFold model of KIF2C, colored as in (C).

Article Snippet: After production and purification of the 6His-Avi-BRCA2 167-260 construct, half of the peptide sample was phosphorylated by adding PLK1 (provided by the recombinant protein platform of Institut Curie, Paris) at a molar ratio of about 200:1, as published [ , ].

Techniques: Phospho-proteomics, Residue, Labeling, Mass Spectrometry, Binding Assay, Variant Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: KIF2C condensation concentrates PLK1 and phosphorylated BRCA2 on kinetochore microtubules in mitosis

doi: 10.1093/nar/gkaf476

Figure Lengend Snippet: KIF2C assembly into condensates depends on its phosphorylated peptide binding capacity as well as the activity of the mitotic kinase PLK1. ( A ) Constructs used for optogenetic experiments, containing KIF2C either WT, deleted from its N -terminal domain, or with point mutations: K52E/K54E, G495A, S715A, and S715E. ( B ) Representative fluorescence images obtained after induction by doxycycline of the expression of Opto-KIF2C mutants and illumination of the cells with blue light (light ON). In the two upper lines, nocodazole was added to the cells (Noc (+)) to observe mostly mitotic cells. In the two lower lines, mitotic cells were analyzed in Noc (-) conditions. Scale bars: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $5\mu$\end{document} m. ( C ) Quantification of the number of foci per cell as measured on images recorded as in panel ( B ). The total number of analyzed nuclei was: in Noc (+) conditions, WT - 86 ( n = 3); \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} ${\mathrm{\Delta }}Nt$\end{document} and K52EK54E ( n = 2); G495A-51 ( n = 2); S715A-88 ( n = 3); S715E-86 ( n = 3) ( P -values: S715E- lower than 0.0001, G495A-0.0394 and S715A-0.6132); in Noc (-) conditions : WT – 54 ( n = 3); \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} $\vartriangle {\mathrm{Nt}}$\end{document} and K52EK54E ( n = 2); G495A-37 ( n = 2); S715A-49 ( n = 3); S715E-47 ( n = 3) ( P -values: G495A and S715E-lower than 0.0001, S715A0.5219). ( D ) Impact of PLK1 inhibition on Opto-KIF2C WT foci formation (light ON Noc (-)). ( E ) Quantification of the number of foci per cell as measured on the images recorded as in panel ( D ). The total number of analyzed nuclei was 131 ( n = 3) for the control and 61 ( n = 2) with the PLK1 inhibitor ( P -value lower than 0.0001).

Article Snippet: After production and purification of the 6His-Avi-BRCA2 167-260 construct, half of the peptide sample was phosphorylated by adding PLK1 (provided by the recombinant protein platform of Institut Curie, Paris) at a molar ratio of about 200:1, as published [ , ].

Techniques: Binding Assay, Activity Assay, Construct, Fluorescence, Expressing, Inhibition, Control

Journal: Nucleic Acids Research

Article Title: KIF2C condensation concentrates PLK1 and phosphorylated BRCA2 on kinetochore microtubules in mitosis

doi: 10.1093/nar/gkaf476

Figure Lengend Snippet: Activated PLK1 is enriched within KIF2C condensates. ( A ) KIF2C construct used for TurbolD experiments. ( B ) Identification of TurbolD-Opto-KIF2C partners in nocodazole-arrested cells by Western Blot. Partners of TurbolD-Opto-KIF2C were biotinylated in different doxycycline and light conditions and purified using streptavidin-coated beads. PLK1, PLK1-pT210 and tubulin were identified as enriched in KIF2C condensates. ( C ) Representative immunofluorescence images of Opto-KIF2C WT, PLK1 or PLK1-pT210 and DNA in nocodazole (Noc (+)) and either Light OFF or Light ON conditions. Scale bars: 5 μm. ( D ) Quantification of the number of PLK1 and PLK1-pT210 foci per cell as measured on the images recorded as in panel ( C ). The total number of observed nuclei for counting PLK1/PLK1-pT210 foci was 79/79 ( n = 3) and 100/79 ( n = 3) in Light OFF and ON conditions, respectively ( P -values: PLK1-0.0098 and PLK1-pT210-0.0057). ( E ) Representative immunofluorescence images obtained as in ( C ) but upon addition of the Aurora B inhibitor ZM447439. Scale bars: 5 μm. ( F ) Quantification of the number of Opto-KIF2C foci observed in panels ( C ) and ( E ). The total number of analyzed nuclei was: Light OFF - ZM447439-80 ( n = 3); Light OFF + ZM447439 ( n = 3); Light ON - ZM447439-91 ( n = 3); Light ON + ZM447439 - 92 ( n = 3). The P -value between the distributions of foci numbers under Light OFF and Light ON conditions is lower than 0.0001, whereas the P -value between Light ON and Light ON + ZM447439 conditions is 0.6951. ( G ) Model for PLK1 regulation in KIF2C condensates. KIF2C condensation is Aurora-B and PLK1-dependent, and through an Aurora B-dependent mechanism, triggers local concentration of PLK1, as well as further phosphorylation of proteins involved in the control of kinetochore-microtubule attachment, including KIF2C-S715 and BRCA2-T207. As KIF2C condensates are next to microtubules, the KIF2C depolymerase activity, enhanced by phosphorylation of S715, might be spatially limited to the periphery of the condensates.

Article Snippet: After production and purification of the 6His-Avi-BRCA2 167-260 construct, half of the peptide sample was phosphorylated by adding PLK1 (provided by the recombinant protein platform of Institut Curie, Paris) at a molar ratio of about 200:1, as published [ , ].

Techniques: Construct, Western Blot, Purification, Immunofluorescence, Concentration Assay, Phospho-proteomics, Control, Activity Assay