Cdt1 diffuses on MTs in vitro and decorates spindle MTs in vivo. (A) Representative Western blot of Cdt1^92–546 (1 µM) cosedimentation with intact (MT) or subtilisin-treated (ST-MT) microtubules at 1 and 4 µM concentrations. (B) Quantification of A (mean ± SD, n = 3). Significance assessed by the two-sided unpaired nonparametric Student’s t test. (C) Quantification of cosedimentation of Cdt1^92–546 (1 µM) with 16 µM MTs in the absence (+MT) and presence (+MT+S; top) of 250 mM NaCl; mean ± SD, n = 3. P < 0.005 represents statistical significance as assessed by the two-sided unpaired nonparametric Student’s t test. Representative Western blot shown (bottom). (D) Single-channel and merged TIR-FM images showing surface immobilized Dylight405-labeled MTs (blue) and GFP-tagged Cdt1^92–546 (green). (E) Kymograph analysis of Cdt1^92–546 interactions with the MT lattice. (F) Cumulative frequency plot and exponential fit of Cdt1^92–546 dwell times. n = 304 molecules, two independent protein preparations. (G) Diffusion coefficient analysis of Cdt1^92–546 on MTs performed using DiaTrack particle tracking Software. Inset graph shows the histogram distribution of number of particles of Cdt1^92–546 that diffused on MTs within the range of 0–1 µm²/s. (H) Untransfected mitotic HeLa cell stained with anti-Cdt1 and either anti-Hec1 antibody (top, panels i and ii) or anti-tubulin antibody (bottom, panels iii–vi). Scale bar, 5 µm. Shown are the representative images for Cdt1 kinetochore (anti-Cdt1/red and anti-Hec1/green) and spindle (anti-Cdt1/red and anti-tubulin/green) staining at different stages of mitosis in HeLa cells as indicated. Orange arrows in panels iv–vi depict Cdt1 staining at the spindle poles; yellow arrow in panel v depicts the Cdt1 staining at the cleavage furrow in late anaphase, and green arrows in panel vi depict Cdt1 localization to the reforming nucleus in telophase. (I and J) Immunostaining of stably transfected (I) or transiently transfected (J) HeLa cells with anti-HA, anti-Hec1, and anti-tubulin antibodies, as indicated. Scale bar, 5 µm. The middle and bottom panels in J depict confocal images of cells in cytokinesis and interphase, respectively, with DAPI staining of chromosomes in blue. (K) Insets from the image stack of the mitotic cell in the top panels of J (left; also see Video 2) with an example from another cell in Video 3 (right). Scale bar, 1 µm. (L) Western blot of mock and transfected lysates to probe for Cdt1 (using anti-HA or anti-Cdt1 as indicated) and mouse tubulin as loading control. Expressed, ectopically expressed (with C-terminal 2×-HA, S- and 12×-His-tags; abbreviated as HASH tags); endogenous, endogenous (without tag) Cdt1.