Partial colocalization between TRAMM and CENP-E occurs during early mitosis. (A) HeLa cells were fixed and stained with anti-TRAMM, anti–CENP-E, and DAPI. During prophase, prometaphase, and metaphase, when the signal for both proteins was punctate, a colocalization channel was used to demonstrate colocalization of the signals. Bars, 5 µm. (B) HeLa cells were transfected with V5-tagged wild-type (WT) TRAMM, TRAMM-5A, or TRAMM-5D. The cells were fixed and stained with mouse anti-V5, ACA, and anti–CENP-E. Fluorescence intensity measurements were calculated for CENP-E signals that colocalized with ACA in V5-positive cells. The results shown are ± SD. Significance was assessed with an unpaired t test, and representative cells for each condition are shown to the right of the bar graph. (C) A model showing the phosphorylation (P) and dephosphorylation of TRAMM during the cell cycle as well as its localization. The localization cycle for CENP-E based on A is also shown. See the text for details. CCAN, constitutive centromere-associated network.