Journal: The Journal of Cell Biology
Article Title: A quantitative map of human Condensins provides new insights into mitotic chromosome architecture
Figure Lengend Snippet: Condensins I and II display different quantitative dynamics during mitosis as determined by automated FCS-calibrated confocal 3D time-lapse imaging. (A) Genome-edited HK cells with homozygously mEGFP-tagged Condensin subunits and chromosomes stained by SiR-DNA were imaged every 90 s for a total of 60 min by 3D confocal microscopy, which was automatically triggered after prophase onset. Images were calibrated by FCS to convert fluorescence intensities ( FI ) into cellular protein concentration maps ( C ) and cellular protein number maps ( N ; for details, see Fig. S1, D–I). For SMC4 (Condensin I/II), fluorescence intensities, cellular protein concentration, and protein number maps are shown. For CAP-D2 and CAP-H (Condensin I) and CAP-D3 and CAP-H2 (Condensin II), protein number maps are depicted. Single z planes are shown for specific mitotic phases comparable between subunits. Condensin II is nuclear/on chromosomes throughout mitosis. Condensin I is cytoplasmic in prophase and gains access to mitotic chromosomes after NEBD in prometaphase. A Gaussian blur (σ = 1) was applied to the images for presentation purposes. Bar, 10 µm. (B) SMC4 protein numbers in specific compartments of mitotic HK cells (cytoplasm [blue-green], nucleus [orange], chromatin bound [beige], and chromatin soluble [turquoise]) are plotted against the mitotic standard time, and the corresponding mitotic phases are indicated. Shown is the mean of 17 cells from four independent experiments. Cellular landmarks were used for 3D segmentation and conversion of compartment-specific protein concentrations into protein numbers (for details, see Fig. S1 I and Materials and methods). Chromosome-bound and soluble chromatin proteins defined as the freely diffusing proteins within the chromatin volume were distinguished until anaphase as described in Materials and methods. For late mitotic stages, the nuclear compartment could not be divided into chromatin-bound and soluble proteins. (C) The numbers of chromosome-bound (prophase to anaphase) and nuclear Condensin subunits (telophase and cytokinesis) are plotted against the mitotic standard time: Condensin II subunits (CAP-D3, dark green; CAP-H2, light green), Condensin I subunits (CAP-D2, dark magenta; CAP-H, light magenta), and shared SMC4 (dark gray) are shown. Means (colored lines) and SD (light gray areas) are shown. (D) The numbers of chromosome-bound Condensin subunits from C are plotted for selected mitotic stages according to the images shown in A. SMC4 subunits (gray) as well as the sum of corresponding HEAT-repeat subunits (CAP-D2, dark magenta, Condensin I; CAP-D3, dark green, Condensin II) and corresponding kleisin subunits (CAP-H, light magenta, Condensin I; CAP-H2, light green, Condensin II) are shown. For C and D, the means of ∼20 cells per subunit (range: 10–36) from three to seven independent experiments are plotted; 36, 22, 10, 17, and 17 cells from seven, three, five, three, and four experiments for CAP-H, CAP-H2, CAP-D2, CAP-D3, and SMC4, respectively, are shown.
Article Snippet: For tagging SMC4 at the C terminus and CAP-H at the N terminus with mEGFP, zinc finger nucleases (ZFNs) containing DNA binding sequences listed in Table S1 were purchased from Sigma-Aldrich.
Techniques: Imaging, Staining, Confocal Microscopy, Fluorescence, Protein Concentration