Figure 7. Quantification of GFP×3-Nup133... | Rockefeller University Press

Figure 7.

$Figure 7. Quantification of GFP×3-Nup133 during the postmitotic nuclear pore assembly. HeLa cells coexpressing GFP×3-Nup133 and H2B-mCherry (not depicted) were chemically fixed and then imaged in 3D. Contours around the chromosome masses and nuclei were established by following the location of the H2B-mCherry fluorescence signal (dotted red lines). (A) Interphase nuclear pores. The image is from a nuclear envelope located at the bottom side of nucleus close to the glass coverslip. Fluorescence intensity distribution of 1,690 GFP×3-Nup133 spots imaged in 16 cells. The data represent the results from 16 cells. (B) Image from the middle section of a cell in anaphase acquired during the initial stages of Nup133 recruitment. The yellow boxes contain diffraction-limited fluorescent spots acquired from (a) rim region, (b) kinetochore region, (c) chromosome mass, (d) cytosol, and (e) control region without objects (background). Nondiffraction-limited objects (arrowheads) were excluded from the analysis. (C) Schematic representation of the regions used for analysis. (D) Fluorescence intensity distribution of diffraction-limited spots from each of the four regions color coded as indicated in C. (E) Image from the middle section of a cell in anaphase before the onset of Nup133 recruitment to the nuclear envelope and fluorescence intensity distribution of diffraction-limited Nup133 spots at the rim of the chromosome mass. (F) Image from the middle section of a cell in telophase and fluorescence intensity distribution of diffraction-limited Nup133 spots at the rim of the chromosome mass. Note the appearance of a population of spots peaking at approximately four GFP×3-Nup133. In B–D, the data represent the results from three cells. Chrom., chromosome. Bars, 10 µm.$

Quantification of GFP×3-Nup133 during the postmitotic nuclear pore assembly. HeLa cells coexpressing GFP×3-Nup133 and H2B-mCherry (not depicted) were chemically fixed and then imaged in 3D. Contours around the chromosome masses and nuclei were established by following the location of the H2B-mCherry fluorescence signal (dotted red lines). (A) Interphase nuclear pores. The image is from a nuclear envelope located at the bottom side of nucleus close to the glass coverslip. Fluorescence intensity distribution of 1,690 GFP×3-Nup133 spots imaged in 16 cells. The data represent the results from 16 cells. (B) Image from the middle section of a cell in anaphase acquired during the initial stages of Nup133 recruitment. The yellow boxes contain diffraction-limited fluorescent spots acquired from (a) rim region, (b) kinetochore region, (c) chromosome mass, (d) cytosol, and (e) control region without objects (background). Nondiffraction-limited objects (arrowheads) were excluded from the analysis. (C) Schematic representation of the regions used for analysis. (D) Fluorescence intensity distribution of diffraction-limited spots from each of the four regions color coded as indicated in C. (E) Image from the middle section of a cell in anaphase before the onset of Nup133 recruitment to the nuclear envelope and fluorescence intensity distribution of diffraction-limited Nup133 spots at the rim of the chromosome mass. (F) Image from the middle section of a cell in telophase and fluorescence intensity distribution of diffraction-limited Nup133 spots at the rim of the chromosome mass. Note the appearance of a population of spots peaking at approximately four GFP×3-Nup133. In B–D, the data represent the results from three cells. Chrom., chromosome. Bars, 10 µm.