These images are two-dimensional projections generated from the image data. Finally, nucleoli (red) have been imaged but are not the focus of this paper due to their extent which makes modelling their location by a point pattern problematic. The nuclear volume was delineated using indirect immunofluorescence directed against lamin B (blue), a protein of the nuclear lamina. PML protein, which localizes to PML nuclear bodies (PML NBs), was also stained via indirect immunofluorescence in all images (green). Nuclei were imaged using indirect immunofluorescence and confocal microscopy in MRC-5 and WI-38 human diploid fibroblasts, and their SV40 T antigen-transformed MRC-5 and WI-38 counterparts. Intensity estimation mammalian fibroblasts nuclear compartments spatial point pattern analysis spatial preference thin-plate spline.įunctional compartments of the nucleus of ( a) MRC-5 and ( b) WI-38 fibroblasts, ( c) SV40-transformed MRC-5 cells and ( d) SV40-transformed WI-38 cells.
This new methodology is thus able to reveal the effect of large-scale perturbation on spatial architecture and preferences that would not be obvious from single cell imaging. In addition, we show that SC35 splicing speckles are excluded from the nuclear boundary and localize throughout the nucleoplasm and in the interchromatin space in non-transformed WI-38 cells. Specifically, the spatial preference of RNA polymerase II is preserved across normal and immortalized cells, whereas PML nuclear bodies exhibit a change in spatial preference from avoiding the centre in normal cells to exhibiting a preference for the centre in immortalized cells. We present two examples of different compartments in mammalian fibroblasts (WI-38 and MRC-5) that demonstrate new knowledge of spatial preference within the cell nucleus. These tools combine replicate images to generate 'aggregate maps' which represent the spatial preferences of nuclear compartments. The concern of this paper is the spatial preference of nuclear compartments, for which we have developed statistical tools to quantitatively study and explore nuclear organization. Such studies typically focus on inter-object distance, rather than spatial location within the nucleus. To date, the study of nuclear organization has often involved simple quantitative procedures that struggle with both the irregularity of the nuclear boundary and the problem of handling replicate images. The nuclei of higher eukaryotic cells display compartmentalization and certain nuclear compartments have been shown to follow a degree of spatial organization.
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