PML nuclear bodies and the spatial analysis of interphase mammalian cell nuclear architecture

Abstract

Promyelocytic leukaemia nuclear bodies (PML NBs) are found within the nucleus of mammalian cells. Numbering between 10 and 30 per nucleus, they are an obvious feature of the nuclear landscape, yet their functions have still to be unambiguously defined. In the mammalian nucleus, compartmentalization of functions is apparent, as reflected in the wide-range of other nuclear compartments that can be identified. Quantification of relationships between PML NBs and other nuclear functional compartments is essential for a complete understanding of PML NB function. Initially, PML size, number, distance relationships, and spatial organisation in relation to each other, and the nuclear boundary and centroid, under the spatial point pattern theory hypothesis of Complete Spatial Randomness (CSR), were investigated in both normal and SV40 transformed MRC5 and WI38 human foetal lung fibroblasts. This was also completed in normal MRC5 cells treated with heat shock, and interferon β (both of which alter PML NB morphometrics), and also serum starvation. PML NBs appeared to locate according to CSR with respect to each other, and inter – PML distances were dependent upon median PML NB number per nucleus. PML NBs did not tend to associate with the nuclear centroid, and were repelled from the nuclear boundary in all cell lines and conditions. The distance and spatial organisation relationships between PML NBs and eleven different nuclear compartments were also compared and contrasted in the cell lines and conditions mentioned previously. PML NBs were shown to share strong distance and spatial organisation relationships with the 11S immunoproteasome regulator, SC35 domains, and transcriptional compartments in normal asynchronous nuclei, and with telomeres in transformed cells, highlighting likely functions for the bodies. Lastly, the three dimensional spatial preference of functional compartments in the nucleus was determined using an aggregate map, which provided a novel means to visualise the nuclear location of functional compartments in relation to each other, and under different cellular conditions. Spatial preference fell into four categories: 1) diffuse, 2) annular, 3) core, and 4) polar. Nucleoli and RNA polymerase maintained their spatial preference across cell lines and conditions, whereas other compartments showed altered spatial preferences. Interestingly, viral transformation led to global disorganisation of the nucleus, where most compartments (including PML NBs) reverted to a diffuse spatial preference.