Structure-function studies on the mammalian nucleoplasmic reticulum

Animal cells separate their genes (DNA) from the rest of the cell inside a nucleus with a complex boundary dotted with tightly regulated ?border crossings?. In many cells the boundary is a simple spherical shape, but we are increasingly detecting cells in which narrow channels of this boundary fence push into the nucleus and form branches. This unusual structure has been called the nucleoplasmic reticulum (NR) and is attracting the attention of scientists because abnormal cells, especially some types of cancer cell, seem to make a much more elaborate version of this structure.

In this project we will use state-of-the-art microscopy techniques to follow the lifecycle of the NR in living cells, both normal and cancer cells. We will examine the formation of these channels as well as their inheritance when cells divide, and we will use a special electron microscopy technique that uses the same method as CT scanning patients in hospital to reconstruct these structures in fine detail. These experiments will be done in cells that naturally lack or contain NR. We will then exploit a recent observation that one of the types of drug used to treat AIDS patients causes a huge increase in NR to repeat our studies in cells forced to make new NR.

Our overall goal is to analyse how these structures assemble and move in normal and cancer cells so that we can understand the significance of increased NR in damaged cells, especially cancer cells.

The nuclear envelope (NE) of many cells contains deviations from a simple spherical outline in the form of deep branching invaginations that give rise to an intranuclear membrane bound arborisation called the nucleoplasmic reticulum (NR). Cell type specific patterns of NR are found, suggesting a non-random organising process but little is known about the mechanisms underlying NR generation or inheritance. NR is significantly increased in many de-differentiated cancer cells; whether this is a causal association remains unknown, although the appearance of NR is already used in
diagnostic staging.

In this project a combination of immunocytochemistry, time lapse imaging, and electron tomography will be used to examine both normal primary cells and transformed tumour cell lines. Particular attention will be paid to the formation of NR and its inheritance through mitosis in proliferating cells. To correlate NR abundance and organisation with changes in gene expression mRNA microarray analysis of transcription will be coupled with high resolution imaging and applied to a range of samples. Comparisons will be made between undifferentiated and differentiated cultured mouse myoblasts (which show increased NR), wild type mouse fibroblasts treated with the HIV protease inhibitor saquinavir (which causes a profound increase in NR) or darunavir (which has no effect on NR abundance).