Dynamics and resolution of the nucleoplasmic reticulum

Animal cells separate their genes (DNA) from the rest of the cell inside a nucleus with a complex boundary made of a pair of membranes dotted with tightly regulated 'border crossings'. Often the overall boundary is a simple spherical shape, but we are increasingly aware that many cells also contain narrow channels of this boundary fence that push into the nucleus and form branches. This unusual structure has been called the nucleoplasmic reticulum (NR) and is attracting the attention of scientists both because it varies during cell development in the foetus and in response to hormonal signalling, and because abnormal cells, especially some types of cancer cell, seem to make a much more elaborate version of this structure.

Adding to our excitement is an appreciation that the boundary between the nucleus and the rest of the cell is not just a fence, but is also a 'communication hub' at which complex signals are considered, compared, stored and acted upon. Thus the formation of NR channels in which this hub invades deep into the nucleus will change the way signals are handled. At the moment we are confident that this happens, because we can see normal physiological processes in which NR forms at specific times and in response to specific signals, but we know almost nothing of the regulation of NR formation and the proteins involved.

We have recently shown that the formation of an NR structure depends upon a particular enzyme that is involved in regulating the production of new membrane in the cell. When we reduce the expression of this enzyme and supply a stimulus that usually induces NR formation we see that no NR is formed. This gives us a new tool to separate effects of the initial signal into three parts; those that regulate NR induction, those that respond to NR formation, and those that are completely independent.

In this project we will study three systems in which we can block NR induction and examine the response of the cell. In two cases we will use normal physiological signals - firstly, embryonic muscle cells that respond to external cues to form muscle fibres and, secondly, endometrial cells that respond like the lining of the uterus to hormonal signals. In both cases, NR formation accompanies these changes. The third system will exploit a recent observation that one of the types of drug used to treat AIDS patients causes a huge increase in NR and a wide range of chemical and protein changes within the cell. We will apply this drug to normal human skin cells in culture and force them to make new NR.

We will use a combination of state-of-the-art microscopy techniques to follow the induction of the NR in these three systems, with detailed studies of all the changes in cellular chemical (the metabolic profile) and gene expression changes. We will examine the formation of NR channels, 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. Having established all of these responses, we will then analyse which of these changes are altered when we prevent formation of the NR. By comparing three very different systems we aim to find components that are part of the general machinery for responding to a signal by building an NR.

Finally, we will check that the proteins we have found are indeed involved in NR formation. We will do this by looking for changes in NR in cells that are forced to make too much or too little of each of these proteins.

Our overall goal is to understand what controls the formation of NR channels, and how this machinery itself is switched on and off. This will give us new insights into the complicated and sophisticated way that cells understand and respond to outside events, whether these are triggers during tissue development in the foetus, hormonal responses in adult tissue or pathological responses in diseases such as cancer.

The nuclear envelope (NE) often includes deep branching invaginations forming 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 regulation. Specific physiological roles for NR are strongly implied by observations of timed, tissue specific induction of this structure - during myoblast differentiation into myotubes, for example, or during endometrial responses to cyclic hormonal stimulus. Pathological NR formation accompanies the structural abnormalities seen in inherited laminopathies, and altered morphology or abundance of NR is sufficiently strongly associated with certain cancers to be useful in diagnostic staging.

In this project a combination of immunocytochemistry, time lapse imaging, electron tomography, microarray analysis of mRNA and miRNA, and metabolic profiling will be used. Two physiological systems in which NR formation accompanies gene expression changes (human myoblast differentiation and endometrial cell steroid response) will be compared with the pathological induction of NR caused by prelamin A accumulation in normal human dermal fibroblasts. We have recently shown that NR induction can be blocked by siRNA knockdown of expression of CTP protein cytidylyltransferase alpha, the rate limiting enzyme for phosphatidylcholine synthesis and a key regulator of de novo membrane production. We will exploit this observation to dissect responses upstream and downstream of NR induction, and compare mRNA, miRNA and metabolite changes between the different systems to identify elements of the common regulatory machinery.

Candidate members of the general NR regulatory machinery will be confirmed by individual over- and under-expression experiments, together with GFP-tagged expression to identify candidates that are structural components of NR rather than regulators.

Who might benefit from this research?

In academia
The proposed project will benefit scientists in the immediate fields of cell and molecular biology, both in the UK and internationally, by providing new knowledge on mechanisms by which gene expression may be modulated by NR formation, by providing large-scale microarray and metabolomic datasets for their own targeted analyses. The work will lay the groundwork for subsequent benefit to the wider field of research pathologists by providing underpinning knowledge of novel cellular signalling events and eventually antibodies capable of decorating NR in histological sections for detecting these structures in normal tissue as well as variations in such signals in pathological states. The work will also benefit scientists in related disciplines, especially that of systems biology. The work will be of relevance as identifying a novel node in signalling pathways between the cytoplasm and the nucleus, especially as the signals are unlike other known epigenetic signals in that they can be followed morphologically by monitoring NR induction. The datasets generated will offer a valuable resource for network theoreticians and mathematical modellers who lack interconnected datasets of signal events and gene expression consequences under a range of experimental conditions.

The benefits will include theoretical advances in our models of how cells respond to change, but also potential material benefits in the form of useful reagents which may have utility far beyond the confines of the present project.

Business/industry
Apart from enhanced knowledge and specific research datasets, the project will identify candidate proteins critically involved in the induction and regulation of a highly novel nuclear structure involved in normal and pathological signaling events. Antibodies generated to these proteins, within this project or commercially will certainly be of great use for basic research, and may play a role in the development of future diagnostic tools. The reagents will be made in conjunction with Covalab Ltd, with whom the applicant has established a productive collaboration. Indeed, a phospho-specific anti-lamin B1 reagent has already been generated in pilot experiments and has already been made commercially available by Covalab.

General public
If the NR regulatory proteins identified in this project are detectable in tissue sections using antibodies, it is possible that the antibodies could become useful diagnostic tools for identifying or staging human disease. Under these circumstances there would be a clear benefit to the wider public in improved health care.

How might they benefit from this research?
The core academic outcome of the proposed research is an enhancement of the knowledge economy by providing a new conceptual framework for a novel layer of regulated gene expression. The concept of the NR as an inducible intranuclear signaling hub is entirely novel, and is likely to have significant impact as a scientific advance in the UK and internationally.

The economic impact of this research will include the further development of a highly trained research scientist, Dr Malhas, enabling him to extend his skills base into metabolomic analysis. It will also have international impact on our collaborators in the US and Europe, especially Dr Chiu Fan Lee, a theoretical physicist who has developed deep insights into complex biological signal networks that he has already carried with him into other subject areas. This collaboration will continue generating inter-disciplinary insights and new and innovative methodologies in visual representation of complex data.

Commercial benefits from the research include development of novel antibody reagents for research use, and potentially human diagnostic use. The outcome of the research has the potential to persist for many years after the project itself.