Structure of the nucleoskeleton and its role in nuclear compartmentalisation

Complex organisms are made up many different types of cells. This is necessary because individual cells are dedicated or specialised to undertake specific functions. Muscle cells and brain cells have quite different functions and their structure reflects this. Despite the fact that different cells can easily be distinguished by their appearance, they all have the same genetic information. This means that in principle almost all cells of our body have the potential to change from one cell type to another. This does not happen, as the genetic information in DNA is controlled so that specific cells only use a small fraction of the genetic information. The process whereby the use of genetic information is restricted in each cell is called differentiation. During differentiation, the genes within a muscle cell are modified so that only genes required for muscle cell function will work. The process of restricting the use of particular genes in defined cell types is extremely complex and is regulated by many different processes. Three of these are of particular importance: 1) Genes work when small proteins called transcription factors are present. Transcription factors bind specific genes and allow theme to be expressed as protein. The protein is then responsible for specific functions. 2) The DNA of genes is bound to proteins called histones that regulate the access of transcription factors to DNA by changing the structure and accessibility of the DNA-protein complex that we call chromatin. 3) Genes only work when they are in the appropriate place in the cell. The genes that dictate specialised cell function are found in the cell nucleus. Even within the nucleus, a gene must be in the correct space in order to work properly. This proposal explores the principles that define nuclear space in human cells. The structural elements that are responsible for defining space include: 1) Structural elements of the nucleus / we call these the nucleoskeleton. 2) The structure of chromosomes, where the genes are stored. 3) The arrangement of the active nuclear compartments where genes must be placed in order to work. We will establish how the nuclear structure is important to gene function. We know that it is important because small alteration in the structural proteins / called nuclear lamins / are associated with a number of different diseases. The easiest mutations to understand make weak nuclear structures, so that cells that are subjected to force during their normal life time / such as muscle cells - tend to be easily damaged and die. The structural proteins form a chicken-wire like mesh of protein filaments just inside the nucleus. The filaments contain as many as five different proteins and are associated with many more. However, we do not know how the different proteins contribute to the strength of the network. The filaments of this structure also pass deep inside the nucleus. The aims of our work are: 1) To understand how the structure of the nuclear lamina determines nuclear function and in particular how genes work. 2) To understand how changes in the nuclear lamina result in functional defects seen in disease. 3) To show how changes in the nuclear lamina influence the organisation of special compartments in the nucleus where genes work / we call these factories. 4) To understand how chromosomes are associated with these special places and how this influences the behaviour of chromosomes and the special structures from which chromosomes are built. We will use some very clever microscopy techniques that define with very high precision where specific molecules are in very complex structures. We will also perform detailed analysis of protein mixtures that are removed from cells when they are treated in ways that gradually destroy the important places. We hope to show which proteins and protein complexes are important in the structure of these sites in living cells.

The regulation of chromatin function in higher eukaryotes is complex and multi-faceted. For example, patterns of expression are regulated at the level of DNA modification, chromatin structure, genome architecture and nuclear organisation. Each aspect of these features of regulation is controlled by complex nuclear systems and each of the systems must interact to define a coherent regulatory network. In proliferating cells, and particularly during development, patterns of gene expression must be preserved during genome duplication and cell division. Hence, the systems that regulate DNA synthesis and repair must also integrate with those that regulate gene expression, so that defined patterns of gene expression are maintained. There is no doubt that chromosome architecture and the spatial nuclear architecture of genes and active nuclear compartments are fundamental regulators of chromatin function. Yet surprisingly little is known about the molecular mechanisms that control the spatial and temporal organisation of genes. With this in mind, this proposal sets out to define the architecture of the key structural network in the nuclei of human cells / the nucleoskeleton. The nucleoskeleton is composed of two components: 1) The nuclear lamina, is a major structural component that underlies the inner nuclear membrane. The nuclear lamina is a major determinant of nuclear structure and is also involved in genome organisation and gene regulation. 2) An internal nucleoskeleton has also been described. This structure has been implicated in the spatial organisation of active centres of chromatin function / DNA replication and transcription factories. Preliminary structural studies suggest that the internal nucloesketon is related to the external elements of the nuclear lamina, but definitive experiments have yet to be performed. The structure and function of the nucleoskeleton will be investigated using two strategies: 1) The structure and distribution of components within the nucleoskeleton will be determined using high-resolution electron microscopy techniques. Changes in the structure will be evaluated using established RNAi techniques and in cell lines with lamin gene mutations that alter the structural integrity of the nuclear lamina. 2) Nuclear deconstruction will be combined with a proteomic analysis to investigate how changes in the structure of the nucleoskeleton influence the organisation of chromosome territories, structural units of chromosome structure / DNA foci / and the major functional nuclear compartments / transcription and replication factories. These studies will employ high-resolution electron microscopy techniques to define the location of specific lamin proteins within the nuclear lamina and internal nuclear network. These structures will be evaluated in a range of cells and cells with reduced (using RNAi) and mutated lamin proteins. Changes in the structure of the nucleoskeleton will be correlated with changes in the organisation of chromosomes and the functional nuclear compartments. Using cell extractions, sophisticated proteomic techniques and live cell imaging, we will evaluate if organisational and functional changes associated with alterations in the nucleoskeleton can reveal how structural nuclear networks influence chromatin function in mammalian cells.