Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

FP6

MITOCHECK Berichtzusammenfassung

Project ID: 503464
Gefördert unter: FP6-LIFESCIHEALTH
Land: Austria

Final Report Summary - MITOCHECK (Regulation of mitosis by phosphorylation - A combined functional genomics, proteomics and chemical biology approach)

The project consortium developed and applied technologies that have enabled a systematic analysis of genes and proteins required for chromosome segregation and cell division in human cells. Human cells, like all eukaryotic cells, pass their genomes from one cell generation to the next by first duplicating their deoxyribonucleic acid (DNA) in S-phase and then segregating the resulting copies during mitosis.

Chromosome segregation during mitosis is an immensely complex process that remains poorly understood at the molecular level. Mistakes during mitosis contribute to cancer, whereas mistakes during meiosis are the leading cause of infertility and mental retardation. Although many proteins required for mitosis had previously been identified, the entire set of proteins that are needed for this process remained unknown.

One major objective of the MITOCHECK consortium was therefore to identify all human proteins that are required for mitosis. To achieve this goal, a genome-wide RNA interference (RNAi) screen was performed by the MITOCHECK Screening Unit, which was formed by the European Molecular Biology Laboratory (EMBL), the German Cancer Research Center (DKFZ) in Heidelberg and Leica Microsystems CMS in Mannheim.

To enable the genome-wide RNAi screen, the MITOCHECK Bioinformatics Unit at the Sanger Institute in Hinxton annotated about 22 000 human genes, all of which were then depleted one by one with several siRNAs in a newly developed solid-phase transfection assay by the MITOCHECK Screening Unit. All siRNA transfected cells were analysed by live cell imaging, resulting in about 260 000 movies. These movies were analysed by automatic image analysis tools that were developed by MITOCHECK, and in the future, all movies and their annotation will be displayed on the MITOCHECK website (http://www.mitocheck.org).

RNAi screening is a powerful technique for identifying genes required for mitosis and other cellular processes. However, detailed molecular analyses are needed to understand the precise function of these genes, among which protein localisation and identification of protein interactions are particularly informative.

In principle, these secondary analyses should proceed at the same rate as phenotypic screens. Indeed, in yeast efficient intrinsic homologous recombination has enabled the modification of most genes at their endogenous loci with tag-coding sequences and has been valuable in allowing systems wide analysis of protein function.

In mammalian cells, large-scale localisation and interaction studies of proteins expressed under control of their own regulatory sequences have so far lagged far behind phenotypic analysis. These analyses are therefore performed on an individual gene basis, precluding systems-scale analysis of protein function. To enable the latter approach in mammalian cells, the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden (CBG), which formed the core of the MITOCHECK Tagging Unit, developed a pipeline for fast and reliable introduction of genes tagged in bacterial artificial chromosomes (BACs) into human tissue culture cells, using previously developed techniques of recombineering. These allowed the stable expression of genes under their own promoters at physiological levels in mammalian cells, thus avoiding artefacts that are due to over-expression.

MITOCHECK applied this "BAC TransgeneOmics" approach to characterise proteins complexes required for mitosis by performing localisation experiments in the Tagging Unit, and by purifying protein complexes and analysing them by mass spectrometry in MITOCHECK's phosphorylation Unit at the Research Institute of Molecular Pathology (IMP) in Vienna. This work led to the characterisation of about 100 protein complexes, many of which had previously not or only incompletely been characterised.

In combination with secondary RNA interference assays, this work led to important new molecular insights into centrosome function, spindle assembly and chromosome segregation. The approaches developed by MITOCHECK will be generally applicable to high-throughput follow-up of phenotypic screens in mammalian cells.

Verwandte Informationen

Reported by

FORSCHUNGSINSTITUT FÜR MOLEKULARE PATHOLOGIE GESELLSCHAFT m.b.H.
VIENNA
Austria
Folgen Sie uns auf: RSS Facebook Twitter YouTube Verwaltet vom Amt für Veröffentlichungen der EU Nach oben