To gain insight into how mitotic proteins function during cell cycle, it is important to determine where these proteins localize inside a cell and with which proteins they form complexes at different stages of cell cycle.

In this Work Package, we are developing techniques to tag a subset of mitotic proteins identified in the previous Work Packages using bacterial artificial chromosomes (BACs). The various tags will enable us to visualize the subcellular localization of the tagged proteins and to affinity purify them and their associated partners as native complex.

1) To establish a strategy and techniques for tagging mitotic proteins

2) To develop technology for high-throughput production of stable cell lines expressing tagged transgenes

3) To determine subcellular localization of mitotic proteins using the GFP tag

Tony Hyman (Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany)

Gene Bridges GmbH (Heidelberg, Germany)

Jan Ellenberg (EMBL, Heidelberg, Germany)

1) Tagging mitotic proteins

A subset of genes that are required for mitosis is presently being tagged with green fluorescent protein (GFP) together with an S-peptide affinity purification tag. The GFP tag enables us to monitor the subcellular localization of the tagged proteins whereas the affinity tag allows purification of the tagged proteins together with their binding partners. These genes will then be stably expressed from their own regulatory elements by using bacterial artificial chromosomes (BACs) in human cell lines.

2) High throughput generation of stable cell lines expressing tagged transgenes

We first established a standard protocol with a few selected genes labeled with various tags. Two types of cell lines were generated with each tagged gene: adherent cells for subcellular localization study and cells in suspension for biochemical analysis.

The standard protocol has been adapted for higher throughput of cell line production. In particular, BACs are prepared in high quality ready for transfection into eukaryotic cells, and cell pools stably-expressing tagged transgenes are generated. So far, more than 100 such cell pools have been generated that express different tagged genes. A local database (http://bac.mpi-cbg.de/) has been set up to keep track of the BAC clones, stable cell pools and cell lines. This database will be linked to the main MitoCheck database in the future.

3) Determine subcellular localization of mitotic proteins by using GFP tags

Studies on a few GFP-tagged transgenes revealed that many of the GFP tagged genes display the same cell cycle-dependent localization patterns as the endogenous genes. We are optimizing the method for higher throughput. Ultimately we will provide subcellular localization information for all the GFP-tagged transgenes MitoCheck will generate.

4) Rescuing mitotic phenotypes with GFP tagged transgenes

The ultimate proof that an RNAi phenotype was caused by the gene targeted for suppression is to rescue the phenotype by expression of an RNAi resistant transgenes. Since we use mouse genes to tag mitotic proteins, these are frequently resistant to suppression by the RNAi designed against the human genes. The human cell lines expressing the tagged mouse gene are then subjected to knockdown of the endogenous human gene and assayed again for the mitotic phenotype. Studies on a few such cell lines have shown that many of the GFP tagged genes are able to completely rescue the mitotic phenotype.