The mitotic proteins identified from the primary screens will be further analyzed in a variety secondary video microscopy assays in which different mitotic processes such as chromosome condensation/segregation and nuclear assembly can be resolved with higher temporal and optical resolution. We are currently developing assays for this study.

1) Primary genome wide RNAi screen to identify majority of genes required for cell division.

2) Secondary RNAi screens: Development of a fully integrated confocal screening system for cell based assays in human cells.

3) Screen data management.

Jan Ellenberg (EMBL, Heidelberg, Germany)

Roland Eils (DKFZ, Heidelberg, Germany)

Frank Sieckmann (Leica Microsystems CMS GmbH, Mannheim, Germany)

1) Genome-wide primary RNAi screen for mitotic genes

By Project Month 36, we have completed the data acquisition and quality control of the genome-wide RNAi screen for mitotic genes using time lapse microscopy. In this comprehensive screen, we depleted all the 22,000 human genes one by one with several siRNAs in living cells and then documented the behavior of the cells using live cell video microscopy, resulting in more than 177,000 movies. All of these data will be displayed on the MitoCheck website once this data set has been analyzed.

2) Secondary RNAi screens

The mitotic proteins identified from the primary screens will be further analyzed in a variety of secondary video microscopy assays in which different mitotic processes such as chromosome condensation/segregation, spindle and nuclear assembly can be resolved with higher temporal and optical resolution. We have already established several of these assays and are currently developing additional assays for this study.

3) Screen data management

The genome-wide primary RNAi screen has generated more than 177,000 video microscopy movies documenting behavior of human cells after targeted suppression of every protein coding genes. This dataset provides a wealth of phenotypic information - mitotic or non-mitotic - about the human genome. It also presents many technical challenges, for example, how to store and transfer a dataset of this scale, how to extract and classify the phenotypic information in an automated way and how to present the data in our database. We are constantly optimizing our data handling methods to better exploit this unique and valuable source of information.

- High-throughput RNAi screening by time-lapse imaging of live human cells..
Neumann B, Held M, Liebel U, Erfle H, Rogers P, Pepperkok R, Ellenberg J Nat Methods. 2006 May;3(5):385-90. (PMID: 16628209)

- High-throughput fluorescence microscopy for systems biology..
Pepperkok R, Ellenberg J. Nat Rev Mol Cell Biol. 2006 Sep;7(9):690-6. (PMID: 16850035)

- Live-Cell Imaging Reveals a Stable Cohesin-Chromatin Interaction after but Not before DNA Replication.
Gerlich, D., Koch, B., Dupeux, F., Peters, J.M., and J. Ellenberg Current Biology 2006;16:1571-1578 (PMID: 16890534)

- Monitoring the permeability of the nuclear envelope during the cell cycle.
Lenart, P., and J. Ellenberg Methods 2006;38:17-24 (PMID: 16343937)

- Reverse transfection on cell arrays for high content screening microscopy.
Erfle H, Neumann B, Liebel U, Rogers P, Held M, Walter T, Ellenberg J, Pepperkok R. Nature Protocols. 2007;2(2):392-9. (PMID: 17406600)

- An RNAi screening platform to identify secretion machinery in mammalian cells.
Simpson, J.C., C. Cetin, H. Erfle, B. Joggerst, U. Liebel, J. Ellenberg, and R. Pepperkok.. J Biotechnol. 2007; 129:352-65. (PMID: 17275941)

- Maximal chromosome compaction occurs by axial shortening in anaphase and depends on Aurora kinase.
Mora-Bermudez, F., Gerlich, D., and J. Ellenberg Nature Cell Biology 2007; 9:822-31 (PMID: 17558394)

- Measuring structural dynamics of chromosomes in living cells by fluorescence microscopy.
Mora-Bermudez, F., and J. Ellenberg Methods 2007; 41:158-167 (PMID: 17189858)