An RNAi screening approach for the identification of factors controlling nuclear envelope brakdown in vertebrates
Final Report Abstract
In eukaryotic cells, the genetic material is stored within the nucleus and thereby separated from cytoplasmic structures. In vertebrates the entire nuclear envelope (NE) has to be removed in the beginning of mitosis, due to an open mechanism of mitosis. The breakdown of the nuclear envelope (nuclear envelope breakdown; NEBD) involves the disassembly of nuclear pore complexes (NPCs), depolymerization and solubilization of the lamina as well as detachment and removal of the nuclear membrane from chromatin. Different models have been put forward, but the exact molecular mechanism of NEBD remains elusive. We proposed an RNAi screening approach to identify new factors controlling NEBD in vertebrates. After a set of proof of principle experiments to assess NEBD with live cell imaging, we used double stable cell lines with two different fluorescently tagged marker proteins (H2B-mCherry and GFP-alpha-Tubulin or GFP-IBB) for our screen. An automated image analysis toolkit developed by our collaborators (Held, NatMeth2010), was used for data analysis. We could show for Plk1 RNAi or inhibition of Plk1 by a small molecule inhibitor that prophase is longer compared to controls before treated cells finally arrest in prometaphase. Furthermore, the Plk1-experiments have shown that the time span between the onset of chromatin condensation and the efflux of IBB-GFP is delayed. This indicates to a function of Plk1 in entry into mitosis. Furthermore, we could show with our developed microscopic assay that the depletion of NIMA related kinases (Neks 2, 6, 7) prolongs prophase and delays nuclear envelope permeabilization during mitotic entry. The RNAi screen using two different cell lines identified several factors previously unknown to contribute to NEBD. To validate these data the screen has to be repeated. Afterwards the hits will be confirmed using secondary microscopic and biochemical assays. The results from the screen will facilitate further mechanistic studies for an in-depth understanding of NEBD, a fundamental process during cell division.
Publications
- Cell. 2011 Feb 18; 144(4): 539-50. Phosphorylation of Nup98 by multiple kinases is crucial for NPC disassembly during mitotic entry
Laurell E, Beck K, Krupina K, Theerthagiri G, Bodenmiller B, Horvath P, Aebersold R, Antonin W, Kutay U
