The genome of eukaryotic cells is compartmentalized inside the nucleus and separated from the cytoplasm allowing a level of regulation which is unprecedented in prokaryotes. This separation is achieved by the nuclear envelope, which is formed by two membranes, the inner and the outer nuclear membrane. Embedded in this two-membrane structure are nuclear pore complexes, the gatekeepers of the nucleus. These are huge macromolecular assemblies of around 125 MDa in vertebrates which allow transport between the cytoplasm and the nucleoplasm.
The nucleus is a highly dynamic structure during the cell cycle. The nuclear envelope including nuclear pore complexes breaks down at the beginning of each mitosis and reforms in the emerging daughter cells around the segregating chromatin. We are interested in the molecular mechanisms of nuclear envelope and nuclear pore complex formation as well as chromatin decondensation. Using a cell free system based on cellular extracts prepared from Xenopus laevis eggs we can reconstitute these processes in a test tube and dissect them biochemically, identify the key components involved and define how they function.
Nuclear pore complex assembly and function
Nuclear pore complexes (NPCs) mediate transport between cytoplasm and the nuclear interior. In most cells, they are the biggest protein complexes with a size of up to 125 MDa. The stepwise coordinated assembly from more than five hundred individual molecules and their integration into the nuclear envelope is a fascinating example of molecular self-organization. Using biochemical and cell biological approaches we delineate the assembly pathway and its function in various transport processes. We study the nuclear membrane interaction of NPC proteins and how it contributes to NPC assembly.
Surprisingly, despite their universal function in all nucleated cells some mutations in NPC proteins have been linked to particular pathologies such as atrial fibrillation, a steroid resistant nephrotic syndrome leading to kidney failure or the AAA-syndrome with patients showing achalasia, Addison disease, and alacrima. We characterize how certain NPC mutations affect very specific cell types and define molecular mechanisms of the underlying pathology.
Vollmer B, Lorenz M, Moreno-Andres D, Bodenhöfer M, De Magistris P, Astrinidis SA, Schooley A, Flötenmeyer M, Leptihn S, and Antonin W (2015). Nup153 recruits the Nup107-160 complex to the inner nuclear membrane for interphasic nuclear pore complex assembly. Developmental Cell, 33 (6): 717-728.
Braun DA, Sadowski CE, Kohl S, Lovric S, Astrinidis SA, Pabst WL, Gee HY, Ashraf S, Lawson JA, Shril S, Airik M, Tan W, Schapiro D, Rao J, Choi WI, Hermle T, Kemper MJ, Pohl M, Ozaltin F, Konrad M, Bogdanovic R, Büscher R, Helmchen U, Serdaroglu E, Lifton RP, Antonin W, Hildebrandt F (2016). Mutations in nuclear pore genes NUP93, NUP205 and XPO5 cause steroid-resistant nephrotic syndrome. Nature Genetics, 48 (4): 457-465.
Weberruss M and Antonin W (2016). Perforating the nuclear boundary - how nuclear pore complexes assemble. Journal of Cell Science 129 (24): 4439-4447
Chromatin decondensation at the end of mitosis
In metazoan the mitotic chromatin is up to fiftyfold more compacted as compared to interphase. Therefore, at the end of mitosis the chromatin decondenses to re-establish the chromatin state competent for transcription and replication. Despite the fact that this is most likely an essential process during cell division we are largely unaware about the factors involved. We use a cell free system to biochemically define and characterize the proteins required for this.
We identify key components of chromatin decondensation by life cell imaging, often combined with RNAi based screening methods. We are interested in the dynamics of chromtin decondensation and how the process is coordinated with other concomitantly happening cellular events such as nuclear envelope reformation.
Magalska, A, Schellhaus, AK, Moreno-Andres, D, Zanini, F, Schooley, A, Sachdev, R, Schwarz, H, Madlung, J, Gerken, J and Antonin, W (2014). RuvB-like ATPases function in chromatin decondensation at the end of mitosis. Developmental Cell, 31 (3): 305-318.
Schellhaus, AK, Magalska, A, Schooley, A, and Antonin, W (2015). A cell free assay to study chromatin decondensation at the end of mitosis. Journal of Visual Experiments (JoVE), 106: doi: 10.3791/53407.
Antonin W and Neumann H (2016). Chromosome condensation and decondensation during mitosis. Current Opinion in Cell Biology, 40:15-22.
Membrane remodeling during herpesvirus nuclear egress
Although nuclear pore complexes are the canonical gatekeepers of the nucleus some transport processes across the nuclear envelope bypass them. Herpesviruses assemble their capsids in the nucleus of infected cells and egress by vesicle-mediated trafficking through the nuclear envelope. This unconventional exit from the nucleus requires budding of viral capsids at the inner nuclear membrane into the lumen of the nuclear envelope. The resulting intralumenal vesicles fuse with the outer nuclear membrane which delivers the viral capsid to the cytoplasm. We study the function of the proteins involved in this process using minimal membrane systems such as giant unilamellar vesicles (GUVs). Here, vesicle budding and scission can be studied and analyzed in a biochemical way.
Lorenz M, Vollmer B, Unsay JD, Klupp BG, García-Sáez AJ, Mettenleiter TC, and Antonin W (2015). A Single Herpesvirus Protein can mediate Vesicle Formation in the Nuclear Envelope. Journal of Biological Chemistry, 290 (11): 6962-6974.
Zeev-Ben-Mordehai, T, Weberruß, M, Lorenz, M, Cheleski, J, Hellberg, T, Whittle, K, El-Omari, K, Vasishtan, D, Dent, K C, Harlos, K, Hagen, W, Klupp, BG, Antonin, W, Mettenleiter, TC, and Grünewald, K (2015). Crystal structure of the herpesvirus nuclear egress complex provides insights into inner nuclear membrane remodelling. Cell Reports, 13 (12): 2645-2652.