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Tinkering with a cellular nanomachine: A minimalist approach to studying nuclear pore complex function by de novo design

Research Project
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01.10.2007
 - 30.06.2011

The separation of the nucleus and the cytoplasm in eukaryotic cells requires that specific biomolecules (e.g. proteins) be trafficked across the double-membraned nuclear envelope (NE) that encloses the nucleus. This phenomenon is known as nucleocytoplasmic transport (NCT), and occurs through ~100 nm-sized perforations in the NE called nuclear pore complexes (NPCs). Interestingly, the NPC does not operate on the principle of size exclusion (i.e. to separate the components of a liquid according to their size and shape). Instead, each NPC employs several "tentacle-like" natively unfolded proteins known as FG-nucleoporins, which collectively act as a barrier to non-specific biomolecules. As the only passageway in and out of the nucleus, proteins known as importins or exportins are required to "unlock" this barrier to allow the access of select cargo through the NPC. Despite its importance in "protecting" the nucleus from unwanted "guests", the manner in which each NPC promotes or prevents cargo exchange essentially remains a fundamental biological mystery. Presently, conventional techniques have been unable to provide for an accurate mechanistic picture of how the NPC regulates transport. This is because NCT occurs over tens of nanometers in and around the NPC. To understand how the NPC works, we have initiated a bottom-up "Frankenstein" approach with the goal of replicating NCT artificially, which will commence in 3 stages: Phase One will involve combining the FG-nucleoporins to gold nanorings in order to emulate certain contextual details of the NPC (e.g. size and pore-like topography). In doing so, we will be able to measure local effects directly with the atomic force microscope (AFM) and correlate the barrier-like function of the FG-nucleoporins to the effect of importins and exportins. More importantly, by fabricating the nanorings on glass slides, we will introduce a novel assay that has the ability to correlate such nanoscopic AFM measurements to conventional macroscopic fluorescence/optical microscope measurements. Phase Two involves developing a de novo designed "artificial NPC" by integrating the FG-nucleoporins with technologically engineered nanopores in order to mimic NCT. This builds logically from the first phase by using the same experimental platform to resolve the dualistic functionality of the NPC (i.e. to promote or prevent transport) with respect to how the interactions between the FG-nucleoporins and importins/exportins directly influence the kinetics of cargo passage (e.g. how fast or slow or not at all) through the artificial NPC. Phase Three will be to identify heuristically the physiological relevance of any de novo effects in situ. This will be accomplished using a combination of AFM and electron microscopy (EM) such as on spread Xenopus (frog) oocyte nuclear envelopes.

Publications

Kowalczyk, Stefan W et al. (2011) ‘Single-molecule transport across an individual biomimetic nuclear pore complex’, Nature Nanotechnology, 6(7), pp. 433–8. Available at: https://doi.org/10.1038/nnano.2011.88.

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Members (2)

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Ueli Aebi

Principal Investigator
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Roderick Lim

Co-Investigator