Biomimetic Nuclear Pore Complexes

In this project, we aim to build a system to mimic biological pores (nuclear pore complex) with solid-state nanopores. In biomimetic nanopores we combine solid state nanopores with key biological proteins, with the aim to understand the fundamental mechanism of the nuclear pore complex (NPC) transport.

In eukaryotic cells the genetic material (DNA) is spatially separated from the rest of the cellular organelles by the nuclear envelope. Both proteins and RNA’s must be imported into and exported out of the nucleus respectively for smooth function of the cell.  The transport events occur through large protein machineries known as nuclear pore complexes, which function as gatekeepers of the nucleus: water, ions and small molecules can freely diffuse through the NPC without hindrance, while larger macromolecules (RNA, Proteins) with diameters of up to ~30nm are selectively transported. The cargoes to be transported are recognized by nuclear transport receptors (NTRs), which travel back and forth between the two compartments (nucleus and cytoplasm) and in doing so they ferry their load, mediating their interaction with the NPC. Both structure and function of the NPC have been studied extensively and are relatively well understood. Nuclear pore proteins (Nups) that consist of natively unfolded domains rich in phenylalanine-glycine repeat motives (FG-domains) play a crucial role in imparting the selective barrier and hence in the translocation process [1]. Several models have been proposed to explain the structural arrangement of the FG-Nups and their interactions with the NTRs, such as: the hydrogel model [2], where FG-Nups are thought to interact forming a gel-like plug, through which NTR-cargo complexes are able to melt and pass through. The polymer brush model [3] asserts instead that FG-Nups are extended brushes, which collapse upon binding to the NTR-cargo complexes, allowing them the passage through the NPC. The reduction of dimensionality model[1][3] proposes that FG-Nups collapse in the presence of NTRs, causing an opening of the central channel. Finally, the forest model [4][5] relies on a combination of the hydrogel and polymer brush models.

Despite the progress made in the field, technologies capable of providing direct observation of these phenomena at the relevant temporal and spatial scales, and ultimately discriminate between the different theories are still missing.

 

BIOMIMETIC

Figure 1. Experimental setup showing a biomimetic nanopore coated with a yeast FG-Nup (Nsp1). The nanopore fabrication technique is already established and based on Si microfabrication technology. a) Nanopores are drilled on free-standing SiN membranes (20nm thick) and coated with purified FG-nup proteins (e.g. Nsp1), by means of Self-assembled monolayer chemistry (SAM). NTRs (e.g. Kap95), supposed to overcome the barrier, and Non-NTRs, supposed to be rejected, are injected into the system. b) Current versus voltage curves showing the change of the nanopore conductance upon Nsp1 coating. Bottom right figure shows TEM images of a 30nm pore before (left) and after (right) coating. c) Typical detected translocation event, from which dwell time (T) and conductance blockade (ΔG) can be measured.

 

Our project aims at understanding the intriguing mechanism of the selective barrier by performing electrophysiological measurement on a minimalistic NPC. For this, we covalently attach purified FG-nups onto the surface of our solid-state nanopores. We then inject NTRs into the system and measure conductance blockade caused by their translocation through the biomimetic pore (Fig 1.C). We can accurately quantify key features of the translocation events, such as conductance drop, translocation time, and event rates. This information allow us to characterize the translocation process in terms of size of the translocating molecules and their interaction with the pore.

The experiments are complemented with MD simulations and cryo-EM imaging carried out by our collaborators. The combination of experiments and simulations aims at providing newer quantitative information on the NPC structure and transport properties, with the ultimate goal of shedding light on the puzzling selectivity mechanism of this sophisticated protein machine.

 

 

[1] Wente, Susan R., and Michael P. Rout. The Nuclear Pore Complex and Nuclear Transport. Cold Spring Harbor Perspectives in Biology 2(10), 2010.

[2] Frey, S.; Richter, R. P.; Görlich, D. FG-Rich Repeats of Nuclear Pore Proteins Form a Three-Dimensional Meshwork with Hydrogel-like Properties. Science, 314, 815–817, 2006.

[3] Lim, R. Y. H.; Fahrenkrog, B.; Köser, J.; Schwarz-Herion, K.; Deng, J.; Aebi, U. Nanomechanical Basis of Selective Gating by the Nuclear Pore Complex. Science, 318, 640–643, 2007.

[4] Peters, R. Translocation through the Nuclear Pore Complex: Selectivity and Speed by Reduction-of-Dimensionality. Traffic, 6, 421–427, 2005.

[5] Yamada, J.; Phillips, J. L.; Patel, S.; Goldfien, G.; Calestagne-Morelli, A.; Huang, H.; Reza, R.; Acheson, J.; Krishnan, V. V; Newsam, S.; et al. A Bimodal Distribution of Two Distinct Categories of Intrinsically Disordered Structures with Separate Functions in FG Nucleoporins. Mol. Cell. Proteomics, 9, 2205–2224, 2010.