Project description
Biomimetic nanopores provide a peek inside the nucleus’ gatekeeper
Cells are the fundamental building blocks of living organisms, and in eukaryotes their activities rely heavily on the compartmentalisation afforded by membrane-bound organelles. The nucleus is no exception, controlling movement in and out with pores. Selectivity of small molecules is enabled by a tangled mesh of intrinsically disordered proteins in a central channel whereas large macromolecules depend on transporter proteins to move across the nuclear membrane. The biophysical mechanisms underlying selectivity and movement are not clear. With the support of the Marie Skłodowska-Curie Actions programme, the NTON project is developing a biomimetic nanopore platform for single-molecule studies to elucidate the enigmatic internal workings of the nuclear pore complex.
Objective
The nuclear pore complex (NPC) is the gatekeeper of the nucleus that regulates the flow of all molecules across the nuclear envelope. Remarkably, this ~40 nm wide pore is capable of efficient and fast transport while remaining highly selective. Its dense central channel is composed of a highly dynamic spaghetti-like mesh of intrinsically disordered proteins that allows small molecules to pass freely, whereas large macromolecules (>40 kDa) rely on specific transporter proteins that ferry their cargo across the pore. Even though various models have been proposed, the fundamental biophysical mechanism of nuclear transport and of the selectivity of the NPC has not been resolved yet. One of the main reasons that have hindered our progress in understanding nuclear transport is the lack of experimental techniques that can probe the structure and dynamics of the disordered proteins and transport receptors inside the NPC channel and during transport with sufficient spatiotemporal resolution. In the proposed action, I aim to establish a modifiable biomimetic platform for single-molecule investigations of nuclear transport. This interdisciplinary and innovative approach combines solid-state nanopores, coated with disordered proteins to mimic the nuclear pore, with optical detection in zero-mode waveguides and DNA origami nanotechnology. The proposed experiments will allow to monitor the translocation of single fluorescently labelled molecules through biomimetic NPCs with excellent sensitivity and specificity. I will use this platform to elucidate the biophysical principles that underlie the structure and selectivity of the NPC channel and the mechanism of active nuclear transport. The direct insights on the single-molecule level obtained by the results of this project will be crucial for our understanding of the molecular principles that govern nuclear transport.
Fields of science
Programme(s)
Funding Scheme
MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF)Coordinator
2628 CN Delft
Netherlands