Periodic Reporting for period 1 - NTON (Unravelling the mechanism of nuclear transport using optical nanopores)
Reporting period: 2021-08-01 to 2023-07-31
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. This process is vital to the well-being of the cell, as the NPC poses a gate between the nucleus, the place of the genetic material and mRNA transcription, and the cytosol, where proteins are synthesized from mRNA by ribosomes - hence posing a bottleneck for the central dogma of molecular biology by regulating the flow of mRNA. At the same time, the NPC protects the genetic material from viral intruders, but needs to allow efficient import of proteins to the nucleus to maintain the structural integrity of the genome, and perform and regulate gene transcription. Given its vital importance, it is unsurprising that mutations of the NPC have been linked to numerous neurodegenerative diseases and various forms of cancer.
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 this project, we have combined nanotechnology with single-molecule fluorescence microscopy to build and study biomimetic, minimal versions of the NPC.
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 this project, we have combined nanotechnology with single-molecule fluorescence microscopy to build and study biomimetic, minimal versions of the NPC.
In this project, we have established a customizable biomimetic platform for single-molecule investigations of nuclear transport by combining solid-state nanopores with optical detection in zero-mode waveguides (see figure). By coating the nanopore with the disordered proteins of the central channel of the NPC (FG nucleoporins), we could reproduce selectivity alike to the NPC and systematically study pores of different sizes, which revealed a loss of selectivity as the pore widens above 60 nm. This has direct implications for the working principles of the NPC, which has recently been shown to dilate up to ~60 nm in vivo. Ongoing work is focused on building NPC mimics using DNA origami nanotechnology, which will enable to study the structure and dynamics within the central channel with excellent control using the single-molecule FRET technique. As a simpler system, we also exploited the propensity of FG nucleoporins to undergo liquid-liquid phase separation. This has allowed us to reconstitute cargo import into condensates of various designer FG nucleoporins and provided direct insights into the sequence-function relationship of these disordered proteins. Overall, the results of this project have shed new light on the biophysical principles that underlie the structure and selectivity of the NPC channel and the mechanism of nuclear transport.
The project has provided novel insights into the working principles of the nuclear pore complex. In particular, we addressed important questions with respect to the recently discovered dilation of the nuclear pore complex in vivo. The various assays and approaches implemented during the project are expected to provide further insights in the near future. The project has also sparked the new development of ZMW nanowells, which have exciting prospects to improve single-molecule experiments in living cells.