Unravelling the mechanism of nuclear transport using optical nanopores

Descripción del proyecto

Unos nanoporos biomiméticos permiten observar el guardián del núcleo

Las células son los componentes básicos de los organismos vivos y, en las células eucariotas, sus funciones dependen en gran medida de la compartimentalización provista por los orgánulos unidos a la membrana. El núcleo no es una excepción, ya que controla el movimiento de entrada y de salida a través de poros. Una enmarañada red de proteínas intrínsecamente desordenadas en un canal central permite la selectividad de pequeñas moléculas, mientras que las macromoléculas dependen de proteínas transportadoras para desplazarse por la membrana nuclear. Los mecanismos biofísicos que subyacen la selectividad y el movimiento no están claros. El equipo del proyecto NTON, que cuenta con el apoyo de las Acciones Marie Skłodowska-Curie, está desarrollando una plataforma de nanoporos biomiméticos para estudios a nivel de molécula única a fin de dilucidar el enigmático funcionamiento interno del complejo poroso nuclear.

Objetivo

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.