A team from the University of California, Berkeley, has shed light on the step-by-step process of capture, filtering, translocation and release of Nuclear Pore Complexes (NPCs), large protein structures that span the nuclear membrane in eukaryotic cells and mediate the exchange of materials between the nucleus and cytoplasm.
Defects in NPC function are implicated in a number of autoimmune diseases, leukaemias and others cancers. Also, nuclear transport plays a pivotal role in viral infections. However, it has been unclear how the NPC facilitates the selective translocation of macromolecules.
Signals from single, protein-functionalised quantum dots cargos (nanocrystals with unique optical properties ranging in size from 2-10nm in diameter) were captured by an Andor iXon camera in a custom-built near-TIR (total internal reflection) microscope as they tracked through human NPCs. This showed that the overall selectivity of the NPC arises from the cumulative action of multiple, reversible sub steps and a final, irreversible exit step.
'With their extraordinary photostability and brightness, quantum dots have established themselves as very useful tools for cellular analysis,' said Karsten Weis at Berkeley. 'Because of their relatively large size, which is comparable to viral particles, the transport of quantum dots across the NPC is quite slow. This, in combination with their photostability, allowed us real-time tracking over extended periods of time and reconstruction of NPC transport events into high-precision transport trajectories.
'Tracking of single dots places real demands on detector technology to perform at significantly higher levels of sensitivity and speed. Electron Multiplying CCD (EMCCD) technology, as seen in the Andor iXon camera, amplifies down to single photons and is ideal for these studies,' concluded Weis.
According to Mark Browne, director of systems at Andor: 'The Andor iXon offers the highest sensitivity from a quantitative scientific digital camera, particularly at fast frame rates.'
