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Controlling Transport through Nuclear Pore Complex
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Nuclear Pore Complexes (NPCs), large protein structures which span the nuclear membrane in eukaryotic cells and mediate the exchange of materials between the nucleus and cytoplasm, play a vital role in many aspects of cellular physiology including gene expression. 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.
Now, a team from the University of California, Berkeley, has shed light on the step-by-step process of capture, filtering, translocation and release. Signals from single, protein-functionalised quantum dots cargos 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," says Karsten Weis, 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," concludes Weis.
The Weis Lab is actively engaged in characterising and analysing the molecular machinery responsible for the transport of macromolecules into and out of the nucleus through a combination of genetic, biochemical and biophysical approaches.
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In the latter they are joined by the Liphardt Lab, which maintains research into super-resolution studies of protein clustering in membranes, and single-molecule studies of transport through biological pores and channels.
Original publication:
Alan R. Lowe, Jake J. Siegel, Petr Kalab, Merek Siu, Karsten Weis & Jan T. Liphardt. "Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking," Nature Vol. 467, 600-604 (2010)
http://www.andor.com
Keywords: Andor Technology EMCCD cameras Karsten Weis Nuclear Pore Complex Quantum Dots TIR Microscope Total Internal Reflection Microscopy University of California
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