Research elucidates individual steps controlling transport through Nuclear Pore Complex
Berkeley research team rely on Andor iXon high sensitivity EMCCD camera to capture dynamic sequences
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.
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. Plus, Andor’s proven UltraVac™ vacuum technology ensures both deep cooling and complete protection of the sensor while Andor iCam software enables market-leading exposure switching.”
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. 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. More information may be found at http://mcb.berkeley.edu/labs/weis/ and http://www.physics.berkeley.edu/research/liphardt/index.html respectively.
Quantum dots are nanocrystals of semiconductor material ranging in size from two to ten nanometres in diameter. Their unique optical properties are different to the bulk material and include the emission of photons under excitation that are visible to the human eye. The wavelength of these emissions depends on the size of the crystal and, since the size of the quantum dots can be precisely controlled at the manufacturing stage, a rainbow of colours is available simply by changing the dot size.
Andor’s microscopy solutions encompass a wide range of high performance digital cameras and associated imaging software, as well as microscopy systems designed to address a broad range of optical microscopy techniques including laser spinning disk confocal microscopy, photo-bleaching, activation, conversion and ablation, TIRF, white light spinning disk confocal, calcium ratio imaging, comet assay and bioluminescence. To learn more about the iXon EMCCD camera and applications in microscopy, please visit the Andor website (http://www.andor.com).
An image was taken from a video and captures the moment when a single quantum dot crosses the membrane through the nuclear pore complex. For a high resolution version of the image above, either click on the image, use this link, or contact John Waite at NetDyaLog Limited. If you would like a copy of the video, please contact John Waite
Reference 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)
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