How Molecules Escape from Cell's Nucleus: Key Advance in Using Microscopy to Reveal Secrets of Living Cells
ScienceDaily (Sep. 27, 2010) — By constructing a microscope apparatus that achieves resolution never before possible in living cells, researchers at Albert Einstein College of Medicine of Yeshiva University have illuminated the molecular interactions that occur during one of the most important "trips" in all of biology: the journey of individual messenger Ribonucleic acid (RNA) molecules from the nucleus into the cytoplasm (the area between the nucleus and cell membrane) so that proteins can be made.
Real-Time mRNA Export: Messenger RNA molecules (green structures) passing through the nuclear pore (red) from the nucleus to the cytoplasm. (Credit: Image copyright Tremani / Courtesy of Albert Einstein College of Medicine)
The results, published in the September 15 online edition ofNature, mark a major advance in the use of microscopes for scientific investigation (microscopy). The findings could lead to treatments for disorders such as myotonic dystrophy in which messenger RNA gets stuck inside the nucleus of cells.
Robert Singer, Ph.D., professor and co-chair of anatomy and structural biology, professor of cell biology and neuroscience and co-director of the Gruss-Lipper Biophotonics Center at Einstein, is the study's senior author. His co-author, David Grünwald, is at the Kavli Institute of Nanoscience at Delft University of Technology, The Netherlands. Prior to their work, the limit of microscopy resolution was 200 nanometers (billionths of a meter), meaning that molecules closer than that could not be distinguished as separate entities in living cells. In this paper, the researchers improved that resolution limit by 10 fold, successfully differentiating molecules only 20 nanometers apart.
Protein synthesis is arguably the most important of all cellular processes. The instructions for making proteins are encoded in the Deoxyribonucleic acid (DNA) of genes, which reside on chromosomes in the nucleus of a cell. In protein synthesis, DNA instructions of a gene are transcribed, or copied, onto messenger RNA; these molecules of messenger RNA must then travel out of the nucleus and into the cytoplasm, where amino acids are linked together to form the specified proteins.
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Nature advance online publication 15 September 2010 | doi:10.1038/nature09438; Received 3 November 2009; Accepted 23 August 2010; Published online 15 September 2010
In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport
David Grünwald1,2 & Robert H. Singer2
Kavli Institute of NanoScience, Department of BioNanoScience, TU Delft, Lorentzweg 1, 2628 CJ Delft, The Netherlands
Albert Einstein College of Medicine, Department of Anatomy and Structural Biology and Gruss Lipper Biophotonics Center 1300 Morris Park Avenue, Bronx, New York 10461, USA
Correspondence to: Robert H. Singer2Email: Robert.Singer@einstein.yu.edu
Abstract
Export of messenger RNA occurs via nuclear pores, which are large nanomachines with diameters of roughly 120 nm that are the only link between the nucleus and cytoplasm1. Hence, mRNA export occurs over distances smaller than the optical resolution of conventional light microscopes. There is extensive knowledge on the physical structure and composition of the nuclear pore complex2, 3, 4, 5, 6, 7, but transport selectivity and the dynamics of mRNA export at nuclear pores remain unknown8. Here we developed a super-registration approach using fluorescence microscopy that can overcome the current limitations of co-localization by means of measuring intermolecular distances of chromatically different fluorescent molecules with nanometre precision. With this method we achieve 20-ms time-precision and at least 26-nm spatial precision, enabling the capture of highly transient interactions in living cells. Using this approach we were able to spatially resolve the kinetics of mRNA transport in mammalian cells and present a three-step model consisting of docking (80 ms), transport (5–20 ms) and release (80 ms), totalling 180 ± 10 ms. Notably, the translocation through the channel was not the rate-limiting step, mRNAs can move bi-directionally in the pore complex and not all pores are equally active.
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