Visualising the inner workings of the living cell

This post was contributed by Clare Sansom, senior associate lecturer at the Department of Biological Sciences

microscopeDr Alan Lowe, who gave the second of the two Science Week lectures on March 25, is a relatively new arrival at Birkbeck. He has been a lecturer at the Institute for Structural Molecular Biology – that is, his post is jointly held between Birkbeck and UCL – for two years.

He obtained his degrees from the universities of Bath and Cambridge and spent several years in California as a postdoctoral researcher before he was appointed to this position. He is researching the development of techniques that allow him to see inside individual, living cells, to identify single molecules, and to follow biochemical processes in ‘real time’.

Lowe started his lecture by citing the so-called central dogma of molecular biology, which can be stated in simplistic terms as ‘DNA makes mRNA makes protein’: or, in other words, that the information in DNA is transformed into the molecules that implement biochemical processes, the proteins, via mRNA.

This, however, has to be very tightly regulated to ensure that the right reactions are carried out in the right cells at the right times: regulation that is out of kilter can cause serious disease.

Metabolism is generally regulated in one of three ways. Taking a simple reaction such as
A + B à C, if you want to limit the amount of C that is produced, you can remove A or B; you can inhibit the enzyme that carries out the reaction; or you can separate A and B into different compartments.

Animal and plant cells contain many specialist compartments, the most important of which is the nucleus that segregates the chromosomes that contain the DNA from the rest of the cell. Proteins that interact with chromosomes can be kept outside the nucleus and therefore inactive until they receive a signal to enter it. Disruption of this signalling or of the nuclear membrane can lead to cancer.

A diagram that shows the distribution of (for example) molecules of one protein within a cell at a given time can be thought of as a map. Like a map, too, this information is limited because it is static. It is more instructive to follow the distribution over time, and that, too, has a geographical equivalent.

Lowe explained that when he was living in California the San Francisco Exploratorium conducted an experiment in which they gave a GPS device to each taxi in the city and followed them all over 24 hours. They could follow one taxi throughout the day or see how the overall patterns changed from hour to hour; the results are still online.

Most of the taxis behaved in a fairly predictable way, but one result could never have been predicted: a taxi landed up in San Francisco Bay. Even now, nine years after the experiment, no one knows exactly why; this is, perhaps, the geographical equivalent of a molecular event that provides one of the steps leading to cancer.

Lowe explained that he would like to be able to put a mini-GPS unit on a molecule within a cell so that it could be tracked in a similar way. This is not quite possible, but it is possible to attach a glowing molecular probe to a molecule. A protein that is isolated from jellyfish and that is known, for obvious reasons, as green fluorescent protein (GFP) can be attached to other proteins to make them glow when exposed to ultra-violet light. Derivatives of this protein have been produced that fluoresce with all the colours of the visible spectrum. It is possible to label interesting proteins with a fluorescent probe and track them through a microscope as they move through the cell, just as the San Francisco taxis were tracked.

One problem with this technique, however, is that optical microscopes and fluorescent probes rely on the visible part of the electro-magnetic spectrum. The protein molecules that we are interested in tracking are smaller than the wavelength of visible light, and, therefore, they will appear fuzzy, with all the fine detail missing. This problem was only solved by the invention of the super-resolved fluorescent microscope: its developers, Eric Betzig, Stefan Hell and William Moerner, were awarded equal shares of the 2014 Nobel Prize in Chemistry.

Lowe went back to the example of proteins entering the cell nucleus to explain this further. The membrane that surrounds the nucleus, which is known as the nuclear envelope, typically contains about 2000 nuclear pore complexes. Each complex has an hourglass-like shape with a narrow aperture through which proteins enter the nucleus, and which is filled with unstructured proteins. Small molecules can diffuse at random into the nucleus through the pore. Large molecules such as proteins, however, may be excluded from the nucleus completely, or, in contrast, they may enter but not leave it.

This is an example of the workings of ‘Maxwell’s demon’, a thought experiment that explains how the Second Law of Thermodynamics – which appears to imply that disorder must always increase – can be violated by imagining a tiny demon at a gate that only lets faster-than-average molecules through.

In the case of the molecular gate in the pore complex, only certain proteins that have been attached to another protein, known as an importin, are allowed into the pore. Lowe and his group bound quantum dots, which are fluorescent nano-particles that are small and bright enough to be visible when attached to a single molecule, to importin molecules, and tracked them as they moved through the pore complex. They found the pore complex channel to go through several stages in selecting cargoes. Most of the molecules are rejected before they enter the complex, others move into the channel before returning to the cytoplasm and only a relatively small fraction enter the nucleus. Nuclear entry requires energy; if energy is removed from the system a ‘gate’ at the bottom of the complex will remain closed.

It is possible to visualise the positions of all the molecules using a technique called single-molecule localisation microscopy (SMLM), in which the fluorescence signal is turned on in one small group of molecules at a time. This enables Lowe and his group to zoom out and look at the nuclei of thousands of cells (as at all the taxis in San Francisco), or, alternatively, to zoom in on a single channel. He used this technique to look at the distribution of proteins at the bottom of the channel through which cargo proteins enter the nucleus.

This structure is composed of tendril-like proteins that reach into the centre of the channel, and these proteins are known to be able to form a solid hydrogel under some circumstances. Lowe mixed them with an importin and showed that the proteins cross-linked to form a material that fell apart when energy was added.

This suggests a molecular mechanism through which the pore may open and close to let cargo into the nucleus. However, many of the details of the system are still unknown; he is developing ways to ‘zoom out’ and combine these images of the cell at the molecular level with larger-scale visualisation of cells that grow and divide.

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