A timeless character once said: ‘Life is like a box of chocolates’ – but perhaps more accurately, life is like a box of chemicals:
All of the processes that make cells what we would consider to be alive (breaking down food for energy, sensing and responding to their environment, reproducing etc.) are just complex interactions of biochemicals.
But while we understand how some of these processes work, capturing them in action is extremely tricky: The chemicals are really small and the reactions are very fast – so being able to ‘see’ these essential facets of life happening for real is impossible even for Sir David Attenborough.
Electron microscopes are one of our best tools for looking at really small things (magnification is up to 5000 times more than typical optical microscopes), but they rely on the sample being ‘frozen’ in time and covered with heavy metals in order to work so can only be used to take individual shots.
But a team of scientists from the US has come up with a neat idea to get around this, taking snapshots of the same biochemical process, but frozen at different points in time.
So, what’s the point?
We can currently find out a heck of a lot about important biological processes: we can work out the chemical sequences, structures and functions of huge, complex biomolecules using a huge range of scientific techniques.
But being able to see precisely how these biomolecules interact with each other is another story entirely: instead we can usually only infer what is going on based on what we know from the molecules’ structures and functions.
So seeing these interactions unfold in sequence will help to add weight to some of our current theories and could also lead to new discoveries.
Being able to see precisely how biochemical processes work has a huge number of uses: understanding the internal mechanisms of our own cells, as well as bacteria and other pathogens, could help us to develop treatments for diseases, for instance.
What did they do?
The research team decided to look at a common bridging process between two large ‘subunits’ known as 30S and 50S. In bacteria (and other prokaryotes), these proteins link together to form the 70S ribosome – a part of the cell’s internal machinery for making proteins.
To make the 70S, 12 ‘bridges’, made from a combination of RNA and protein, are formed between the 30S and 50S subunits. The researchers hoped they could find a way to view the order in which these bridges are made, to better understand the mechanism behind 70S formation.
To do this, they used a technique long-windedly called ‘time-resolved cryo-electron microscopy’. The basic idea is to mix together a load of reagents (in this case, 30S, 50S and a load of other biochemicals) and then spray them onto a carbon grid which is then plunged into liquid ethane (less than 88oC).
This way, the subunits and bridges are frozen in place (hopefully mid-reaction) and can be looked at using an electron microscope.
By varying the amount of time the reagents are mixed together before spraying them onto the grid, the researchers were able to view the positions of each reagent at different points in time to figure out what was going on.
So rather than making a film by taking lots of shots of one ribosome coming together, they have made one by taking single shots of lots of ribosomes, but at different times.
Because they only needed to prove their concept worked, they ended up taking snapshots at only 2 different mixing-times: 9.4 and 43 milliseconds, which they also compared to a ‘preassembled’ ribosome. So what they ended up with was similar to a sequence of ‘before’ and ‘after’ photos.
In the end they took 303 micrographs (still pictures from the electron microscope), of which 258 were ‘deemed suitable for further analysis’. These were then used to create computer-renderings of the full ribosome complex at different times.
Did they prove anything?
From their images, they could see that 8 of the bridges were present in all of the samples, suggesting they are formed very quickly (less than 9.4 ms). The remaining 4 bridges were present in the preassembled ribosome, but not the 9.4 or 43 ms samples, suggesting that these bridges take longer than 43 ms to form.
The scientists claim that this shows the bridges form in ‘sequential order’, and that it is the first time (to their knowledge) that anyone has shown that these bridges appear to form on this timescale.
So, what does it mean?
As a proof of concept, the researchers appear to have shown that their kit has worked, although it perhaps would have been cooler to take snapshots at a range of other times so we can see the bridge formation unfolding more obviously.
Another criticism is that the control (pre-assembled ribosome) may not necessarily reflect the conditions of their equipment – they perhaps should prove that their mixing/spraying method has not interfered with the formation of the ribosome (i.e. maybe the bridges haven’t formed properly because of the mixing, rather than because they take longer than 43 ms).
One of the simplest ways of doing this would be to compare the pre-assembled ribosome to one that has been mixed in their system for a long time (say, several minutes). If they match, then the equipment hasn’t affected the ribosome formation. If they don’t, then it could be a problem.
Obviously, constraints such as time and money are probably an issue, and the authors point to other evidence that supports the idea that the B2c and B4 bridges are ‘not vital but, rather, are formed at a later stage… to help stabilize the 70S structure’, which corroborate their claims to an extent.
In any case, the idea for ‘time-resolving’ electron microscope images of biochemical interactions in this way is pretty neat and could well prove a valuable method for viewing how these interactions might occur.
The authors suggest that it can be used to view a range of other ‘dynamic functional events’ involving proteins in living organisms. But before amateur film-makers get too excited about the prospect of shooting images of the weird, tiny chemicals that give us life and pondering on the pseudo-metaphors they could represent, bear in mind that an electron microscope will set you back tens, if not hundreds of thousands of dollars (although you could always try building one yourself!)
Original article in PNAS Jun 2014
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