Feature: Using 3D electron microscopy to explore cell biology

When Professor Rob Parton was studying his PhD in biochemistry in the UK, he started dabbling with electron microscopy (EM) as one tool to aid him with his research. “In those days it was more or less standard type thin sections, just a two dimensional view of a very small part of the cell,” says Parton.

Yet these early experiences sparked an interest in EM that has led since him to develop new and innovative ways of using the technology to explore the ultrastructure of cells.

Since 1996 he has been deputy director of the Centre for Microscopy and Microanalysis at the Institute for Molecular Bioscience (IMB) at the University of Queensland, and also heads up his own research group within the IMB. His most recent work involves building three-dimensional images of cells using electron tomography.

“What we can do now is move from a 2D view to a 3D view,” he says. “We can take our materials and cut a relatively thick section, then we put that in the electron microscope. We produce a tomogram by doing a tilt series, taking images at several different angles with the specimen tilted with respect to the electron beam.”

Parton is particularly interested in using this electron tomography to gain a deeper understanding of caveolae, a structure found in the plasma membrane of cells that has long been somewhat of a mystery to cell biologists.

With the help of his new EM approaches, he and his team are beginning to reveal some of the key functions of this complex and intriguing structure, as well as what happens when they malfunction.

“We’re trying to work out what caveolae do, and why having mutations in caveolae is so harmful to patients,” says Parton.

“We’re building a picture in 3D of how the caveolae form using all our different systems, including EM and our new molecular markers, and we’re putting that together with functions that we can then assess in knockout mice, zebra fish or in cell cultures.”

Little caves

The earliest practitioners of electron microscopy in the 1950s – the first to peer at the minute inner workings of the cell – noticed amongst the mitochondrion, endoplasmic reticulum, golgi and endocytic vesicles, another flask-shaped structure, one that almost looked like small caves: the caveolae.

“By electron microscopy you see these beautiful arrays of these structures on the surface. They are the most spectacular structures, their morphology is absolutely unique,” says Parton. The caveolae are so populous that some cells can have 40 per cent of the surface covered by caveolae. “They are one of the most abundant structures you can see on the surface of any mammalian cell.”

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The decades that followed their discovery saw a tremendous amount of information revealed about the other cellular components, but caveolae remained somewhat of a mystery.

Some believed they were primarily involved in cellular endocytosis, due to their resemblance to clathrin-coated vesicles and their movement between the plasma membrane and the interior of the cell. But, while they do appear to play a role in endocytosis, that didn’t appear to be their sole function.

One clue as to their function comes from observing the impact of blocking the protein caveolin, which is responsible for the formation of caveolae. “If you don’t have the caveolin protein, and you lose the caveolae, then you have problems storing lipids, or you can have muscle diseases.

But we’re still trying to work out at the molecular level what the caveolae are actually doing. “Our general idea is that the caveolae sense changes that occur on the cell surface. That could be a tension on cell surface – in a muscle cell, for example – and if there’s a tension on the surface it would translate that into a signal in the cell.”

So it was that Parton sought to develop new EM techniques in order to view the caveolae in more detail, understand how they’re formed by the caveolin protein, and see what happens when they go wrong.

Frozen in time

One of the innovations developed by Parton and his team is a method of preparing samples for viewing in the electron microscope that allows for a better, less adulterated, view of the cell.

The challenge with fixatives is finding a way to effectively stop the processes within the cell – to freeze them in time, if you will – in a way that enables the sample to be viewed with minimal distortion or decay of the structures to be analysed.

The problem with traditional fixatives, such as formaldehyde, is that they can result in damage to the cell membranes and organelles. As an alternative to traditional fixatives, Parton and his team employ a rapid freezing technique, which preserves the sample in close to a pristine state.

“We freeze our specimens very rapidly under conditions where we get no ice crystal formation,” says Parton. “We then either look at the material in the vitreous state at low temperature in the microscope. Or we can go through a scheme called free substitution, where we embed the specimen in plastic in very specific conditions, so we retain our antigens in their native form but also retain the structure as well.”

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Parton uses a high pressure freezer that is able to snap freeze the specimens very quickly without risking damage from the formation of ice crystals. They then store the samples in liquid nitrogen.

From there they can embed the sample in a layer of vitreous ice, which is a form of amorphous ice that has been rapidly frozen, making it like smooth glass. “This allows us to get high resolution information in the most native state possible,” says Parton. “This is real cryo-EM, where we have as little perturbation as possible.”

Another technique used by Parton is freeze substitution, which is where water is removed from the sample at very low temperature and embedded in a resin that has been polymerised by ultra violet light. This approach has proven particularly useful in working with green fluorescent protein.

“We actually keep the fluorescence of the green fluorescent protein, even in the resin. So, using a light microscope, we can find the area we’re interested in and then look at it in the electron microscope, and we can also immunolabel the same specimen.

This is quite an advance in the field, because it means we can use green fluorescent protein as a nice marker – we can do light microscopy, electron microscopy, immuno-gold labelling and tomography all in the same specimen.”

Moving depth

One of the puzzles with caveolae has been how they’re formed by the caveolin protein. Parton has been investigating this problem by building detailed 3D images of the caveolae using electron tomography, and then putting a series of images together into an animation that moves through the specimen in 3D.

“With these kinds of methods we’re starting to actually see the monomeric units, and how the caveolin generates the caveolae,” says Parton. “We don’t yet understand how they form, but we’re getting closer by looking at the high resolution structure, to the point where we can see the molecules that generate the caveolae.

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“Putting that together with other information on the lipid composition of the structures that form, we’re starting to get to the stage where we’ll be able to actually model how caveolin and the lipid molecules work together to generate a caveola.”

While Parton’s team has made some significant advances using EM, there’s still a long way to go in perfecting the technology. “The challenge in all EM is to get to the stage where we can get a large amount of data from a whole cell or whole tissue, and then be able to go in at higher magnification in as native a state as possible.

“In an ideal world we’d be able to recognise the shape of a caveolin molecule, and then take a whole cell and have a look at where these molecules are, and see how they are changed in different disease states.

“The challenge is getting that 3D information at the cellular scale, but also to go to the very high resolution scale where we can see the molecular structure. We’re making progress in all these directions, not with a single technique, but by combining a lot of different techniques. There’s no right or wrong technique. There are many different approaches to tackle the same problem, and every approach has its problems.”

Parton also has a paper that appeared as the cover article in the August 23 issue of the Journal of Cell Biology where he details how he and his team have used a host of EM techniques, along with biochemistry and functional experiments, to characterise another of his interests, the clathrin-independent carrier, or CLIC.