Seeing the correlative light


Rebecca Pool

Thursday, January 9, 2020 - 10:45
Image: Paul Verkade with the Leica EMPACT2 and his Rapid Transfer Stage for fast-freezing CLEM samples.
When it comes to imaging, Professor Paul Verkade’s first love was electron microscopy at a time when it seemed the world was edging towards confocal methods.
It was the late 1980s, the University of Bristol Professor of Bioimaging and academic lead of advanced electron microscopy at the Wolfson Bioimaging Facility was about to embark on his PhD, and the biological marker, green fluorescent protein, was rising in popularity amongst biologists.
“This was when many electron microscopy labs were actually closing as GFP was coming online,” he says. “Many researchers thought ‘we don’t need electron microscopy anymore’ and I was going to be the only [researcher] using electron microscopy in my laboratory.”
The young Verkade was studying biology at the University of Utrecht, The Netherlands, and had just finished an undergraduate project that relied heavily on electron microscopy.
He now wanted to use electron microscopy to study neuroscience, as following on from this first foray with this high resolution imaging method, he was, in his words, “gripped”.
“I was just so taken with this visualisation of life and the level of detail you could get from this kind of microscopy,” he says. “I knew that this was my thing and I wanted to continue with it.”
He remained at Utrecht and joined the now-late structural biologist and electron microscopy enthusiast, Professor Arie Verkleij, to study neuronal regeneration.
Protein puzzle
At the time, Verkleij was part of a team investigating the Growth Associated Protein, GAP-43, in a bid to understand how this protein was involved in the development and regeneration of neurons. Importantly for Verkade, Verkleij was an inspiration.
“He really was my guiding light and throughout my PhD encouraged me to continue with electron microscopy,” says Verkade.
At the time and using quantitative immunoelectron microscopy, Verkade was able to look at the subcellular distribution of the protein in axons from the sciatic nerves of rats. And from 1995 to 1997, he, Verkleij and colleagues published a number of papers elucidating how the protein was transported along the sciatic nerve.
“An entire department was working on this protein,” says Verkade. “But with electron microscopy, I was the only one that could really look at where this protein was localised and how it moved from the cell body, where it’s synthesised, to the neurons.”
Following his successes here, and with a PhD in hand, Verkade left Utrecht and moved over to the European Molecular Biology Laboratory, Heidelberg, Germany, in 1996.
Verkade’s supervisor, Verkleij, had been working with cell biologist and EMBL co-founder, Kai Simons, who wanted an electron microscopist. Verkade was thrilled.
“Kai said to me, ‘I could really use an electron microscopist so come over and I will have a salary for you,” he says. “This was an amazing experience – there are a lot of passionate people there, so it is a very competitive environment, but it was really exciting and very international.”
Now firmly focusing on sorting mechanisms in intracellular transport pathways, Verkade quickly set to work on using electron microscopy to look at the structure of lipids within the plasma membranes of cells.
These assemblies were thought to play a key role in cellular processes and together with Simons and colleagues, Verkade helped to unravel a lipid-mediated mechanism critical to intracellular membrane sorting, cell signalling processes and more.
Breakthrough results were published in The Journal of Cell Biology in 1998, and at nearly 1300 citations, Lipid Domain Structure of the Plasma Membrane revealed by Patching Membrane Components is now Verkade’s second most highly-cited paper.
Still, as Verkade puts it: “At the time, you don’t always realise the impact your research has had, especially when you’ve been working on it for a year or so, and of course, at EMBL, it’s almost the norm to have papers such as this one.”
Application of advanced Electron Microscopy to Synthetic Biology. By expression of a single protein from a Bacterial MicroCompartment together with synthetic peptides, bacteria can be made to make a cytoscaffold (in red). A model generated fromelectron tomographic reconstructions is shown. Data from Lee et al., paper.  
Come 2000, Verkade was ready to move on. Three EMBL group leaders were establishing the new Max Planck Institute for Molecular Cell Biology in Dresden, Germany, and Verkade accompanied them to set up a new electron microscopy department.
“This was such a unique opportunity at the time... I was designing a whole new facility and was also involved in selecting which new instruments to buy,” he says.
Verkade chose two FEI TEMs, which together cost some €1m at the time and then got to work collaborating with architects on laboratory design.
“The Institute had trams passing by it and these microscopes needed a very stable platform so we used a concrete block to deal with the vibrations,” he says. “Working with architects was really interesting – they were coming from a completely different world to mine, but we still had to speak each other’s language.”
With electron microscopes in tow, Verkade joined forces with Teymuras Kurzchalia – also investigating lipids in cell functions – and a host of colleagues from the Max-Delbrück Center for Molecular Medicine, Hannover Medical School and the Technical University of Dresden, to study the role of the plasma membrane proteins, caveolae, in cellular processes.
Crucially for the world of cellular transport, the researchers had worked out how to generate mice without caveolae, so they could study the impact of this missing subcellular organelle on cell transport, signalling and tumour suppression. And Verkade’s electron microscopy expertise proved instrumental.
“You needed electron microscopy to recognise the caveolae as these were just too small to see using fluorescence microscopy,” he says. “So we used genetics to show that the protein was missing, electron microscopy to show what the proteins may do and then studied the phenotypical outputs of the mice... and the most interesting part of this was using this wide variety of techniques.”
The researchers discovered that caveolae played a fundamental role in organising multiple signalling pathways in the cell and published their findings in Science in 2001. And with 1600 citations, Loss of Caveolae, Vascular Dysfunction and Pulmonary Defects in Caveolin-1 Gene-Disrupted Mice is now Verkade’s most highly cited paper.
For Verkade, the interdisciplinary nature of this research was critical.
As he puts it: “An interdisciplinary approach has been, and still is, so important to research.”
“As researchers we have our own expertise, technologies, ideas but by ourselves we can’t solve the complete problem,” he adds. “Buy by putting the pieces of a puzzle together, then suddenly everything will come together.”
From collaboration to CLEM
In line with Verkade’s enthusiasm for collaboration across disciplines and on methods, he was now becoming increasingly interested in correlative light electron microscopy (CLEM).
Alexander Mironov, Alberto Luini and colleagues from the Mario Negri Institute for Pharmacological Research, Italy, had just published seminal papers on combining GFP technology with correlative light-electron microscopy to image intracellular traffic. Verkade went to visit them to learn more and was soon hooked.
CLEM WORKFLOW for the localisation of ESCRT-III during cell division. ESCRT-III localises for 90 seconds on an approximate 24h cell cycle to the reforming nucleus (blue nuclear dye, 1). These specific cells can be traced back in the EM using CLEM (2), and an overlay of the ESCRT-III on the ultrastructure can be made (3). The nanogold is silver-enhanced and localises (4) to small membrane connections between the cytoplasmic (bottom) and the nuclear side (top) of the nucelar membrane. An Electron Tomography model (5) shows one of the small remaining membrane pores that will be sealed by the ESCRT-III complex (yellow dots). Data with Lorna Hodgson, Judith Mantell, and Jeremy Carlton, adapted from Olmos et al., Nature.
On returning to the Max Planck Institute, Verkade continued to study intracellular trafficking using correlative light-electron microscopy but was having problems with maintaining sample integrity during chemical fixation.
He and colleagues needed a better way to fix samples, and quickly realised that freezing was the answer.
“We had access to high pressure freezers but it would have taken minutes to get our samples to these,” says Verkade. “So we needed [a system] to take a sample from the confocal microscope and freeze it within around five seconds.”
With this in mind, Verkade developed the automated Rapid Transfer System (RTS) that could fast-freeze samples in less than five seconds.
Designed to attach to Leica’s EM PACT2 high-pressure freezer, researchers could now, for the first time, use high-pressure freezing in combination with high time-resolution correlative light and electron microscopy.
“The RTS made it possible to capture specific events occurring live in the cell as observed by light microscopy, cryofix that sample and its event within 4 s, and then analyse the event at high resolution in the electron microscope with excellent preservation of ultrastructure,” highlights Verkade.
What’s more, the RTS could also be used with conventional methods.
As Verkade adds: “I also felt this was a much easier [set-up] to use for high pressure freezing than what was around at the time, as this automated system took away a lot of the human involvement and human error that comes about when processing a sample.”
CLEM approach applied to SAGE particles. Synthetic SAGE particles can be tagged with fluorescent molecules to study their uptake into cells (left: fluorescence overlay of green fluorescent SAGEs and blue nuclear dye on top of the EM overview structure). The zoom in on the right shows the higher resolution ultrastructure where SAGEs (arrows) are taken up into endosomes. Data with Lorna Hodgson and Dek Woolfson.
By now, Verkade was looking for a permanent academic position. His EMBL and Max Planck Institute posts were time-limited with rolling contracts, and in his words, “given the RTS I was quite well known at this point, so it was a good time to move on”.
At the time, the University of Bristol, UK, had just secured significant funds from The Wolfson Foundation and other funding bodies, and combined with in-house investment, was set to build an integrated light and electron microscopy centre.
So, in 2006, Verkade moved to Bristol to take up the position of senior lecturer in Biochemistry and Physiology & Pharmacology, and also to set up the electron microscopy unit as part of what is now known as the Wolfson Bioimaging Facility.
“When I moved, there was a very small electron microscopy department here as well as a light microscopy department, but these were on different floors,” says Verkade. “So one of the things that I really insisted on for the new electron microscopy facility, was to bring these two departments together in a single facility.”
“This was so essential and I think it’s been one of the most significant things we have done, and a lot of other imaging facilities have since followed our example,” he adds.
Progress has been rapid, and over the years the facility has grown enormously and is now home to 22 imaging systems covering a broad range of advanced fluorescence and electron microscopy techniques.
“Investment in people is important – it’s not good enough to buy the machines, we need the people too.” Professor Paul Verkade
The latest methods include multiphoton, super-resolution and fluorescence lifetime imaging alongside a high resolution TEM with STEM and cryo-EM facilities and serial block face SEM.
“Our significant investment gave us a lot of momentum to invest in equipment and this really cemented our place as a world-leading facility, and of course, with something like this, you generate more investments,” says Verkade.
Indeed, by 2016, the facility had almost doubled in size.
“We actually had too many instruments, so we went back to the Wolfson Foundation and said, ‘we’ve been so successful that we need more space, are you willing to provide us with the investment to accommodate that?’,” says Verkade. “The Foundation did and that’s what we call phase II of the facility.”
Along the way, Verkade has also grown a thriving research group, which remains focused on intracellular membrane transport but also develops routes to deliver cargo to specific destinations in
the cell.
CLEM and 3D electron tomography are now fundamental to this research, and crucially, Verkade and colleagues develop software and hardware for experiments, from image segmentation algorithms to fluorescent markers.
As a result, Verkade’s research has also become increasingly interdisciplinary. For example, in 2013, Verkade worked with colleagues from the fields of chemistry, biochemistry, physics, materials science and mathematics to develop self-assembling peptide-based cages (SAGEs) to mimic protein assembly for drug development.
Research was published in Science together with Prof. Dek Woolfson and as Verkade says: “This long-standing collaboration was very significant and such a highlight – and brought a lot of research together.”
Meanwhile, in 2015, cell biologist, Dr Jeremy Carlton from King’s College London, used CLEM fluoronanogold probes, used in Verkade’s lab, to unravel the role that endosomal sorting complexes required for transport (ESCRT) played in cell division.
“Only by applying our technology could Jeremy answer his biological question, which culminated in our Nature paper and a very satisfying collaboration,” says Verkade. “We get the best collaborations when all groups involved are willing to share knowledge.”
Future Hopes
Clearly CLEM has expanded tremendously from a mere smattering of researchers when Verkade first started experimenting with the method to an international playground.
Indeed, just last year, Verkade and a host of researchers from 32 universities and organisations from around the world published the ‘The 2018 correlative microscopy techniques roadmap’.
Now Professor of Bioimaging at Bristol, Verkade is excited about the European-funded consortium, COMULIS, (Correlated Multimodal Imaging in Life Sciences) that aims to fuel collaborations in the field.
“We put this book together almost as a political tool to ask people, ‘OK, where is this field going and where do we need to invest?’,” says Verkade.
Areas ripe for development include correlative imaging with force microscopy and soft X-ray tomography, volume electron microscopy methods, and as always, the perfect CLEM probes. 
Verkade is also keen to highlight data issues and the importance of investing in people.
“One of our biggest challenges is going to be the amount of data we generate and this is where quite a bit of future investment should be taking place,” he says. “Investment in people is equally important – it’s not good enough to buy the machines, we need the people too.”
So with people in mind, what does Verkade see as being important to the microscopists of tomorrow?
“I’m actually a tutor to our first year biochemistry students, and they ask me questions about all kinds of things,” he says. “I always say, follow your own path, and don’t follow the crowd – it’s worked well for me.”
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