Lorne special: Cell polarity and the T-cell

This feature appeared in the January/February 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.

Sarah Russell describes herself as a hard-core biologist of the signalling variety. She spent her early career doing straight biochemistry to get a handle on the signalling pathways that make lymphocytes - specifically T cells - do their stuff.

However, in recent years, Russell has seen the light. Literally. She now spends much of her time addressing the same core biological questions but through the lens of a microscope. And it’s certainly one of the best times in the history of microscopy to be just that, with new advances in fluorescence and laser capabilities for viewing and analysing cells appearing almost monthly.

The change in Russell’s experimental approach to T cell research really kicked off about a decade ago. After a highly successful postdoctoral stint at the National Institutes of Health (NIH) in the U.S. working on the pathways that control IL2-receptor signalling to mediate the immune response, she returned to Australia and started looking for different ways to further understand these immune signalling pathways and complexes.

“I had a fabulous time at the NIH, identifying players in the IL2-receptor complexes and so on. But at the end of my postdoc we had a large collection of proteins, and couldn’t really work out what was doing what. Then I realised that simply finding out what sticks together, from a biochemical viewpoint, was not enough to elucidate the signalling pathways.”

Once back in Australia, Russell became interested in the idea that cellular context and compartmentalisation were going to be defining factors in the control of signalling responses. In other words, she needed to find out exactly where, and with what other proteins, her proteins of interest reside in the cell at any given stage in the T cell’s many functions.

She also realised that microscopy was really the only way to look at the pathways and components that she needed in the context of cell compartments. One of the first technologies that piqued her interest for investigating the bank of candidate protein-protein interactions emerging from her biochemical and molecular biology data was Fluorescence Resonance Energy Transfer, or FRET microscopy. FRET gives information about whether two or more fluorescently labelled proteins that are close together in a cell might actually be interacting.

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“At that time, I was at the Austin Research Institute in Melbourne, and we didn’t have the facilities to do FRET. So I went looking around for somebody who could help me.”

That quest brought her to a talk describing multiphoton laser imaging given by a student of Professor Min Gu, who is the current director of the Centre for Microphotonics at Swinburne University, in Melbourne. Russell was immediately hooked.

“I realised the potential power of laser technology for addressing the kinds of biological questions I have always been interested in and, soon after, I started working with Min’s group.”

Even after Russell moved from the Austin Research Institute to the Peter MacCallum Cancer Centre in 2000, her relationship with Swinburne grew to become a second home, and eventually, a flourishing cooperative venture. “Basically, the physicists at Swinburne enable me to do lots of wonderful and different things to address the core biological issues I am interested in.”

Russell has since taken this collaborative esprit to the next level by formally establishing a second research group at Swinburne University of Technology in Melbourne, in addition to her existing and well-established lab at Peter Mac. She even has an ARC Future Fellowship that is centred on the partnership between the two institutions.

Russell is now officially part of the Centre for MicroPhotonics at Swinburne and heads an already sizeable joint initiative between there and Peter Mac. The idea is to develop new imaging technologies in-house such as multiphoton intravital microscopy, microfabrication for live imaging, and laser tweezers, and then apply these technologies to Russell’s ongoing studies into T cell lymphocyte function.

According to Russell, the process of forming the collaborative venture was not instant. “But with a lot of commitment on both sides, including trust between the two different institutions – which is no small thing, involving arranging truly joint appointments, sharing IP etc. – both parties are coming to realise the enormous benefits that could go both ways. I think that we are now to the point where everybody is very positive about it.”

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Polarity and Scribble

Russell defines her major biological question as being how cell compartmentalisation and polarity impacts on T cell fate. In more general biological terms, how cell polarity describes the programmed movement of cellular components and machinery to set up specialised cell regions or surfaces. This process is crucial for normal differentiation, proliferation and development of all cellular organisms, and dysregulated polarity can cause developmental disorders and cancer.

In immune cells, such as T lymphocytes, polarity describes the means by which that cell reorganises its cellular components and functions to adapt to its environment, be it to migrate towards an incoming signal, reorganise its internal transport system to respond to that signal, or marshal the appropriate signalling troops to be in the right place at the right time.

Thus polarity dictates T cell shape via the selective recruitment of molecules to different regions of the cell and is integral to every aspect of T cell function, from migration to cytotoxicity.

“For us, one of the most interesting aspects of T cell polarity – and the thing that is unique about the polarity proteins compared to other sorts of signalling players – is that they antagonise each other,” Russell explains. It seems that these polarity proteins actually push each other away in ways that are tightly regulated and highly related to the functional compartmentalisation of T cells. But the details of how this occurs remains largely a mystery to Russell and others in the field. “That is what I would really like to understand, and I hope that some of our new technical approaches will help us get there.”

Russell is hoping to illuminate some recent intriguing results concerning her favourite family of polarity proteins, containing members such as Scribble, Par, Discs large and Lethal giant larvae.

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“Up until recently, a lot of the ideas about polarity came from Drosophila experiments and was mostly about apicobasolateral polarity (typical of epithelial cells), which is really fairly static and involves two cell ends that are quite different from each other.”

The thought was always that polarity proteins push each other to opposite ends of the cell. “But we found that in the more dynamic T cells, you quite often see these antagonistic polarity proteins transiently together in the same site, such as at the leading edge of a migrating T cell or at the contact edge with the antigen-presenting cell [APC].”

So, a key question is: Are these supposedly antagonistic proteins clustering together because they are now functioning cooperatively, and these proteins can transiently swap from antagonising to cooperating? Or are they still performing the same antagonistic function but doing it in separate regions or microdomains? It’s the latter option that Russell is leaning towards.

It is clear from other cell types that many different functions happen at the same time at the leading edge of a migrating cell, such as a fibroblast, and so it makes sense that in all of these morphological reorganisations, there are many smaller, but specific, sites where these different processes and interactions are going on within the one region of the cell.

“I suspect that the Par and Scribble complexes are important components of these microdomains in T cells for different functional activities in the one region,” she says. “Of course, there will be many factors and controllers involved, but I think that the unique antagonistic abilities of these polarity proteins are probably a pre-determining step in a lot of these processes and location-specific interactions. So, answering these types of big questions really needs the newer approaches in microscopy to see it all a lot better, and that is where my colleagues at Swinburne really come into their own.”

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Herding cells

“Some of what we do together involves microfabrication to facilitate the live imaging of our T cells over long periods of time,” explains Russell when giving examples of what her partnership with the group at Swinburne has yielded.

“One thing that the physicists at Swinburne recently created are these T cell traps, called cell paddocks, which are basically an array of walls about 60 microns high and arrayed in two dimensions on a microscope slide to form grids about 100 micron square. You seed your cells at the bottom of this chamber slide where they settle and basically stay inside the walls.”

This was an attempt to counter one of the major problems with imaging cells like lymphocytes, which are notorious for moving around very fast and clumping, both of which are the natural enemies of time-lapse microscopy. Using these cell paddocks, T cells are kept happily in normal growing conditions and in contact with their lymphocyte companions via contiguous medium, but they do not go anywhere.

“We can do experiments in the paddocks, such as placing an antigen presenting dendritic cell with a T cell and watch what goes on over multiple generations, holding the cells in place without disrupting their biology,” says Russell. “What we dream about doing next is to combine the paddock chambers with microfluidic technology and so be able to shunt cells and reagents such as siRNAs in and out of the cell environment. This really is an incredibly enabling technology and has absolutely changed what we can do. Compared with the sophisticated structures our Swinburne collaborators can design and make, these paddocks were relatively simple, but for biologists, it is magic,” she says.

“The other really exciting thing about our partnership is that with all the recent advances in fluorescence microscopy and laser technology – and the know-how at Swinburne – we can basically get any kind of microscope built that we need. We just have to work out just which approach is best for addressing our questions.”

One project that is definitely going ahead is to build a super-resolution photo-activated localisation microscopy (PALM) microscope.

What Russell would ideally like to do is combine the super-resolution microscopy with some of the new techniques in correlation spectroscopy, and specifically one called raster image cross-correlation spectroscopy, or RICCS.

“The idea would be to image your cell pixel by pixel and eventually measure the fluctuations in fluorescence signal across the entire cell. This gives you a sense of molecular diffusion. So, how much and where your protein or proteins of interest have diffused over a given time. For instance, you can monitor one part of the cell compared to a different compartment or area of the cell and trace your proteins of interest in and/or out. Then, of course, once you start doing that kind of thing, you could plug other players into the system known to activate certain T cell functions or states and really start to find out what goes where and when and why, and of course capture it all on film.”

This feature appeared in the January/February 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.