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Looking at the structure inside cells

February 16, 2016, 7:51 am
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How complex and structured is the inside of a cell? It's hard to imagine, but the internal organisation of cells is typically precisely controlled by molecular skeletons and scaffolds, giving cells the shape they need to function.

We can discover the 3D organisation of the inside of cells using electron tomography; a process where you capture a series of images with an electron microscope, with the sample tilted at a slightly different angle for each image. This can then be used to calculate the 3D shape of the sample, using the same maths as for an X-ray CT scan.

Leishmania parasites are exquisitely structured. While they are only 2 micrometres wide (100 would fit across a human hair) they have a precise internal organisation which they faithfully replicate each time they divide. One of the distinctive parts of this organisation is the flagellar pocket, where the cell membrane folds in on itself at the base of the whip-like flagellum that the cell uses to swim.

In my latest paper, "Flagellar pocket restructuring through the Leishmania life cycle involves a discrete flagellum attachment zone", I used electron tomography to reconstruct the three-dimensional organisation of the Leishmania flagellar pocket. The structure in this area of the cell is incredible, and the journal picked a rendering of it for the cover image.


Volume covered in this 3D reconstruction is only 3 by 2 by 1 micrometres, about the size of a typical bacterial cell, but has enormous complexity. I have shown the microtubules (which make up most of the cytoskeleton) in red and membranes in blue. Each microtubule is only about 5 molecules wide, and is about 10,000 times narrower than a human hair! Some other specialised parts of the cytoskeleton are in green.

You can download the paper for free here to take a look at the structures in this area of the cell in more detail.

Software used:
IMod: Electron tomography structure
Blender: Tidying and rendering of the 3D structure
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TrypTag.org

September 5, 2016, 5:24 am
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The website for one of my new major research projects is now live!

TrypTag.org



TrypTag is a project to tag every gene in the trypanosome genome with a fluorescent marker to see where it goes in the cell.

Do you have no idea what I'm talking about? Read on to see what all that jargon means!

Trypanosomes are one of the parasites my research involves. They are single cell parasites that live in the blood, and they cause the diseases sleeping sickness in humans and nagana in livestock across Africa. All in all, not very nice.

Like all cells, trypanosomes are made up of protein machinery. Each protein is encoded by a gene in genome. A first step in finding a protein's function is finding where it goes in the cell. If you can map it to a particular structure then you have a good idea it's going to function there too.

To find where a protein goes in a cell we genetically modify the cell, sticking a fluorescent marker to the protein so we can see where it goes using a microscope. This is the process of tagging.

We are going to tag every protein gene in the genome, around 8000 genes, and build a complete map of the protein composition of the cell.

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Molecular Cell Biology of Protozoan Parasites - Ghana 2017

January 28, 2017, 8:35 am
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Things change fast in Ghana! Three years ago, I helped teach a course for young African scientists in the University of Ghana in Accra. This January I got the chance to do the same again, and it has been fantastic. The University of Ghana is becoming a centre of great science in West Africa, with huge contributions by Gordon Awandare and his West African Centre for Cell Biology of Infectious Pathogens (WACCBIP) course, funded by the World Bank.


This time around, our teaching course was made possible by our TrypTag project, funded by the Wellcome Trust. We applied for extra funds so we could transfer the genetic engineering technologies we developed for TrypTag into the hands of African researchers; to get the research techniques to the parts of the world that really suffer from parasitic diseases.

Our course focused on tools for analysing parasites and what makes them tick, particularly using genetic tools. We mostly looked at Plasmodium (malaria), Trypanosoma (sleeping sickness) and Leishmania (leishmaniasis). To teach the malaria side of the course we had the excellent Kirk Deitsch (Cornell University, New York) and Oliver Billker (Sanger Institute, Cambridge). On the trypanosome and Leishmania side we had Keith Gull (University of Oxford) and Sue Vaughan (Oxford Brookes University), along with the TrypTag team: Jack Sunter, Sam Dean and me!

So what was the course all about?

We focused on the tools to help young African scientists (starting their Master's or PhDs) take control of their research - from learning about free genome data and bioinformatics experiments, to computational and genetic tools to make discoveries about parasite biology.

A major part of the course was tools for handling DNA: PCR for detecting genes in a sample, amplifying DNA to clone it into a plasmid, and working with software (ApE) to design cloning strategies for gene tagging, deletion and RNA interference/siRNA knockdown. Teaching how to design a PCR or cloning experiment, rather than just teaching how to do the experimental technique, was very popular.



We also taught how to use the sequence resources you need for working with DNA: How to get the most from genome databases, like PlasmoDB for malaria and TriTrypDB for trypanosomes and Leishmania. The course also covered bioinformatics experiments, thinking how to test a biological hypothesis using existing data from genome data. The students quickly recognised the power of this approach, particularly given genome sequence resources are free! Many were immediately applying these ideas to their areas of research.



We made sure there was a big push towards critical thinking. The student's loved critical reading of articles in the journal clubs, and thinking about how to apply this critical assessment to their own experiments to make them as good as possible.


We also tried, for the first time ever, using the TrypTag.org website as the start point for a bioinformatics experiment. The students were challenged to start with a protein localisation patterns to identify protiens likely involved in particular aspects of parasite energy metabolism, then test whether any of these were unique to trypanosomes making them a potential drug target.



This was the perfect stress test for the new TrypTag.org website and server. It coped with up to a page view per second, and downloads of 10 images per second, with no problems. All over a slightly unreliable internet connection in Ghana! Many thanks to the scientific computing at the Sir William Dunn School of Pathology in Oxford for helping make this happen.


Overall the course was a great success, with very positive feedback from the students and local research staff. The students were smart, engaged and hard-working. It will be exciting to see what these young people can achieve over the next few years.
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Moving in a straight line - sounds simple, right?

February 2, 2017, 12:02 pm
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One hundred years ago Asa Schaeffer blindfolded his friend and challenged him to walk in a straight line. He did three loops of a spiral, before tripping over a tree stump. This wasn't a cruel prank, it was an experiment, and all people are surprisingly bad at this simple challenge.




More modern experiments showed it is lack of external reference points that trips people up. Blindfolded in a desert? You walk in circles. Not blindfolded in a desert? Straight lines. Forest on a sunny day? Straight lines. Forest on an overcast day? Circles. Current Biology, DOI: 10.1016/j.cub.2009.07.053

People need some external reference point, a distant hill, the sun, or even the direction of shadows, to manage a straight line. Why they drift into circles without a reference isn't clear. Is it asymmetric leg strength? A handedness bias? Or some psychological miss-correction? What is clear is that it is a universal problem.

Navigation without reference points is always difficult. This is why spin stabilisation is widely used to stop flying objects, from bullets to rugby balls, curving off course in the air. Even advanced tools like inertial navigation systems, which use dead reckoning from measuring acceleration, always drift off course. 

So how about swimming cells? Many swimming cells and microorganisms have the ability to swim in a straight line. Most have no ability to look at a reference point, they can only perceive the liquid they are in immediate contact with. They are also typically top small to be affected by gravity, so have no way of using up or down for reference. They are essentially blind.

The trick for straight line swimming in cells seems to be some kind of spin stabilisation. Way back in 1901, H.S. Jennings noted that many microorganisms spin as they swim, and thought this could be a spin stabilisation somewhat like a spinning bullet. The problem is it can't be. Rugby balls, bullets and spacecraft use spin stabilisation which depends on rotational inertia to keep them spinning and stable, similar to a spinning top. If you made a top that was the size of a cell, and span it in water, it would stop spinning immediately; there is too much friction. The American Naturalist, 35(413):369-379


It turns out the mechanism is just geometry. A person walking is a back-forward/left-right, a 2D, situation. If you curve the walking path it makes it into a looping circle. For a cell swimming there is another direction to think about; in 3D there are two ways to curve the swimming path. The first which curls the path into a circle, and a second which twists the circle to elongate it into a helix-shaped path. Elongate the helix far enough and it turns into a straight line, with the cell rotating as it swims.


The interesting property of the helical swimming paths is their stability. If a cell deliberately twists its swimming path into a helix then small asymmetries (the cellular equivalent of having one leg stronger than the other) won't bend the swimming path into a circle. Instead it just slightly alters the shape of the helix. It can ensure the cell swims in a dead straight line.

My latest paper is all about how cells manage straight line swimming, looking at trypanosome and Leishmania human parasites. Each aspect of swimming has been looked at before, but I believe has never previously been put together as a full story: from mutants with altered cell shape (to add more or less twist), measuring the effect on swimming, and matching this to simulation of how cells achieve their straight line swimming. PLOS Computational Biology, DOI: 10.1371/journal.pcbi.1005353

There are still big unanswered questions though: Why do parasites need to swim in a straight line? And what are they swimming towards? This is an area of active research, with several major trypanosome research groups (especially Kent Hill and Markus Engstler) interested in addressing these questions.
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How to be a parasite

October 30, 2017, 10:21 am
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For an organism to become a parasite it has to adapt to live in a host. This might mean it needs to grow faster, invade cells or tissues, or avoid being killed by the immune system. It can also 'forget' how to live outside a host. It can forget how to search for food, how to survive the cold, and how to avoid drying out.

We can learn how parasites adapt to infect hosts by looking at the nearest free living relatives. What had the parasite lost relative to the free living cousin, because it just doesn't need it to survive in a host? Or vice versa, what has it kept because it's useful for infecting a host?

I've looked at this question for trypanosomatid parasites. They are protozoan (single cell) parasites, like the malaria parasite, and cause several deadly tropical diseases.

A few hundreds of millions of years ago, they weren't parasites at all. The ancestors of these parasites were free living, probably swimming in ponds, lakes and seas. Then, at some point, some evolved the ability to infect insects.

Millions of years passed. Then, several tens of millions of years ago, some managed to get transmitted from their host insect into a vertebrate host. And, most importantly, they survived and could get transmited back to the fly.

This adaptation to infect animals probably happened on three separate occasions. Those parasites kept evolving and adapting, and are now the three human trypanosomatid-caused diseases: Sleeping sickness, Chagas disease and leishmaniasis.

My most recent paper is all about this. Julius Lukeš has discovered a species of trypanosomatid that only infects insects, and looks like it hasn't changed much from the first ancestor ever to infect insects.



With Tomáš and Eva, we looked at what this species tells us about how these parasites adapted to infect flies: How does the cell grow, adapts its shape, and stick to surfaces? How does its internal organisation adjust to allow these changes? How does its metabolism change for different energy sources? And, how has the genome, which encodes the proteins that drive these functions, changed to achieve this?



Using this information, we could then get insights into what is important for human infective parasites. What aspects of shape, structure and metabolism adaptations have they 'forgotten'? And which have they kept? The ones they have kept are the ones important for infecting, and killing, people.

Want to read more? You can get a copy of the paper from my website: richardwheeler.net

Skalický T*, Dobáková E*,1, Wheeler RJ*, Tesařová M, Flegontova P, Jirsová D, Votýpkaa J, Yurchenkoa V, Ayalag FJ, Lukeš J (2017) "Extensive flagellar remodeling during the complex life cycle of Paratrypanosoma, an early-branching trypanosomatid"PNAS doi:10.1073/pnas.1712311114
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Impossible Pipes

May 1, 2018, 2:36 pm
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How happy are your HeLa cells?

September 14, 2018, 6:43 am
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Figure 1. HeLa cell nucleus happiness scale. HeLa cells expressing FUS eGFP were asked how they felt. The morphology of the nucleus and nucleolus, visible via FUS eGFP localisation, was used to infer happiness. The response was ranked by two independent experts, and used to build a HeLa happiness scale.
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How does a cell swim fowards?

September 18, 2018, 2:02 am
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“Why are they swimming backwards?” This is one of the most common questions I get asked whenever I show a video of Leishmania parasites swimming.

Swimming Leishmania at 200 frames per second (8× slower than actual speed)

If you look at Leishmana with a high speed video it's easy to see they tend to swim 'tail-first', with the flagellum sticking forward into the direction of travel. Sperm, probably the best-known swimming cells, do the opposite, and swim 'head-first'.

Sperm on the left, Leishmania on the right

Of course, if you could ask the Leishmania, they'd say that they're swimming forwards and it's the sperm which are swimming backwards. This raises the question of how does a swimming cell decide which direction it should swim? Which direction is forwards?

The direction a cell swims depends on the waves which travel down the flagellum. If they start at the base and go towards the tip then the cell will swim head-first. If they start at the tip and go towards the base then the cell will swim tail tail first. So how do they choose where the waves start?

We answered this question taking advantage of a useful behaviour of Leishmania. If you look closely at the video above you can see one cell is swimming forwards (the cell on the left) and the other is turning on the spot (the cell on the right). And if you look very closely you'll see that the waves in the cell on the left start at the flagellum tip, and the waves in the cell on the right start at the base of the flagellum.


Tip-to-base on the left and base-to-tip on the right.

This let us use genetic tools to try to break one direction of flagellum wave without affecting the other and tease apart how the flagellum movement might be chosen by the cell. To cut a long story short, we found differences in the motor proteins between the base and tip which seem responsible. Interestingly, similar differences turn up in many organisms, including human sperm, suggesting it might be a general pmechanism. You can read all about this in our recent paper: At the PNAS website or as a PDF.

https://doi:10.1073/pnas.1805827115
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Tryp/Leish Stickers!

October 15, 2019, 4:09 am
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It's been a busy year starting my lab... But in more important news, T. brucei, T. cruzi and Leishmania stickers!

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Trypanosomatid cell structures

May 25, 2021, 11:01 am
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These are my oft-requested diagrams of trypanosomatid parasites. They're heavily inspired by the amazing illustrations of trypanosomatids by Keith Vickerman, like this classic, though with a more modern twist.

Both aim to be fully accurate diagrams, targeting a specialist text book or review paper level. They're drawn based on on lots of papers, both by others and by me, plus many hours on electron microscopes looking at them! They are slightly 'cartoony' though, exaggerating the size of some of the organelles/structures for clarity.


Trypanosoma brucei procyclic trypomastigote form - as found in the tsetse fly midgut. This diagram is featured in my annual review with Keith Gull and Jack Sunter: Coordination of the Cell Cycle in Trypanosomes

Leishmania mexicana procyclic promastigote form - as found in the sandfly midgut. Drawn in the same style as T. brucei, but not published.

Trypanosomatid parasites are wonderfully structured and intrinsically beautiful cells. They've been the subject of many of my illustrations over the years, you'll find them around, and give a track record of my illustration skill... for better or for worse.

Please ask before using these - they represent a big investment of time - but they are made to be used and belong in lecture slides! Drop me a message if you need really high resolution versions, vector versions (pdf/svg), etc.
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