How does a cell swim fowards?

“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

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.

Looking at the structure inside cells

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

Cheeky

Human cheek cells are a classic subject of school microscopy. It is easy to collect some by gently scraping the inside of your cheek. This is a high resolution phase contrast image of one of my cheek cells, put together using focus stacking of a 4 by 4 montage of 57 focus slices using one of my ImageJ macros. The detail of the nuclear structure, the granular contents of the cytoplasm and the structured surface of the cell really jump out.

This cell is quite large for mammalian cells, about 75 μm across, and around 10 times larger than the single cell Leishmania parasite I currently do much of my research on. If you have sharp eyesight you can even see human cheek cells by eye (although only just) when they are spread on a slide.

Like most mammalian cells, cheek cells are essentially transparent. If you use a microscope in the most basic way, essentially as a giant magnifying glass, shining light straight through the sample towards your eye, you see something like this:

Bright field micrograph of a human cheek cell.

This picture has even had the contrast artificially enhanced. Practically it is tough to even find the cells on the slide and get them in focus!

For many years the best alternative was oblique or dark field microscopy. Here you deliberately avoid shining light straight through the sample, and instead make sure that only light scattered by structures in the sample can collected by the objective lens and get up to your eye.

Dark field micrograph of a human cheek cell.

Images by dark field microscopy can be hard to interpret, and are typically limited to fairly low resolution.

More complex methods based on interference of light travelling through the sample were developed in the 20th Century. These methods, phase contrast and differential interference contrast, were a revolution. They allowed completely new approaches for looking at the biology of cells, particularly live cells and dynamic processes like cell division. They were such a revolution that the inventor of phase contrast microscopy, Frits Zernik, was awarded the Nobel prize in Physics in 1953 for this work.

Phase contrast micrograph of a human cheek cell.

DIC micrograph of a human cheek cell.

It was not until the development of the famous green fluorescence protein, for fluorescence microscopy in live cells in the 1990s, that there was another discovery which improved the capacity for live cell microscopy to the same extent as phase contrast and DIC.

Software used:
ImageJ

Micro 3D Scanning - 1 Focal Depth

3D scanning is a very powerful tool, and it's value isn't limited to the objects and scenes you interact with in everyday life. The ability to precisely determine the 3D shape of tiny (even microscopic) objects can also be really useful.

 The 3D reconstructed shape of a tiny (0.8 by 0.3 mm) surface mount resistor on a printed circuit board. This was made using only a microscope; no fancy laser scanning required!

3D scanning through a microscope is a bit different to normal 3D scanning; mostly because when you look down a microscope at an object it looks very different to what you might expect from day-to-day life. The most immediately obvious effect is that out of focus areas are very out of focus, often to the point where you can barely see what is there. This effect comes down to the angle over which light is collected by the lens capturing the image; your eye or a camera lens in everyday life, or an objective lens when using a microscope.

Three images of the surface mount resistor. The three pictures are taken at different focus distances so different parts of the image are clear and others blurred. The blurred parts are very blurred!

In every-day-life when using a camera or your eyes distance from the lens to the object is normally long, it may be several metres or more. As a result the camera/your eye only collects light over a small of angle, often less than one degree. In comparison microscopes collect light from an extremely large range of angles, often up to 45 degrees. The angle must be this large because the objective lens sits so close to the sample. A wider angle of light collection makes out of focus objects appear more blurred. In photography terms the angle of light collection is related to the f-number, and large f-numbers (which have a large angle of light collection) famously have very blurred out of focus portions of the image.

The upshot of this is that in a microscope image the in focus parts of an image are those which lie very near (often within a few micrometers) to the focal plane. It is quite easy to automatically detect in focus parts of an image by using local image contrast (this is actually how autofocus works in many cameras) to map which parts of a microscope image are perfectly in focus.

In this series of images the most in-focus one is image 6 because it has the highest local contrast...

 ... using edge detection to emphasise local contrast in the image really highlights which one is perfectly in focus.

In this series of images the most in-focus one is image 55 instead.

The trick for focus 3D scanning down a microscope is taking the ability to detect which parts of an image are in focus, and using this to reconstruct the 3D shape of the sample. Going to the 3D scan is actually really easy:

1. Capture a series of images with the focus set to different distances.
2. Map which parts of each of these images are perfectly in focus.
3. Translate this back to the focus distance used to capture the image.

This concept is very simple; if you know one part of an object is perfectly in focus when the focus distance is set to 1mm, that means it is positioned exactly 1mm from the lens. If a different part is perfectly in focus when the focus distance is 2mm, then it must be positioned 2mm from the lens. Simple!

It may be a simple idea, but this method gives a high quality 3D reconstruction of the object.

The reconstructed 3D shape of the resistor, using 60 images focused 0.01mm apart, mapped to a depth map image. The lighter bits stick out more from the surface, and the darker bits stick out less.

Using the depth map to reconstruct the resistor reconstructed in full colour in 3D! Pretty cool for something less than 1 mm long...

Does that seem impressive? Then check out the videos:

A video of the original focus series of images captured of the resistor.

The reconstructed 3D shape.

A 3D view of the resistor, fully textured.

This approach is, roughly speaking, how most 3D light microscopy is done in biological and medical research. It is very common practice to capture a focal series like this (often called a "z stack") to get this 3D information from the sample. 3D imaging is most useful in very thick samples where you want to be able to analyse the structure in all three dimensions, an example might be analysing the structure of a tumour. My research on Leishmania parasites inside white blood cells uses this approach a lot too. The scanning confocal fluorescence microscope was actually designed to maximise the value of this 3D effect by not only blurring out of focus parts of the image, but also eliminating the light all together by blocking it from reaching the camera.

Software used:

ImageJ: Image analysis.

Blender: 3D viewing and rendering.

Cell Biology of Infectious Pathogens - Ghana 2013

For the last four years there has been a cell biology workshop in West Africa, organised by Dick McIntosh, an intense two week course aiming to help young African scientists around the master's degree stage of their careers. This course ran again this year, and was the first organised by Kirk Deitsch (malaria expert and a regular from the previous courses) and I was fortunate enough to be invited to teach the trypanosome half of the course. For its fifth incarnation the course returned to a location where it has previously been held, the Department of Biochemistry, Cell and Molecular Biology in the University of Ghana, and was organised with Gordon Awandare.

[Interactive 360° Panorama]

The Department of Biochemistry, Cell and Molecular Biology - University of Ghana, Accra.

The focus this year was teaching basic cell biology and the associated lab techniques, emphasising how this helps understand and fight some of the major parasitic diseases in Africa: African trypanosomiasis (sleeping sickness), leishmaniasis and malaria. All three of these diseases impact Ghana and the surrounding countries and these diseases are of enormous interest to students embarking on a scientific career in Africa.

[Interactive 360° Panorama]

The teaching lab in The Department of Biochemistry, Cell and Molecular Biology.

Of the three diseases we were teaching about malaria is by far the most well known, both locally and internationally. It is caused by Plasmodium parasites (which are single cell organisms) which force themselves inside the red cells in the blood to hide from the host immune system. Malaria is often viewed as the iconic neglected tropical disease, however in the last 10 years or so the understanding of the disease and efforts to find a vaccine and new drugs has improved vastly. Unfortunately it is still very common (we had one case in the participants on the course in the two weeks), drug resistance is rising, and it places a huge cost and health burden on the affected countries. It also impacts a huge area; almost all of sub-Saharan Africa is at risk.

Looking at Leishmania. One of the lab practicals was making light microscopy samples from non-human infective Leishmania using Giemsa stain.

Leishmaniasis and trypanosomiasis are caused by two related groups of parasites, Leishmania and trypanosomes (also single cell organisms), and if malaria is a neglected tropical disease the these are severely neglected tropical diseases. The two parasites live in different areas in the host, trypanosomes swim in the blood while Leishmania live inside macrophages, a type of white blood cell that should normally eat and kill parasites. In comparison to malaria fewer drugs are available, the drugs are less effective and several have severe side effects. Even diagnosis is thought to often be inaccurate. The impact of these diseases is less than malaria; human trypanosomiasis is thought to be relatively rate and leishmaniasis is confined to a semi-desert band just to the south of the Sahara. Trypanosomiasis does have a huge economic impact though, as it infects cattle and prevents milk and meat production, and cases of leishmaniasis are probably under-reported.

Staining trypanosomes. One practical was making immunofluorescence samples. In this sample the flagellum of trypanosomes was stained fluorescent green using the antibody L8C4.

So what did we teach?

The teaching was a mixture of lectures, small group discussions, lab practicals and lab demonstrations and we taught for 14 hours a day for 11 days; we could cover a lot of material! All the teaching was focused on linking basic cell biology to parasites and to practical lab techniques. Topics taught included how parasites avoid the host immune system, molecular tools to determine parasite species, light microscopy techniques, using yeast as tool to analyse cell biology of proteins from other species, host cell interaction of parasites, and many more.

Detecting human-infective trypanosomes. This gel of PCR products shows whether the template DNA was from a human-infective or non-human infective subspecies of T. brucei. If there was a DNA product of the correct size (glowing green) then the sample was human-infective.

A great example of how all the teaching tied together was polymerase chain reaction (PCR) to determine species. Human-infective trypanosomes have a single extra gene which lets them resist an innate immune factor in human blood which would otherwise kill them, and I taught about why this is important for understanding the disease and how it was discovered. This gene can be detected by PCR, and this technique is used to tell if a particular trypanosome sample could infect people. We ran a practical actually doing this in the lab.

PCR is a simple, adaptable and easy technique for checking any parasite for a particular species-defining or drug resistance gene, and we also taught how to use online genome sequence data to design PCR assays. We even worked through PCR assay design for many individual participant's personal research projects, really transferring the skills we were teaching to their current research. Finally we looked at papers using PCR techniques to critically analyse the experiment and assay design to help people avoid pitfalls in their own work.

This was a great demonstration of how basic cell biology and lab techniques can have real practical application with medical samples and help with surveillance of a disease. We designed all of the teaching to have this kind of practical application.

7x speed timelapse video of fish melanophores responding to adrenaline.

One practical with massive visual impact was the response of fish melanophores to adrenaline/epinephrine. Fish normally use these cells to change colour in response to stimuli and melanin particles (melanosomes) inside specialised cells (melanophores) run along the microtubules which make up a large portion of the cell cytoskeleton. We used it to demonstrate signalling; adrenaline can be used to stimulate movement of the melanosomes towards the centrosome.

This is really flexible experimental system for demonstrating the functions of the cytoskeleton, motor proteins and signalling pathways because the output (movement of the pigment particles) is so easy to observe with a cheap microscope or even a magnifying glass. This experiment was particularly chosen as it is a useful and accessible teaching tool for cell and molecular biology, and many of the course participants had teaching obligation in addition to their research.

Western blots in Western Africa. 100x timelapse of loading and running a SDS-PAGE gel.

We aimed to cover all the major molecular and cell biology techniques and had practicals doing microscopy, immunofluorescence, growing a microorganism (in this case yeast), PCR, agarose gel electrophoresis SDS-PAGE and Western blotting. The yeast practicals were particularly cool; using genetically modified cell lines the students analysed the function of p53, a transcription factor with a major role in recognising genetic damage and avoiding cancer, and how well it promotes transcription from different promoter sequences. These practicals taught growing yeast, temperature sensitive mutants, several types of reporter proteins in yeast and Western blotting, all concerning a transcription factor with huge clinical relevance in cancer!

Exploring DNA and protein structures through PyMol in a bioinformatics session.

Practicals weren't just limited to lab practicals though. We also ran interactive bioinformatics sessions looking at the kinds of data which are freely available in genome and protein structure databases online. These were also very popular, especially as so much data is available for free online.

All in all the course was a great success. The participants were all extremely enthusiastic, hard working and scarily smart! Feedback so far has also been very positive. I feel that courses like this can have a huge impact on the careers of young African scientists, and I sincerely hope that funding can be secured to continue running this type of course in the future.

You can also read more about this course at the ASCB website.

Software used:

ImageJ: Image processing and timelapse video creation.

Tasker for Android: Timelapse video capture.

Pymol: Protein structure analysis

Cell Picture Show

One of my scanning electron microscope images from my research into the division of Leishmania parasites is currently featured in the Cell journal picture show on parasites and vectors. You can check it out (image number 11) here.

A montage of seven scanning electron micrographs of L. mexicana.

"Leishmania mexicana promastigotes, which normally inhabit the gut of the sand fly vector, are shown at different stages of the cell cycle. Cells are arranged by cell-cycle progression, increasing in a clockwise direction. L. mexicana mainly causes a mild form of cutaneous leishmaniasis, forming ulcers at the bite site of an infected sand fly."

Software used:

ImageJ: Micrograph processing