I Know What I Like

 

I guess most of us at some time or other will have stood gazing at a painting for a while before muttering ‘Wow, that’s awesome’ or words to that effect if we’re not into the modern argot. Some combination of subject, style and colour has turned our crank and left us thinking we wouldn’t mind having that on our kitchen wall.

Given the thousands of years of man’s daubing and the zillions of forms that have appeared from pre-historic cave paintings through Eastern painting, the Italian Renaissance, Impressionism, Dadaism and the rest to Pop Art, it’s amazing that everyone isn’t a fanatic for one sort or another. The sane might say the field’s given itself a bad name by passing off tins of baked beans, stuff thrown at a canvas and unmade beds as ‘art’ but, even so, it seems odd that it remains a minority obsession.

Can science help?

Science is wonderful, as we all know, but the notion that it might arouse the collective artistic lust seems fanciful. Nevertheless, unnoticed by practically everyone, our vast smorgasbord of smears has been surreptitiously joined over the last 30 years by a new form: an ever-expanding avalanche of pics created by biologists trying to pin down how animals work at the molecular level. The crucial technical development has been the application of fluorescence in the life sciences: flags that glow when you shine light on them and can be stuck on to molecules to track what goes on in cells and tissues. The pioneer of this field was Roger Tsien who died, aged 64, in 2016.

Because this has totally transformed cell biology we’ve run into lots of brilliant examples in these pages — recently in Shifting the Genetic Furniture, in Caveat Emptor and John Sulston: Biologist, Geneticist and Guardian of our Heritage and in the use of red and green tags for picking out individual types of proteins that mark mini-cells within cells in Lorenzo’s Oil for Nervous Breakdowns.

To mark the New Year this piece looks at science from a different angle by focussing not on the scientific story but on the beauty that has become a by-product of this pursuit of knowledge.

Step this way: entrance free

So let’s take a stroll through our science gallery and gaze at just a few, randomly selected works of art.

  1. Cells grown in culture:

This was one of the first experiments in my laboratory using fluorescently labelled antibodies, carried out by a student, Emily Hayes, so long ago that she now has a Ph.D., a husband and two children. The cells are endothelial cells (that line blood vessels). Blue: nuclei; green: F-actin; red: Von Willebrand factor, a protein marker for endothelium.

 

  1. Two very recent images taken by my colleague Roderik Kortlever of a senescent mouse fibroblast and of mouse breast tissue:

 

 

 

 

 

 

3. Waves of calcium in firing neurons:

One of my fondest memories is helping to do the first experiment that measured the level of calcium within a cell, carried out with my colleague the late Roger Tsien and two other friends. I only grew the cells: Roger had designed and made the molecule, quin2. We didn’t know it at the time but Roger’s wonder molecule was the first of many intracellular ‘reporters.’ Roger shared the 2008 Nobel Prize in Chemistry for his discovery and development of the green fluorescent protein with organic chemist Osamu Shimomura and neurobiologist Martin Chalfie.

This wonderful video of a descendant of quin2 in nerve cells was made in Dr. Sakaguchi’s lab at Iowa State University.

 

4. Calcium wave flooding a fertilized egg: Taro Kaneuchi and colleagues at the Tokyo Metropolitan University:

Click for a time-lapse movie of an egg cell that has been artificially stimulated to show the kind of calcium change that happens at fertilization. In this time-lapse movie the calcium level reaches a maximum signal intensity after about 30 min before gradually decreasing to the basal level.

 

5. The restless cell (1):

This movie shows how protein filaments in cells can continuously break down and reform – called treadmilling. Visualised in HeLa cells using a green fluorescent protein that sticks to microtubules (tubular polymers made up of the protein tubulin) by HAMAMATSU PHOTONICS.

 

6. The restless cell (2):

This movie shows how mitochondria (organelles within the cell) are continuously changing shape and moving within the cell’s interior (cytosol). Red marks the mitochondria; green DNA within the nucleus. HAMAMATSU PHOTONICS.

 

7. Cell division:

Pig kidney cells undergoing mitosis. Red marks DNA (nucleus); green is tubulin: HAMAMATSU PHOTONICS.

 

8. DNA portrait of Sir John Sulston by Marc Quinn commissioned by the National Portrait Gallery:This image looks a bit drab in the present context but in some ways it’s the most dramatic of all. John Sulston shared the 2002 Nobel Prize in Physiology or Medicine with Sydney Brenner and Robert Horvitz for working out the cell lineage of the roundworm Caenorhabditis elegans (i.e. how it develops from a single, fertilized egg to an adult). He went on to sequence the entire DNA of C. elegans. Published in 1998, it was the first complete genome sequence of an animal — an important proof-of-principle for the Human Genome Project that followed and for which Sulston directed the British contribution at the Sanger Centre in Cambridgeshire, England. The project was completed in 2003.

The portrait shows colonies of bacteria in a jelly that, together, carry all Sulston’s DNA. This represents DNA cloning in which DNA fragments, taken up by bacteria after insertion into a circular piece of DNA (a plasmid), are multiplied to give many identical copies for sequencing.

 

9. “Brainbow” mice by Tamily Weissman at Harvard University:

The science behind this astonishing image builds on the work of Roger Tsien. Mice are genetically engineered to carry three different fluorescent proteins corresponding to the primary colours red, yellow and blue. Within each cell recombination occurs randomly, giving rise to different colours. The principle of mixing primary colours is the same as used in colour televisions.  In this view individual neurons in the brain (specifically a layer of the hippocampus) project their dendrites into the outer layer. Other magnificent pictures can be seen in the Cell Picture Show.

It’s certainly science – but is it art?

A few years ago the Fitzwilliam Museum in Cambridge staged Vermeer’s Women, an exhibition of key works by Johannes Vermeer and over thirty other masterpieces from the Dutch ‘Golden Age’. I tried the experiment of standing in the middle of each room and picking out the one painting that, from a distance, most caught my amateur eye. Funny thing was: not one turned out to be by the eponymous star of the show! Wondrous though Vermeer’s paintings were, the ones that really took my fancy were by Pieter de Hooch, Samuel van Hoogstraten and Nicolaes Maes, guys I’d never heard of.

Which made the point that you don’t need to be a big cheese to make a splash and that in the new Dutch Republic of the 17th century, the most prosperous nation in Europe, there was enough money to keep a small army of splodgers in palettes and paint. Skillful and incredibly patient though these chaps were, they simply used the tools available to paint what they saw in the world before them — as for the most part have artists down the ages.

But hang on! Isn’t that what we’ve just been on about? Scientists applying enormous skill and patience in using the tools they’ve developed to visualize life — to image what Nature lays before them. So the only difference between the considerable army of biological scientists around the world making a new art form and the Old Masters is that the newcomers are unveiling life — as opposed to the immortalizing a rather dopy-looking aristocrat learning to play the virginal or some-such.

Controversial?

Not really. Let’s leave the last word to Roger Tsien. In our final picture there are eight bacterial colonies each expressing a different colour of fluorescent protein arranged to grow as a San Diego beach scene in a Petri dish. It became the logo of Roger’s laboratory.

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Now You See It

 

In the pages of this blog we’ve often highlighted the power of fluorescent tags to track molecules and see what they’re up to. It’s a method largely pioneered by the late Roger Tsien and it has revolutionized cell biology over the last 20 years.

In parallel with molecular tagging has come genetic engineering that permits novel genes, usually carried by viruses, to be introduced to cells and animals. As we saw in Gosh! Wonderful GOSH and Blowing Up Cancer, various ‘virotherapy’ approaches have been used with some success to treat leukemias and skin cancers and a trial is underway in China treating metastatic non-small cell lung cancer.

A major aim of genetic engineering is to be able to control the expression of novel genes (i.e. protein production from the encoding DNA sequence) that have been introduced into an animal — in the jargon, to ‘switch’ on or off at will. That can be done but only by administering a drug or some other regulator, either in drinking water, by injection or squirting directly into the lungs. An ideal would be something that’s more controlled and less invasive. How about shining a light on the relevant spot?!

Wacky or what?

That may sound as though we’re veering towards science fiction but reflect for a moment that every animal with vision, however rudimentary, sees by transforming light entering the eyes into electrical signals that the brain turns into a picture of the world around them. This relies on photoreceptor proteins that span the membranes of retinal cells.

How vision works. Light passes through the lens and falls on the retina at the back of the eye. The photoreceptor cells it activates are rod cells (that respond to low light levels — there’s about 100 million of them) and cone cells (stimulated by bright light). Sitting across the membranes of these cells are photoreceptor proteins — rhodopsin in rods and photopsin in cones. Photoreceptor proteins change shape when light falls on them — the driver for this being a small chemical attached to the proteins called retinal, one of the many forms of vitamin A. This shape change allows the proteins to ‘talk’ to the inside of the cell, i.e. to interact with other proteins to switch on enzymes and change the level of ions (sodium and calcium). The upshot is that the signal is passed through neural cells in the optic nerve to the brain where the incoming light signals are processed into the images that we perceive. © Arizona Board of Regents / ASU Ask A Biologist.

The seemingly far-fetched notion of controlling genes by light was floated by Francis Crick in 1999. The field was launched in 2002 by Boris Zemelman and Gero Miesenböck who engineered neurons to express one form of rhodopsin. This gave birth to the subject of optogenetics — using light to control cells in living tissues that have been genetically modified to express light-sensitive ion channels such as rhodopsin. By 2010 optogenetics had advanced to being the ‘Method of the Year’ according to the research journal Nature Methods.

Dropping like flies

One of the most dramatic demonstrations of the power of optogenetics has come from Robert Kittel and colleagues in Würzburg and Göttingen who made a mutant form of a protein called channelrhodopsin-1 (found in green algae) and expressed it in fruit flies (Drosophila melanogaster). The mutant protein (ChR2-XXL) carries very large photocurrents of ions (critically sodium and calcium) with the result that photostimulation can drastically change the behaviour of freely moving flies.

Light-induced stimulation of motor neurons in adult flies expressing a mutant form of rhodopsin ChR2-XXL. Click to run movie.

Left hand tube: Activation of ChR2-XXL in motor neurons with white light LEDs caused reversible immobilization of adult flies. In contrast (right hand tube) flies expressing normal (wild-type) channelrhodopsin-2 showed no response. From Dawydow et al., 2014.

Other optogenetic experiments on flies can be viewed on You Tube, e.g., the TED talk of Gero Miesenböck and the Manchester Fly Facility video of fly maggots, engineered to have a channel protein (channelrhodopsin) in their neurons, responding to blue light.

Of flies … and mice … and men

This is stunning science and it’s opened a new vista in neurobiology. But what about the things we’re concerned with in these pages — treating diseases like diabetes and cancer?

Scheme showing how genetic engineering can make the release of insulin from cells controllable by light. Normally cells of the pancreas (beta cells) take up glucose when its level in the circulation rises (via a glucose transporter protein). The rise in glucose triggers ATP production in the cell. This in turn causes potassium channels in the membrane to close (called depolarization) and this opens calcium channels. The increase in calcium in the cell drives insulin secretion. From Kushibiki et al., 2015.

The left-hand scheme above shows how glucose triggers the pancreas to produce the hormone insulin. Diabetes occurs when either the pancreas doesn’t make enough insulin or when cells of the body don’t respond properly to insulin by taking up glucose.

As a first step to see whether optogenetic regulation of calcium levels in pancreatic cells could trigger insulin release, Toshihiro Kushibiki and colleagues at the National Defense Medical College in Saitama, Japan engineered the channelrhodopsin-1 protein into mouse cells and hit them with laser light of the appropriate frequency. An hour after a short burst of light (a few seconds) the insulin levels had doubled.

The photo below shows a clump of these cells: the nuclei are blue and the channel protein (yellow) can be seen sitting across the cell membranes.

 

Cells expressing a fluorescently tagged channelrhodopsin protein (yellow). Nuclei are blue. From Kushibiki et al., 2015.

 

 

To show that this could work in animals they suspended the engineered cells in a gel and inoculated blobs of the goo under the skin of diabetic mice. Laser burst again: blood glucose levels fell and they showed this was due to the irradiated, implanted cells producing insulin.

Fast forward three years

Those brilliant results highlighted the potential of optogenetic technology as a completely novel approach to a disease that afflicts over 300 million people worldwide.

Scheme showing a Smartphone can be used to regulate the release of insulin from engineered cells implanted in a mouse with diabetes. The key events in the cell are that the light-activated receptor turns on an enzyme (BphS) that in turn controls a transcription regulator (FRTA) that binds to a DNA construct to switch on the Gene Of Interest (GOI) — in this case encoding insulin. (shGLP1, short human glucagon-like peptide 1, is a hormone that has the opposite effect to insulin). From Shao et al., 2017.

In a remarkable confluence of technologies Jiawei Shao and colleagues from a number of institutes in Shanghai, including the Shanghai Academy of Spaceflight Technology, and from ETH Zürich have recently published work that takes the application of optogenetics well and truly into the twenty-first century.

They figured that, as these days nearly everyone lives with their smartphone, the world could use a diabetes app. Essentially they designed a home server SmartController to process wireless signals so that a smartphone could control insulin production by cells in gel capsules implanted in mice. There are differences in the genetic engineering of these cells from those used by Kushibiki’s group but the critical point is unchanged: laser light stimulates insulin release. The capsules carry wirelessly powered LEDs.

The only other thing needed is to know glucose levels. Because mice are only little and they’ve already got their gel capsule, rather than implanting a monitor they took a drop of blood from the tail and used a glucometer. However, looking ahead to human applications, continuous glucose monitors are now available that, placed under the skin, can transmit a radio signal to the controller and, ultimately, it will be possible for the gel capsules to have a built-in battery plus glucose sensor and the whole thing could work automatically.

Any chance of illuminating cancer?

This science is so breathtaking it seems cheeky to ask but, well, I’d say ‘yes but not just yet.’ So long as the ‘drug’ you wish to use can be made biologically (i.e. from DNA by the machinery of the cell), rather than by chemical synthesis, Shao’s Smartphone set-up can readily be adapted to deliver anti-cancer drugs. This might be hugely preferable to the procedures currently in use and would offer an additional advantage by administering drugs in short bursts of lower concentration — a regimen that in some mouse cancer models at least is more effective.

References

Dawydow, A., Kittel, R.J. et al., 2014. Channelrhodopsin-2–XXL, a powerful optogenetic tool for low-light applications. PNAS 111, 13972-13977.

Kushibiki et al., (2015). Optogenetic control of insulin secretion by pancreatic beta-cells in vitro and in vivo. Gene Therapy 22, 553-559.

Shao, J. et al., 2017. Smartphone-controlled optogenetically engineered cells enable semiautomatic glucose homeostasis in diabetic mice. Science Translational Medicine 9, Issue 387, eaal2298.