28 Oct 2014

Turning on proteins with light

Just like for married couples, communication is fundamental for cells. When an embryo is developing, its cells need to tell one another who and where they are, so every tissue and organ grows in the right place and at the right time. Our neurons are constantly talking to each other to control our thoughts, feelings and behaviours. Even single-cell organisms like bacteria can exchange information to decide, for example, how many times they should multiply.

But how do cells communicate? Scientists have a good understanding of the key proteins involved in cell communication, or cell signalling. Typically, a cell sends out a chemical signal (or electrical, in the case of neurons) that sticks to a specific receptor protein on the surface of the neighbouring cells. We then say the receptor is ‘activated’, because it can trigger a cascade of molecular events that ultimately leads to a cellular response. For instance, the cell might start moving in a particular direction, or a specific gene gets translated into protein.

There is, however, quite a lot we still don’t know about cell signalling. What would happen if we could activate a receptor only at the tip of a moving cell? Would the cell change the direction of migration? And what if we could activate a receptor repeatedly, or at different time intervals? Would the cell responses be different? Questions like these have been bugging scientists for decades, but they simply lacked the tools to address them.

Now, a research team led by Harald Janovjak at the Institute of Science and Technology (Austria) has developed a new method to study the fine temporal and spatial regulation of cell signalling using proteins activated by light. This work opens the way for the development of powerful approaches to manipulate cell behaviour in health and disease.

Human cells illuminated in a pattern depicting the letters IST. The cells carry a reporter gene that 'glows' when it is triggered 
with light-activated receptor tyrosine kinases (Credit: Medical University of Vienna).
The optogenetics revolution
Scientists have been using engineered light-activated proteins to manipulate cell activity for about a decade or so, a technique that has been named ‘optogenetics’. The first light-activated proteins, or photoreceptors, applied in optogenetics belonged to the microbial opsin family. These opsin photoreceptors are useful because they can move ions across cell membranes in response to light, a process similar to what triggers neuron activation. In these initial studies, channelrhodopsins (a type of opsin photoreceptor) were removed from algae and inserted into particular neuronal cell types in mice. Upon exposure to light, the neurons containing these proteins started to fire, and depending on which neurons were activated in this way, a different behaviour was observed in the mice; in one study, the mice’s levels of anxiety increased, and in another they started going round in circles.

The reason why optogenetics has been coined a ‘revolutionary technique’ (and why it is tipped for a Nobel prize) is that it allows scientists to control the activity of particular cell types or proteins with an unprecedented level of precision, both in a temporal and spatial manner. And this, sure enough, comes very handy for cell signalling research. It is a bit complicated though, to build optogenetic tools for that purpose.

“The main challenges are the same as for many engineering problems. For example, you want the signalling receptor to be completely inactive in the “OFF” condition (no light), and to be as much active as if the natural chemical signal is added in the “ON” condition (light),” says Janovjak.

This fine level of receptor manipulation is very hard to achieve with conventional optogenetics tools, so Janovjak and colleagues decided to build signalling receptors activated by light from scratch, by taking bits and pieces from several proteins and then sticking them together.

They focused on cell-surface receptors of the receptor tyrosine kinase (RTK) family, which sense growth factors and hormones and have been involved in a variety of cellular processes. When an RTK receptor is activated by a chemical signal, let’s say a growth factor, it attaches to another receptor in what is called ‘dimerisation’. It is this contact between two RTK receptor molecules that triggers the molecular events leading to a cell response, or in other words, that activates RTK signalling. Janovjak and colleagues knew this, so they looked in bacteria, fungi and plants for proteins that dimerise in response to light, and then fused them to an RTK receptor skeleton. In theory, these engineered RTK receptors should dimerise—and therefore become activated—upon light exposure.

“We were quite beautifully able to do this. In our study cancer cells with RTKs under optical control quantitatively respond to light and the growth factor! This is nothing short of amazing and the basis for all future work by us and others,” says Janovjak.

Manipulating cell signalling with light
The team showed that when engineered RTKs are inserted into several cell types, including cancer cells, they can be efficiently activated by light and induce the predicted cell response very quickly and within a tiny spatial range.

Morgan Huse, an expert on cell signalling at the Sloan Kettering Institute (US) says “This study represents the first time that homodimerising [light-activated] protein domains have been used to activate RTK signalling. The results are quite significant.”

These new optogenetic tools will be invaluable for understanding cell signalling, and could also be adapted to study other cellular processes. In the future, Janovjak’s team will use these tools to investigate regeneration.

“Our research will focus on regeneration. In essence, growth factors are known to be efficacious in disease animal models, including diabetes and Parkinson’s disease. However, delivery of these growth factors is a real issue because they can induce side effects like (but not limited to) cancer, and growth factors often can’t reach the desired cells (for example in the brain). Maybe optogenetics can help”.

Grusch M., R. Riedler, E. Reichhart, C. Differ, W. Berger, A. Ingles-Prieto; H. Janovjak (2014). Spatio-temporally precise activation of engineered receptor tyrosine kinases by light, The EMBO Journal, 33 (15) 1713-1726. DOI: http://dx.doi.org/10.15252/embj.201387695

This article was published in Lab Times on the 22-08-2014. You can read it here