Scientists have long estimated the average stress produced by muscle or other cells, and more recently have been able to measure the stress from isolated molecular motor proteins. However, developing tools to measure the effect of mechanical stress on intact cells has been challenging.
To study the network of mechanical stresses and biochemical communication, or the mechanosome, that act on a cell, investigators at the State University of New York at Buffalo have developed an optical sensor that can be inserted into various structural protein hosts.
The probe, developed by UB professor Frederick Sachs and colleagues, is a fluorescence energy transfer cassette, call stFRET, comprising the green fluorescence protein pair Cerulean and Venus with a stable α-helix.
The investigators found that the cassette expressed well by itself in human embryonic kidney cells (HEK-239) and was concentrated in the nucleus. However, when properly incorporated into large host proteins such as collagen-19, alpha-actinin, non-erythrocyte spectrin, and filamin A in HEK-239 and 3T3 cell lines, stFRET expressed well and located similarly in the unmodified host.
The researchers concluded that stFRET is a good probe of mechanical stress within large host proteins. Their work was published this month in the FEBS Journal, the journal of the Federation of European Biochemical Societies.
Sachs, SUNY distinguished professor and a member of the Center for Single Molecule Biophysics in the department of physiology and biophysical sciences at UB, spoke this week with CBA News about the work, its applicability in drug discovery, and the next phase of the project.
Maybe you can give me little background on this work?
We discovered, about 20-something years ago, ion channels that can be activated by mechanical stress. We found them actually in skeletal muscle, which one does not think of as a sensory organ. However, it turns out that they are present in all cells — all cells have mechanically activated ion channels.
It is very difficult to determine what applies the mechanical stress to the channel, because it is not just a lipid bilayer in a patch-clamp experiment. You have the lipid bilayer, you have the cytoskeleton, and extracellular material, and when you apply force, the question becomes, “What is holding the force?”
You do not know where the stimulus is coming from, because you have to know where the stressors are. We would stress the patch, for example, by applying suction to the patch. That would stretch it, and the channels would become activated.
You realize that you do not know how much force the channel actually sees, you only know how much pressure you are applying to the pipette. There are forces acting on these channels in three dimensions. But which ones are they?
In the region of the cell membrane, there are thousands of proteins, but which one bears the force? If I distort a cell, which one is pushing back? Is it actin, tubulin, dystrophin, keratin, et cetera?
The cells always have internal forces acting upon them, even if they are not doing anything. That was mentioned in the paper. But it had not been possible before to ask, “What is it that is developing these stresses?”
We intrinsically think actin, myosin, or tubilin, but there are also many other molecules in the cell besides those. The mechanical forces modulate many things in cells. Those forces are distributed over thousands of components. How do you know what they are?
You can change the rates of biochemical reactions enormously by distorting their catalytic enzymes mechanically. All the enzymatic reactions in a cell, except for things that are soluble, are going to be modulated by mechanical stress.
The whole of physiology and biochemistry is modulated by mechanical stress. How much? That is a good question!
Can you describe what you did in this paper?
In this paper we developed a probe to measure the stress in specific proteins. It was as if we had a chain and stuck a strain meter in the middle of it. That is basically what we did. You pull on the chain, and see what the strain meter says.
Now, if I have a whole system of chains, cables, and ropes, but I only have the sensor in the chain, when I stretch everything, I can determine how much of the force is in the chain, and how much is not.
Exactly how does the probe work?
It works by fluorescence resonance electron transfer. Here, the energy transferred between the donor and acceptor varies with the sixth power of the distance between them, so if you change the distance, you change the energy transfer. The donor will get brighter or dimmer, depending on how much you stretch it.
We took a donor and a helix attached to an acceptor, and then we inserted those into the genes for different proteins, such as spectrin, filamin, actinin, and collagen.
How did you use this to probe cultured cells?
We transfected the cells with a gene for a protein having the probe, called stFRET, in the middle of it. If you stick the probe in the middle of the gene for the protein, the cell seems to think it is a normal gene for a protein.
How would this be used in drug discovery?
What are you looking for in a drug discovery program? What do you want to measure? If you happen to know that the thing you are looking for is mechanical stress, you’d better decide how you will measure it.
I think you will find that any reaction in the cell will respond to mechanical stress in the cell. Among other things, intracellular calcium levels increase in response to mechanical stress. This changes many things, including the mechanics of the cell.
For example, the heart hypertrophies when stretched, as do skeletal muscles. What is stretched? Which molecules?
In muscular dystrophy, loss of the protein dystrophin causes reorganization and atrophy of the muscles. What absorbs the stress when dystrophin is not present? Boys with dystrophy survive because other proteins step in and redistribute the excess stress.
Why do muscles get bigger with exercise? Which proteins are stressed to stimulate muscle protein synthesis?
The cytoskeleton of the endothelial cells of the blood vessels is controlled by shear stress from the blood flow. Which proteins in the cells are stressed by the flow? Which pathologies are the result of a change in mechanical stress?
Aneurysms tend to appear in bifurcations of the vessels where the fluid stresses of the blood have large gradients. Which proteins in the blood vessels are modified by these stresses?
Bones atrophy without load, as astronauts know. Which proteins in bone cells are stressed with a load? Is there a change in stress in particular proteins associated with osteoporosis?
Which proteins in pathogenic protozoa are under stress? Are there dugs that can modify this stress and be used to kill infection?
Which proteins in an erythrocyte are stressed in sickle cell attacks or malaria?
Edema is a major component of many diseases of the circulatory system and kidneys. Which proteins are stressed in edema?
Collagen is the most common proteins in animals, and it serves to distribute and communicate the presence of mechanical stress. What happens in diseases of collagen? Where does the stress go, and what is the result?
All fibrous proteins such as keratin, elastin, and fibrin are stressed. How is stress distributed in blood clots? How do defects in the components affect the stress? Which ones communicate stress to the cofactors in the clot? What happens with defects in the genes of those proteins?
How do genetic defects redistribute stress in all cells? What is the stress in the various membranes in the ear and how are they affected by congenital forms of deafness or other hearing defects?
Resolution of mechanical stresses is as important to living things as the properties of water. Not knowing mechanical stress is the same as not knowing the concentration of chemicals in cells.
Molecular dynamics simulations are the explicit study of the mechanics of molecules. We have not had a tool to ask about the stresses in vivo before. This opens the door to a much more complete understanding of cell and organism physiology and pathology.
Do you think that pharmaceutical companies are looking at mechanical stress in their drug discovery assays?
No. Because there have been no tools. I know that some companies may be, but most are not, because they depend on readily available tools. If calcium levels change, that’s one thing, but mechanical stress has even broader effects than calcium does. I suspect that once people start looking, they will identify and develop tools to study the effects of mechanical stress on cells.
One thing you can do is grow cells on a rubber sheet, stretch it, and see if the drug effects you are looking for are mechanically sensitive, and ask, ‘If it is mechanically sensitive, who is bearing the stress?’
In what areas of drug discovery would mechanical stress be a factor, or be most relevant?
Cardiology, any place where there is flow and stress, such as the blood vessels or kidneys. I think that fundamentally, there is no limit, any more than anyone would rule out calcium effects.
That is why I say it is very general. There are very few things in the cell that are not mechanically sensitive.
What would be the next step in this work for your lab?
We are trying to make some smaller probes that we can put in smaller proteins. We are also looking at the effect of mechanical stress in the cell cortex, in terms of, which proteins are stressed, and how do the stress-sensitive ion channels react?
Is this technology something you plan to commercialize?
No. UB did not want to patent it.