Wednesday, August 24, 2011

;-) ;-) (wink, wink)

No living thing is in equilibrium with its environment. I mean this in a physical-chemical sense; ecologists will discuss how ecosystems are at equilibrium, meaning that populations stay constant. However, to a biochemist, equilibrium is the same thing as death. A system at equilibrium has zero energy, and since life by any definition requires energy, equilibrium is death. It follows that if you can measure how far out of equilibrium a living thing is, you can get a clue as to how much energy it has at its disposal.

The way almost all living things get energy is sort of like the way energy is stored in a battery:

In a battery, there’s too many electrons over at one end, and not enough at the other. This potential difference is a form of energy. We can let the battery get to equilibrium by letting the charges become uniformly distributed, and we can use that movement to do some work for us:

If it achieves equilibrium—an equal concentration of charges on either end—no more work can be done.

A living cell functions almost exactly the same way. The only changes are that the charge difference is between inside and outside, and the charged particles are (positively-charged) protons (H+) instead of (negatively charged) electrons.

And, just like a battery, this potential difference stores energy. If we let the cell get to equilibrium by letting the charges become uniformly distributed, the cell can use that movement to do some work. Instead of powering light bulbs, cells have enzymes in their membranes that use that energy to make a chemical form of energy, ATP, or wiggle their flagella around so they can move, or pack food into the cell and expel wastes.

Also like a battery, equilibrium means no more work can be done.

Establishing the disequilibrium in a battery takes work, and making the proton gradient across a cell membrane also takes work. Respiring cells use the oxidation of food to move protons out of the cell. Cells that can use light for energy have evolved a couple of mechanisms to move protons out of the cell. The simplest of these is called proteorhodopsin. It’s an enzyme that straddles the cell membrane, like so:

When it’s hit by light...

it can use that energy to change its chape, grab a proton from inside the cell and force it out of the cell (and away from equilibrium). Like so:

To understand the physiology of a cell, it’s nice to know the charge difference, or membrane potential, of the cell. Keeping track of membrane potential is pretty straightforward in cells like ours. Our cells are large enough that we can make tools that can investigate to our cell membranes the way that a stethoscope can check in on a guinea pig. However, bacterial cells are much too small for this approach, and their membranes are covered up by cell walls. So until recently, we haven’t been able to probe the membrane potential of individual bacterial cells, despite knowing that membrane potential is essential for the well-being of the cell.

A bunch of researchers at Harvard, led by Adam Cohen, have devised (and are in the process of patenting) and exceedingly clever way to look at membrane potential in single bacterial cells. Remember proteorhodopsin, which absorbs light energy and uses it to pull a proton out of the cell? The reason it’s able to absorb light energy is that it has a color. Grabbing onto a proton changes the energy state of the molecule, so it changes its color a little bit. So, Cohen and his gang figured out a way to modify proteorhodopsin, essentially reversing its normal behavior: if a proton were forced onto this modified enzyme, it would not only change color, it would fluoresce. How can a proton be forced onto this modified proteorhodopsin? Simply have a higher-than-normal concentration of protons inside the cell. So, here's a normal cell, and the engineered proteorhodpsin is dark:

And here's a depolarized cell. The relatively high concentration of protons inside the cell forces one onto the engineered bacteriorhodopsin, and it fluoresces:

Bear in mind that what triggers the light is not the concentration of protons, just the difference in concentration across the cell membrane.

This designed membrane protein doesn’t help the cell out any—it can’t do what normal proteorhodopsin does—but it does serve as a sensitive monitor of the membrane potential of a single bacterial cell. It is, in effect, a very sensitive tool. Cohen’s group embedded single bacterial cells in a gel matrix, hooked one end of the gel matrix up to the positive end of a battery and the other end up to the negative end. The electrical field pushed all of the protons in the matrix towards one end of the cell, depolarizing the other end. Sure enough, that end of the cell lit up like a Christmas tree. Here it is in cartoon form...

and the actual microscope view, as they flipped the polarity from one end of the cell to the other:

So far, this is just a cute tool. Unlike our cells, which are so large that small patches of the membrane can depolarize, bacterial cells are so large that they essentially can’t have any regionalized depolarization under normal conditions. Also, this modified enzyme only lights up when there’s essentially no difference in charge across the cell’s membrane—which condition, if sustained, is the same thing as death. So, what’s the point?

Here’s where this gets interesting, and actually tells us something new and unexpected about how bacterial cells live. When Cohen looked at a bunch of these cells under the microscope, he saw them “blink” at him at irregular intervals. Whatever the reason, something about the cells’ membrane potential would periodically just give out. (This movie is sped up four times actual speed.)

The modified bacteriorhodopsin essentially senses the difference in proton concentration across the cell membrane. So, there’s a couple ways that a blink could happen—either too many protons inside the cell, or not enough protons outside the cell. “Concentration of protons” is simply a long way to say “pH,” so to test this possibility, Cohen loaded his cells with a pH indicator (like Litmus paper, only in convenient liquid form!). When a cell “blinked,” its internal pH stayed the same.

The cell uses this proton concentration difference to do stuff—make ATP, and rotate its flagella, among other things. It’s kind of a rude trick, but if you glue a cell’s flagellum down, the whole cell rotates…

…as long as there is a proton gradient. (By the way, that movie is actually slowed down three times!) When Cohen’s group examined a cell that was “blinking,” they found that the blink correlated with the cell’s rotation slowing or stopping. Since the concentration of protons inside the cell is remaining constant, the concentration of protons outside the cell must drop during a blink. (I’m not going to include a movie of a cell…not moving.)

So why would a cell blink? Why would it periodically deprive itself of energy? There’s one thing that it’s much easier for a cell to do if it no longer has a membrane potential, and that is push positively charged substances out of the cell. There are some toxins and metals that are harmful to the cell that are positively charged, and cells are known to spit these out of the cell under stressful conditions. So, perhaps this is happening during a blink: the cell deliberately depolarizes, and purges itself of toxins. Cohen tried feeding his cells a toxic dye, and sure enough, it could be observed flowing out of the cell only during a blink.

So, this Cohen’s cleverly modified protein ends up being more than just an example of clever engineering; it tells us something new about how cells work. Until very recently, the only way we could study the physiology of bacterial cells was by studying the average properties of billions of cells all at once. Very useful information can be gained that way (just ask the US Census Bureau), but vital information about how individuals work is lost. This modified proteorhodpsin is one of several neat tools that have emerged in the last decade that allow us to study single cells or even single proteins. Just as doing interviews with individuals would change your views of census data, these new tools are changing how we view the lives of bacteria.

Joel M. Kralj, Daniel R. Hochbaum, Adam D. Douglass, and Adam E. Cohen (2011). Electrical Spiking in Escherichia coli Probed with a Fluorescent Voltage-Indicating Protein. Science 333: 345-348.


  1. Thank you for this excellent summary of our recent paper in Science. It is a wonderfully clear read, and the illustrations are very nice.

    There is a small technical point I want to correct. During the blinks, neither the concentration of protons inside the cell nor outside the cell changes substantially. The membrane voltage is mostly determined by other ions, potassium and sodium, and the conductivity of the membrane for these ions. When the inside of the cell is electrically negative, the protons in the cytoplasm are free to wander throughout the cell. But when the inside of the cell becomes positive, the positively charged protons are repelled by the positively charged interior of the cell, so they try to run away. They can't leave, however, because of the cell membrane. So they pile up on the inside of the membrane, and some of them get captured by the proteorhodopsin molecules, which then start to glow.

    Thanks again for a great summary!

    Adam Cohen

  2. Dr. Cohen--
    Thanks for the note (and notice). I'm especially glad that my summary/simplification meets with some approval from you.

    In writing for an audience with an intro bio-level of understanding, I'm always vexed by finding the line between simplification and misrepresentation. I sorta fudged on this by pretending that all cations are protons, figuring it would be easier for an audience vaguely familiar with pH and ATPase to swallow--but now I definitely have a teachable moment to work from; thanks!

    By the way, I'm wondering...By analogy with "tuning" Green Fluorescent Protein to different wavelengths by amino acid substitutions in the beta-barrel, have you considered "tuning" different PROPS to different pKa's? Could be interesting for looking at physiology of alkalaphiles and acidophiles.

    Thanks again, and congrats on a *most* interesting paper.