Fluorescent protein labeling: a cautionary tale

June 29, 2012 § 1 Comment

There was a time when we viewed bacterial cells as mere bags of randomly mixed molecules.  Lacking the obvious compartmentalization of eukaryotic cells, bacteria were viewed as being completely unstructured.  But increasing numbers of studies seem to show clearly defined localization patterns for proteins in bacteria.  One example is that the main proteases responsible for regulated proteolysis in bacteria — the Clp proteases (pronounced “clip”) — have been observed in several studies to form a single bright proteolytic focus, detected by fluorescent protein labeling.

The Paulsson lab spotted these observations and became intrigued.  One of the major interests in the lab is variation between individual cells at the RNA and protein level, and this looks like a potentially significant place where variation may happen.  If all proteolysis in a cell is localized into a single spot, then when a cell divides something interesting must happen: either the spot also divides, or one of the two daughter cells gets all of the Clp proteases in the cell while the other daughter gets nothing.  The second option would lead to a potentially enormous difference in the ability of the two daughters to perform proteolysis.  So a graduate student, Dirk Landgraf, set out to look at whether this difference exists, and if so how long it lasts (Landgraf et al. 2012, Segregation of molecules at cell division reveals native protein localization.  Nature methods doi: 10.1038/nmeth.1955).

The first step was to ask what happens to the proteolytic focus at cell division.  Landgraf et al. made movies of cells carrying fusions of a Clp family member, ClpP, with two different fluorescent proteins, Venus and superfolder GFP.  In each case they saw a single focus of fluorescence, and when the cell divided the whole fluorescent focus went to one daughter.  After a few generations, fluorescent foci (one per cell) reappeared in the line of cells descending from the other daughter.  This strongly suggested that there should be significant variation in the level of proteolysis going on in different cells. If regulated proteolysis is an important function for the cell — which we believe it is — this seems odd, and therefore interesting.  So the authors tested this possibility directly using another fluorescent tag (mCherry)  fused to a Clp substrate, allowing them to measure the variation in the degradation of the substrate in pairs of daughter cells from a single division event.

Clp foci (green) are present in only one of the two daughter cells after cell division.

This is where things get surprising, not to say shocking.  Yes, the lines in which ClpP was labeled with Venus or superfolder GFP showed very significant daughter-to-daughter variation.  But in the wild type strain, in which the ClpP was unmodified, very little daughter-to-daughter variation was seen.  The inescapable conclusion is that the fluorescent protein tags are changing the behavior of the protein being studied.  And this is not a small change: the whole notion that ClpP self-organizes into a single localized focus, which has led for example to the idea that protein degradation needs to be compartmentalized, appears to be an artifact.

Fluorescent proteins have swept the world of cell biology. What better way could there be to study the behavior of your favorite protein than to put a brightly glowing tag on it and watch it going about its normal business?  The images you get are beautiful and compelling, and make great figures in your paper. We’ve become so comfortable with the essential benignity of fluorescent protein fusions that we barely bother to worry about whether adding an extra 238 amino acids to a protein changes its behavior.  Partly this is because we can see so much with fluorescent protein fusions that we could never see before, so there is no easy way to be sure that the behavior of the protein under study hasn’t changed. But partly, too, it’s because the standard in the field has shifted.  Fluorescent proteins are the gold standard now.  If your results from an older and apparently cruder technique, such as immunofluorescence, don’t match the results from live-cell imaging using fluorescent proteins, then the immediate suspicion is that the older technique is wrong.  And probably this is often true.  What Landgraf et al. show, however, is that in the case of the Clp family the older methods are the better methods.  Immunofluorescent staining shows many small Clp foci, probably corresponding to individual protease complexes, located throughout the cell in the wild type, but also detects the large single clump induced when fluorescent tags are added.   « Read the rest of this entry »

Clumping is good; controlled clumping is better.

August 26, 2011 § 1 Comment

When I pouted last week about the fact that other writers had beaten me to the punch in discussing an interesting recent paper on the fitness benefits of clumping in yeast, I had somehow failed to notice that another, similarly fascinating, paper on a related topic had just come out from the Bassler lab (Nadell and Bassler 2011. A fitness trade-off between local competition and dispersal in Vibrio cholerae biofilms.  PNAS doi:10.1073/pnas.1111147108).  This paper is looking at the formation of biofilms in the bacterium Vibrio cholerae, a nasty little bug that has been a major evolutionary force in the development of modern sewage systems.  One of the factors that makes V. cholerae hard to get rid of is the fact that it can, when it chooses, grow in biofilms; it can produce a structural matrix called extracellular polysaccharide (EPS) in which the bacterial cells are embedded. EPS production has a number of benefits, including offering bacteria from many species the opportunity to collaborate and behave as a community.  The puzzling thing, though, is that these community benefits are available to everyone, not just the bacteria who do the work of producing the EPS.  This is a classic set-up for “cheating”; in theory, if some bacteria can gain the benefits of EPS production without paying the price for it, then those “cheating” bacteria would be expected to grow faster than the poor exploited EPS producers.  At some point, the EPS producers (still struggling to build community, no doubt) would die out, and the whole system would collapse.  The theoretical arguments seem very persuasive, but actually EPS-producing bacteria show no signs of going away.  So clearly we need a new theory.

Kevin Foster and colleagues (including Carey Nadell, the first author of this paper) have been working for some time now on the possibility that EPS production, in addition to its benefits for the community, offers direct benefits to the cells that produce it.  If you simulate the growth of EPS-producing microbes in three dimensions, including the way that nutrients and oxygen diffuse and are consumed, you can see that producing EPS can help a lineage of cells to push itself above the masses and get access to better conditions, incidentally suffocating non-EPS producing cells.  This line of argument suggests that, far from being a happy “all for one, one for all” type commune, microbial biofilms are a balancing act between cooperation and competition — much like some other societies you might be aware of.  It also suggests that, though there are some conditions in which “cheaters” (non-EPS producing cells, though now they look lazy and stupid rather than cunning) can win, especially when a group of cells is colonizing a new area, if a biofilm persists for a long time the EPS-producing cells have a strong advantage.  And a particularly clear prediction from the 3D modeling is that, in a mixture of EPS-producers and non-producers, the EPS-producing lines should end up in skyscraper-like towers (reaching towards better oxygen conditions), suffocating the cheaters. « Read the rest of this entry »

Sources + sinks = swarmers + stalks: a signaling activity gradient within a bacterial cell

January 6, 2011 § Leave a comment

You’re probably familiar with the idea that gradients of signaling molecules determine cell fate in early animal embryos.  Now, a recent paper from the Laub lab (Chen et al. 2010.  Spatial gradient of protein phosphorylation underlies replicative asymmetry in a bacterium, PNAS doi:10.1073/pnas.1015397108) has discovered gradients of active signaling molecules within a single bacterial cell.  The existence of this gradient had been missed until now because the signaling protein itself, CtrA, is evenly distributed across the dividing cell.  But, Chen et al. find, the active phosphorylated form of the protein, CtrA-P, is not.  You’ll have to read a little further to find out why not.

First, the model system.  Caulobacter crescentus is unusual among the familiar bacterial model organisms, in that it divides asymmetrically; it’s used as a model system for studying cell differentiation.  There are two forms, called “stalked” and “swarmer”: the stalked form is able to copy its DNA and divide, producing one stalked daughter and one swarmer daughter; later, the swarmer daughter may differentiate and become stalked, and then have daughters of its own.  The protein that stops swarmers from undergoing replication is CtrA, a transcription factor that, when activated by phosphorylation, binds to the bacterial replication origin and prevents the initiation of DNA synthesis.  So, in theory, one way you could give stalked and swarmer cells different fates is to wait until the cell divides into two (cytokinesis), then selectively dephosphorylate CtrA-P in stalked cells.  By making CtrA-P levels high in swarmer cells and low in stalked cells, you would block division in swarmers but not in stalked cells. « Read the rest of this entry »

Guest Post: The cellular postal system: more postmen don’t spoil the delivery

October 12, 2010 § Leave a comment

Bodo Stern writes:

Remember the children’s game “Telephone” in which the first participant whispers a phrase to their neighbor who in turn whispers what they believe to have heard to the next player, and so on? The phrase announced by the final player often differs substantially and in hilarious ways from the original message. Luckily, molecular cascades are more accurate than these human chains: not only are they able to carry information faithfully, they can introduce specificity and integrate several inputs. An interesting recent discovery of such a molecular chain comes from Vlad Denic’s laboratory at the Department for Molecular and Cellular Biology at Harvard University. The work, published in Molecular Cell, identifies new components in a pathway that delivers so-called tail-anchored (TA) proteins to their destination in the endoplasmatic reticulum (ER) membrane.

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