Fluorescent protein labeling: a cautionary tale

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.  

The authors concluded that a natural tendency in the Clp proteins to self-associate has been exacerbated and distorted by a similar, but weak, tendency in the Venus and superfolder GFP proteins. They then tested a number of other fluorescent proteins and other tags.  This is where even the most partisan of fluorescent protein defenders will have to throw in the towel, because different fluorescent proteins behave differently, and not all of them cause the single focus to appear.  However, all the fluorescent proteins tested did distort proteolysis to some degree, presumably because the tag is large relative to the target.  The least distorting tag was the SNAP tag, which is not fluorescent; among the fluorescent proteins, mGFPmut3, Dronpa and Dendra2 seemed to be the least distorting in this system. The authors tell me that mGFPmut3 is not the only GFP variant that works, however; they’ve tested more proteins since the study was published.  Dirk Landgraf will be happy to tell you which ones.

Does this tendency of (some) fluorescent proteins to bring proteins together into clusters only matter for those who study Clps?  Sadly, no.  The authors tested five E. coli proteins (Hfq, PepP, IbpA, FruK and MviM) that had been repeatedly reported to form bright foci when tagged with fluorescent proteins, and found that all five of them behaved quite differently when tagged with the very monomeric GFP they had identified, mGFPmut3.  It’s very likely that a good deal of the protein clustering that’s been reported is either exaggerated or just plain wrong.  For many proteins, it will be possible to use the type of assay Landgraf et al. introduce to check whether the cell-to-cell variation in protein function after cell division is affected by the fluorescent protein tag.

Note that this clustering is not mere aggregation, which would have been easier to spot.  This is a different phenomenon: the proteins in the clusters are still functional, but they are brought together in large clusters because both the protein of interest and the fluorescent protein have a weak tendency to associate. The authors suggest that this results from the avidity effect: when you have multiple binding sites that can all bind simultaneously, it is much harder for a monomer to escape a complex because all of the binding sites have to be released at once for the monomer to diffuse away.

The moral of this story is not that we should stop using fluorescent proteins.  The power (and beauty) of fluorescence imaging isn’t changed by this study.  But this is a salutary reminder that no technique is so powerful that you don’t need confirmation of what it shows you.  Every act of observation, in biology as in physics, changes the thing observed.

Landgraf D, Okumus B, Chien P, Baker TA, & Paulsson J (2012). Segregation of molecules at cell division reveals native protein localization. Nature Methods PMID: 22484850