We have a new preprint out – it is a cautionary tale about using GFP nanobodies in cells. This short post gives a bit of background to the work. Please read the paper if you are interested in using GFP nanobodies in cells, you can find it here.
Paper in a nutshell: Caution is needed when using GFP nanobodies because they can inhibit their target protein in cells.
People who did the work: Cansu Küey did most of the work for the paper. She discovered the inhibition side effect of the dongles. Gabrielle Larocque contributed a figure where she compared dongle-knocksideways with regular knocksideways. The project was initiated by Nick Clarke who made our first set of dongles and tested which fluorescent proteins the nanobody binds in cells. Lab people and their profiles can be found here.
Background: Many other labs have shown that nanobodies can be functionalised so that you can stick new protein domains onto GFP-tagged proteins to do new things. This is useful because it means you can “retrofit” an existing GFP knock-in cell line or organism to do new things like knocksideways without making new lines. In fact there was a recent preprint which described a suite of functionalised nanobodies that can confer all kinds of functions to GFP.
Like many other labs we were working on this method. We thought functionalised GFP nanobodies resembled “dongles” – those adaptors that Apple makes so much money from – that convert one port to another.
A while back we made several different dongles. We were most interested in a GFP nanobodies with an additional FKBP domain that would allow us to do knocksideways (or FerriTagging) in GFP knock-in cells. For those that don’t know, knocksideways is not a knockout or a knockdown, but a way of putting a protein somewhere else in the cell to inactivate it. The most common place to send a protein is to the mitochondria.
We found that dongle-knocksideways works really well and we were very excited about this method. Here we are removing GFP-clathrin from the mitotic spindle in seconds using dongle knocksideways.
Since there are no specific inhibitors of endocytosis, we thought dongle knocksideways would be cool to try in cells with dynamin-2 tagged with GFP at both alleles. There is a line from David Drubin’s lab which is widely used. This would mean we could put the dongle plasmids on Addgene and everyone could inhibit endocytosis on-demand!
Initial results were encouraging. We could put dynamin onto mitochondria alright.
But we hit a problem. It turns out that dongle binding to dynamin inhibits endocytosis. So we have unintended inhibition of the target protein. This is a big problem because the power of knocksideways comes from being able to observe normal function and then rapidly switch it off. So if there is inhibition before knocksideways, the method is useless.
Now, this problem could be specific to dynamin or it might be a general problem with all targets of dongles. Either way, we’ve switched from this method and wrote this manuscript to alert others to the side effects of dongles. We discuss possible ways forward for this method and also point out some applications of the nanobody technology that are unaffected by our observations.
The post title comes from “Not What You Want” by Sleater-Kinney from their wonderful Dig Me Out record.