Here's one for Joolya.
I've been growing some cells that express a splicing factor (probably. But that's a different story) tagged with green fluorescent protein (GFP). For the non-biologists, GFP is a protein that naturally fluoresces green in response to certain wavelengths of light. This is useful for sub-cellular localization of proteins, because it means you can fuse your favourite protein gene to the gene for GFP, stick that into cells, and see where your protein lives. It's incredibly useful, and pretty to boot.
Now, my protein lives in the nucleus, so when it's tagged with GFP the nuclei of my cultured cells glow green. And after the cells have been growing a while, all sorts of weird things happen, but mainly I get multinucleate cells: The nucleus of a cell divides but then something goes wrong and you don't get daughter cells. Most cells have one nucleus each, but these guys (gals?) are forming syncytia, although the term is usually used to describe the fusion of existing cells, rather than what I think is happening here.
This is what it looks like:
In the two images above, the left is the fluorescent image (looking at the glowing GFP) and the right is (what passes for) normal light — what the cells actually look like down the microscope. See how there are many green blobs in the huge cells? They are multiple nuclei in a single cell. Here are parts of the two different image types (normal and fluorescent light) overlaid:
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Maybe 2% of the total cell population is affected, but they stand out because they are so big. I am growing up some control cells (transfected with GFP fused to the SV40-NLS) and I hope to see if the effect is due to my protein or is an artifact. Either way, something very, very odd is going on with these cells.
PS/Update I didn't realize until this morning that the bloody sidebar is covering up the phase images. Just open the image in a new window to see the full splendour of it all.
Comments
Have you tested (or treated) for mycoplasma? Should be your first move whenever your cells do anything odd.
Posted by: Ian | May 25, 2007 11:30 PM
Those are very pretty images.
For the TC-illiterate: what kind of cells are they? I presume they're not just differentiating?
Posted by: Whiffling syncytially | May 26, 2007 01:12 AM
Ian - we routinely check for mycoplasma, and have found no evidence for it. I get this effect whenever I transfect (variations on) that particular construct. I should have enough cells to do the control experiment soon.
R'pus: They're HEK — human embryonal kidney — cells. They're immortalized by adenovirus DNA, and I've just come across http://www.mbi.ufl.edu/~shaw/293.html which suggests that they express neuronal markers. First thing I thought when I saw that big one was 'neuron!', so . . . my spider sense is tingling. If My Protein is persuading these suckers to differentiate into neurons, then I have stumbled across something exciting. It's probably just the CMV promoter, though.
Posted by: BK | May 26, 2007 11:38 AM
The cells do not look very healthy, they are quite rounded up. Sick cells often round up and leave just a few attacj=hments onto the pates. Do they always look like this? It could be the expressed construct, or perhaps something else is going on?
Posted by: helorime | May 26, 2007 12:48 PM
They don't usually look quite that bad (aside from these bizzaro fellows, they usually look just like the parental line), but these particular ones had cooled down by the time I got around to taking the photos, and I think they were sulking.
Posted by: BK | May 26, 2007 04:41 PM
Oooh, looking at those cells my cell culture withdrawal flares up once more!
Posted by: beta gal | May 28, 2007 01:35 PM
There are two variants of HEK. One,HEKtsa201 is a "neuronal" version. Do you know which you're using? If its tsa201, maybe swiching to regulars might help?
Posted by: tideliar | May 29, 2007 05:50 AM
Don't make me larf, Tidy.
Posted by: BK | May 29, 2007 06:37 AM
I wonder if some of them are hitting the senescence wall (or some differentiation machinery), or else have got some mutation that affects cytokinesis. I use MEFs (mouse embryonic fibroblasts, although "fibroblasts" is to be used loosely) and they are often bi- or multinucleate - especially after they have been passaged a few times and/or I have transfected them. Generally a few % of the population will be these huuuuuuuge multinucleate guys. I have seen this in 3T3 cells, too. Sometimes on timelapse I can see a cell try to cytokinese and then not make it and turn into a binucleate cell.
Posted by: Joolya | July 6, 2007 09:37 AM