When we image cells transfected with live celllight-GFP proteins we notice a photobleaching when we stimulate cells with ATP but not other proteins like EGF. Can anyone help!
I'll tell you what I am thinking. First, this is not photobleaching. When you photobleach GFP, its fluorescence does not come back. Things move around and the fluorescence intensity comes back some, but this is due to the diffusion of unbleached GFP. I believe that what you are seeing is the result of the remodeling of the cytoskeleton, which is a well-documented consequence of the activation of P2Y receptors and is mediated by Rho GTPases. I surmise that your observations indicate a redistribution of the fluorescence, not fluorescence loss. If you could integrate the whole cell fluorescence (like doing a maximal projection of a confocal Z-stack), I bet the numbers would be identical with or without the nucleotide. You don't see it with EGF because the EGFR does different things in different cells. The EGFR also affects Rho GTPases, but in some cells it activates Rho, while in other cells the effect is the opposite.
It would help to know which probe, which fluorescent protein and what imaging conditions you are using. GFP does photobleach, but not nearly as much as most other probes. My lab has used GFP photobleaching as a tool to look at protein dynamics since the mid 1990s. However, some flavors (colors) of GFP photobleach much easier than others. For example, the red-shifted yellow varieties of GFP are much more light sensitive. Furthermore, there are other phenomena that will cause reduction in GFP fluorescence w/o photobleaching. For example, GFP fluorescence is pH-dependent, or can be quenched by chromophores that absorb in the same excitation range.
We are using the cell light bacman actin and talin RFP and GFP probes. As I mentioned before this does not happen when the cells are stimulated with EGF only when cells are stimulated with ATP and now we see it with UTP. Images are collected every 30 sec for 20 min. Interestingly if you look 10 min after one stops imaging the fluorescence level appears to have returned. Ideas?
I'll tell you what I am thinking. First, this is not photobleaching. When you photobleach GFP, its fluorescence does not come back. Things move around and the fluorescence intensity comes back some, but this is due to the diffusion of unbleached GFP. I believe that what you are seeing is the result of the remodeling of the cytoskeleton, which is a well-documented consequence of the activation of P2Y receptors and is mediated by Rho GTPases. I surmise that your observations indicate a redistribution of the fluorescence, not fluorescence loss. If you could integrate the whole cell fluorescence (like doing a maximal projection of a confocal Z-stack), I bet the numbers would be identical with or without the nucleotide. You don't see it with EGF because the EGFR does different things in different cells. The EGFR also affects Rho GTPases, but in some cells it activates Rho, while in other cells the effect is the opposite.
Yes they have abaundant P2Y and P2X receptors
thank you for your insight!
George, I am kind of overwhelmed by your reply and the great wealth of information it contains. However, as I said above, it is unlikely that Vickery is seeing any photobleaching.
By the way, we have imaged GFP/CFP/YFP/RFP and all kinds of other derivatives for about 20 years in live cells, and never bothered to multimerize, eliminate flavins, or use any of the specialized media you describe. Phenol red free medium works perfectly fine for everything we have done.
Emerald GFPs in the cell lights are notable for their reversible photoswitching (see Shaner NC, Steinbach PA, Tsien RY. A guide to choosing fluorescent proteins. Nat Methods. 2005;2(12):905-9. doi: 10.1038/nmeth819.) It's a weird thing that is intermittent by nature and it is not well understood how to mitigate it in cell preparations. Unfortunately, if you are having problems with it, the only solution is to switch to a different fluorescent protein variant.
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