As soluble, untagged GFP is not membrane bound and doesn't contain targeting or signal sequences, one might expect it to be synthesized by free ribosomes and not bound ribosomes. That would mean less GFP expression and fluorescence in the lumen of the endomembrane system compared to the cytosol, which might be detected in reduced fluorescence. But I've never noticed that phenomenon or seen that effect reported. Has anyone looked at whether free GFP ends up in the lumen of the endoplasmic system, particularly in neurons? Electron microscopy, super-resolution imaging, protein subcellular fractionation each seem like they would detect such a phenomenon, but I can't seem to find any research on it. If anyone has any leads or references that would be helpful. Thanks!
I worked with very high-resolution confocal microscopy and I didn't notice any reduction in fluorescence of free GFP in Golgi or ER. I don't remember about other internal structures though. Also, note that free GFP tends to accumulate in the nucleus for some reason (nuclear fluorescence is a bit stronger). Maybe it could get from the nucleus to Golgi/ER, assuming there are communication channels between them and the nucleus.
Thanks Yulia Panina ! Thats interesting. I wonder how GFP gets into the Golgi and ER so efficiently. Presumably this means it is translated by rough ER as well as free ribosomes as that is how proteins get into the lumen of the ER and Golgi, no? I've seen references about free GFP accumulating in the nucleus, but my understanding is that there is not free movement between the two organelles.
Jeremy Linsley Well, I will not draw any references (I have two horrible deadlines approaching) so I will just tell you from memory. Potentially this is good stuff, lots of questions nobody really asked:) First of all, I remember distinctly that I tried to find out how exactly is ER/Golgi separated from nucleus and whether they are connected or not. There seem to be no works about it. Thus, the question of how the nucleus is connected to them is open. Golgi/ER never really move separately from the nucleus. I did a lot of filming of cells (time-lapse) and you can definitely see there that Golgi/ER is a structure that always is attached to the nucleus - never separated. That makes you wonder why, right?
So, GFP could get there from the nucleus.
On the other hand, why GFP accumulates in the nucleus? To answer this question, you need to read some papers about the hydrophobic properties of the nuclear pore complex. I think this really has to do something with the hydrophobicity of the molecules overall. An interesting start would be https://jcs.biologists.org/content/125/21/4979
See, some molecules can be just more likely to pass into the nucleus thanks to their hydrophobicity properties - not to some "homing signal". And that may be the reason GFP goes to nucleus.
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