Here are the talks on Sensor and Probe development, all on the last day of the conference (Part 1)
Amy Palmer probes the cellular ionosome
First up today, Amy Palmer develops new probes to investigate the ‘ionosome’* – the complex but under-recognised flow of metal ions within living cells. This is a relatively untapped field – 30 % of proteins in cells require a metal cofactor to function, yet only really Calcium has been addressed with fluorescent probes, or even recognised as a regulator of cell function.
Zinc ions are particularly interesting, and that’s the focus for today’s talk. It’s level is sensed by cells, which adjust their metabolism in response. 10% of proteins require Zinc, so it does a lot of different jobs.
OK so first of all the probe made by Amy and her team. This is a FRET probe, composed of two Zinc binding domains between the donor and acceptor. When Zinc binds, conformational change occurs and the probes come within the magic distance, facilitating energy transfer which can be detected. In addition, a signal sequence can be added to direct the construct to somewhere specific eg – membrane, nucleus, cytoplasm.
To prove the probe works, the group carried out in situ calibrations – within the same cells they will be testing. To soak up all the Zinc for a low signal readout, they used a chelator (in this case TPEN) and to get a high Zinc signal they used a Zinc carrier (Zinc Pyrithione). This works pretty nicely, and their yellow FRET signal is ratiometric with reference to the green donor signal alone. There also doesn’t seem to be any perturbation of endogenous Zinc concentration due to the probe itself, which is nice.
Next they looked at the kD values for binding of Zinc to some of the proteins it regulates, and it turns out that they are not fully occupied under physiological conditions. This is important as it points towards Zinc indeed being a regulator, functioning by binding on and off to the protein of interest.
One example of this is CDK2 in the cell cycle. Zn fluctuations accompany high CDK2 cytoplasmic recruitment/activity, when the cell has just exited mitosis. The group found that these zinc fluctuations are required for the decision to translocate CDK2 from nucleus (inactive) to cytoplasm (active).
One of the next steps for the group might be to target the probes to particular organelles, to investigate Zinc’s role there. Very interesting stuff.
*me and Amy agreed that this was a cooler name for this network than metallosome – what do you think?
Takeharu Nagai (part 1/2) – Acid resistant fluorescent protein for super resolution
Takeharu’s group looks in strange places for new fluorophores.
First up is the work of Hajime Shinoda, a “very handsome and cool student” in Take’s words. He has developed a new fluorescent protein, called Gamillus isolated from a flower hat jellyfish, which survives at low pH, where EGFP and others lose their signal.
That’s because it has a trans-isomeric conformation, instead of a cis one. It can’t gain H+ ions in a way that would disrupt the aromatic rings in the chromophores of the rest of the green cis proteins, so it is compatible for imaging low pH environments, like the inside of lysosomes. A nice control is shown: use GFP, you can’t see lysosomes, use Gamillus, and suddenly little polkadots appear in strategic cellular locations. It works!
Next, handsome Hajime altered the protein, so that it might be compatible for super resolution. He used the very problem that Gamillus solves, transition to cis isomerism, to achieve photoswitchability. Reversibly switchable Gamillus blinks on exposure to UV light, by switching between the inactive cis and active trans form. (Shinoda, unpublished data).