Takeharu Nagai (part 2/2): Singularities in cells: leaders, followers and citizens
Takeharu describes a familiar situation. Our results, whether its molecular behaviour or cellular behaviour are generally an averaged description of phenomena. We get an idea of what’s going on, but ignore the minority phenomena: the singularities that lead to group behaviours.
After a brief rundown of things he considers singularities: the big bang, the benign to malignant switch, formation of iPS stem cells with only 4 genes altered, populist fascism under Trump etc, he sets out to investigate this idea armed with a plethora of new fluorescent probes, and some cool model systems.
Here, he focuses on cAMP signalling in social amoeba, which transition from single celled entities to an intercommunicating mass, a multicellular being. To initiate this switch, all you have to do is starve them of nutrients.
By using two markers, Flamindo2 (Odaka) and R-FlincA (Horikawa, in press) he derives a ratiometric measurement for cAMP activity, known to be associated with this switch in amoeba behaviour. Combining this with a high speed microscope tiling technique, he is able to look at the behaviour of both single cells and the whole population of cells.
What he finds is amazing – single amoeba cells become leaders, displaying a burst of cAMP, before setting off their neighbours. “Early followers” then signal to “late followers”, and within a matter of hours, to “citizens”, setting off a continuous spiral of cAMP signalling which causes the amoeba to group together and become multicellular. The fluorescence videos of this are quite amazing – and while the paper isn’t out yet, you can view some similar behaviours online at Take’s website.
Interestingly, several leaders seem to be selected, but only one gains ultimate dominance as the seed of the spiral of signalling. One of the goals of the Nagai lab now is to find out how this leader is selected.
Okay, next up, Sua Myong
Sua Myong – How does RNAi actually work?
Sua employs FISH, a super resolution technique, to view RNA interference in single cells. What she finds is the first insight into the function of RNAi with reference to biologically relevant miRNAs since Fire and Mello won the nobel prize for the work and revolutionised the field.
The investigative technique works by targeting particular mRNAs with search RNAi strands loaded with 30 to 40 fluorophores each. By watching the transient binding of these RNAs in TIRF, the PSFs can be localised and the relative number of RNAs between conditions can be quantified.
The group tried to figure out which parts of shRNA were important in terms of its structure and its interactions with DICER and RISC, the proteins that bind it to genes of interest and cut the genes, respectively. To do this they altered the shRNA supplied to the cell by first changing the size of the hairpin loop, before measuring silencing using the FISH technique described above.
Longer loop size improved silencing, and it was found that this was dependent on better association with the DICER protein.
Introducing mismatches in the nucleotides was also trialled, as this is common in biological settings – many endogenous microRNAs have these mismatches which prevent them from binding the target gene perfectly.
What they found was that the altered shRNA had not problem binding DICER, but was inhibited in its handover to the RISC complex.
This is important, because it may be a way for cells to control the power of microRNAs, in cases where protein translation must be fine tuned.
Thanks for reading – that’s me over and out for this meeting.
Michael Shannon (Dylan Owen lab, KCL)