... we wanted to easily label neighbouring cells without having to engineer them with label-uptake machinery

So, Tamina found a neat trick. Positively-supercharged fluorescent protein interacts with negatively-charged cell membranes to enable efficient receptor-independent delivery into cells (see PMID: 19307578)

And….
2/n

… we wanted ultra-bright sensitive & reliable neighbour labelling

So, Tamina used HaloTags to amplify signal…. this is BRILLIANT because it also lets us label neighbours in any colour we like, just by changing the choice of Halo dye! My favourite colour is orange.

Look at those orange sparkles decorating neighbours of red secretors!

(orange here is actually far-red – this is gentle on the cells during live imaging, so we had lots of fun using it for time-lapse experiments).

AND...
3/n

...if you don’t want to use Halo dyes we have a version of PUFFFIN with a built-in Ultra-Bright mNeon-green amplifier (Tamina’s favourite colour is green).

Just for fun, Tamina made a pretty green stripe across a group of cells by making them crash into a crowd of PUFFFIN-secretors.

AND....

4/n

....Tamina has designed a modular single-plasmid system – we can switch components in and out of the PUFFFIN plasmid like Lego-bricks

This is thanks to the amazing EMMA assembly method of Martella et al https://pubs.acs.org/doi/10.1021/acssynbio.7b00016

We particularly like the fact that we can slot in a transgene to induce differentiation in one cell while at the same time applying fluorescent label to its neighbours..

.. This is super-useful because…

5/n

......PUFFFIN labelling allows us to unambiguously identify labelled neighbours of PUFFFIN secretors using flow cytometry.

It is easy to distinguish labelled neighbours from unlabelled cells and from secretor cells.

So we can sort and profile all three populations to uncover neighbour-responses.

6/n

We are very excited to see where PUFFFIN takes us in our explorations of how cells influence their neighbours during development, homeostasis and disease.

Please let us know if you think it could be useful for your experiments too!

7/n

Congratulations to the innovative and creative scientist that is Tamina Lebek!

And big thanks to Mattias Malaguti for expert guidance and advice at every step on the path to PUFFFIN

And finally.....

8/n

Tamina has a great story about how the concept behind PUFFFIN was inspired by the unusual behaviour of some real-life puffins… should we update our preprint with a Supplemental Anecdote?

(anecdote-unrelated picture of a small Lowell pointing at a small puffin)

9/9

An update on our PUFFFIN preprint (see preceding thread), now published in @embojournal via #ReviewCommons
https://www.embopress.org/doi/full/10.1038/s44318-024-00154-w

A constructive review process led to two major additions to the paper since preprint, which are...

10/n