Almost forgot to thank @reneinckemann.bsky.social for helping us set up the chloroplast transformation system we relied so heavily upon! Go read his cool paper here:
www.nature.com/articles/s41...

Lastly, if you prefer to see this as a poster, you can find that here:
james-r-barrett.github.io/presentation...

Huge thanks to @yorkbioscience.bsky.social for outstanding facilities and support as always!

None of this would be possible without the pioneering works of some previous pyrenoid pros (to list but a few):
www.pnas.org/doi/10.1073/...
www.pnas.org/doi/10.1073/...
www.cell.com/cell/fulltex...
www.nature.com/articles/s41...
www.nature.com/articles/s41...
journals.aps.org/prxlife/abst...

This was a great collaboration with bsky-less Gaurav, @cellarchlab.com, @phaips.vd.st, @mariahondele.bsky.social and a product of the York Physics of Pyrenoids Project (YP3) with @bpsiyork.bsky.social, @mjplevin.bsky.social, @alexpaynedwyer.bsky.social and many others!

In summary we used a whole bunch of techniques across in vitro and in vivo contexts to connect valency to condensate formation and fitness. We argue this is a readily tuneable parameter for condensates that determines their properties.

Using this we showed that the linker with 9x stickers outcompetes the other strains, as well as the WT background! This experiment connects linker valency to condensate function in vivo.

We wanted to see what effect this has on CO2-fixing efficiency of the pyrenoid, but conventional spot tests weren’t sensitive enough. We developed a flow cytometry-based competition assay to unequivocally determine the phenotypes of the strains:

We also noticed that Rubisco condensation happens quicker with more stickers.

The great thing about working with Rubisco condensates is that we can see individual Rubiscos using cryo-electron tomography #TeamTomo! Using this approach @phaips.vd.st was able to show that higher linker valency increases Rubisco concentration.

We expressed the same constructs in the chloroplast of Chlamydomonas to observe the effects on pyrenoid formation, where we see a clear threshold for Rubisco condensation and a dependency on sticker number for Rubisco partitioning.

We observed similar effects in simulations which were parameterised using our biophysical measurements of Rubisco and linkers.

See our parameterisation here:
journals.aps.org/prl/abstract...

In vitro, we see a clear dependence on sticker number for droplet critical concentration and partitioning of Rubisco.

We explored across length scales in vitro, in silico and in vivo.

We varied the number of stickers (valency) in the linker protein to understand the effects on pyrenoid formation, properties and function, using Chlamydomonas as our model system.

Pyrenoids are found in diverse algae and mediate a large proportion of global carbon fixation. At the molecular level, the pyrenoid is a condensate of the CO2-fixing enzyme Rubisco, which is phase-separated by a multivalent linker protein with stickers and spacers.