Super excited to share the new preprint from @LaskerKeren and me. TL/DR: We studied a bacterial protein droplet, found that material properties tune fitness, and we re-engineered this protein into a platform for designer condensates. A thread(1/n) https://www.biorxiv.org/content/10.1101/2021.02.03.429226v1

We all know that eukaryote cells have membraneless organelles and lots of condensates. But prokaryotes are more than just bags of ribosomes. @LaskerKeren has been working for the last few years on the PopZ microdomain in the bacterium Caulobacter to figure out what it does. (2/n)

Caulobacter divides by asymmetric cell division, and PopZ is the key regulator of this event. But what is this "microdomain"? Well, in bacteria it's just a tiny spot at the cell pole. So can we make these things bigger and study them in more detail? (3/n)

We decided to express the PopZ protein in human cells, and to our surprise the protein formed these large liquid droplets in the cytoplasm (yellow). More importantly, when coexpressing G3BP1 (red), we see that PopZ droplets do not interfere with human stress granules. (4/n)

So, we can use human cells as an orthogonal system to study PopZ droplets, and it seems PopZ uses a molecular grammar that is different from human condensates. (5/n)

. @LaskerKeren talks in her thread more about the biophysics of PopZ phase sep. In brief, there is a helical oligomerization domain that pulls, and a negatively charged IDR that pushes. This push-pull mechanism is what drives PopZ condensation and tunes material state (6/n)

We rationally designed an array of mutants that span the material state spectrum. From very fluid to viscous to solid-like. Since we know that PopZ regulates cell division in Caulobacter we could now ask a super important question: Material properties = function? (7/n)

Imo this is the most important finding of our paper. If PopZ behaves too fluid, bacteria fail to divide properly and grow into these long filaments. If PopZ becomes too solid the same thing happens! Hence, material properties tune biological fitness. (8/n)

Details on why and how we think material properties affect PopZ function you can find in the manuscript, but it seems to -in part- relate to how cells can correctly localize the PopZ microdomain to the cell pole. (9/n)

Ok, that was Caulobacter biology. Now what about these designer condensates that I promised? PopZ does form condensates in human cells, does not seem to interfere with endogenous condensates, and has a highly modular architecture. (10/n)

We re-engineered PopZ into the PopTag module. By fusing this module to "actor" modules one can create condensates of different functionality. We can make everything from enzymatic reactors to selective sinks. Find the module on @FASEdesign (11/n) https://designercondensates.org/poptag 

Our favorite application so far is the NanoPop system, which consists of fusing the PopTag to nanobodies. This allows for the specific recruitment of target proteins into these cytoplasmic condensates. Check out the following examples using a GFP nanobody. (12/n)

Want to use the PopTag or NanoPop systems for your project? Reach out and we gladly help/work with you! (13/n)

Thanks to LS and ADG who have been so incredibly supportive of our work, @czbiohub @StanfordMed, @alexholehouse @TheVillaLab and all other collaborators, @idpsig @IDPseminars and everyone else who gave us feedback. Hope you enjoy the read and we welcome any comments! (14/14)