Researchers in Nobel prize winner W.E. Moerner’s laboratory at the Stanford Department of Chemistry and the Lucy Shapiro lab at the Stanford School of Medicine and the Chan Zuckerberg Biohub joined together to study how protein concentration and diffusion rates in membraneless compartments in bacteria influence signaling rates and subcellular organization.[1]
Bacterial cells organize their functions into subcellular regions just like eukaryotic cells, but generally these are not enclosed by membranes. Membraneless compartments are unique regions in a wide range of cell types. These nanoscale regions are interesting to researchers who investigate signaling rates, since they allow signals to be transmitted on a faster time scale than cellular organelles enclosed by a lipid membrane.
In this paper, researchers used asymmetrically dividing Caulobacter crescentus bacteria to observe membraneless compartments at each pole of the cell before, during, and after cell division. Like human stem cells, Caulobacter daughter cells undergo different cell fates after division. Caulobacter uses a different composition of proteins at each pole to prime asymmetric division, but the dynamics of how these protein signals communicate with the remainder of the cell was unknown. In order to understand polar dwell time, diffusion rates, and to measure protein concentrations in vivo, they fluorescently tagged proteins CckA, CtrA, ChpT and PopZ, combining 3D single-molecule tracking with super-resolution microscopy, enabled by Double Helix Optics’ Light Engineering™ technology, to track individual proteins and observe their co-localization at the poles during cell division.
Using their novel single-molecule and intensity profiling measurements, the researchers calculated the concentrations of CckA, the source and sink of cell-differentiation signal, at the new pole, old pole, and cell body. CckA distributions confirmed in vitroCckA concentrations. Using 3D single molecule tracking with super-resolution microscopy in vivo, they attained high enough precision to image protein distributions in the 200 nm pole region, allowing them to measure differential CckA density between the two microdomains. In addition, the downstream signaling proteins were tracked leaving the selected cellular region, travelling over 1.5 µm from the cell pole.
Their work demonstrated the importance of nanoscale protein assemblies for modulation of signal propagation and the cell fate of daughter cells. Additionally, they demonstrated the important role microdomain condensates play during development and gene regulation, with implications for many species including humans. The observed localized switching between diffusive states studied here supports cell biology research on how “a wide range of eukaryotic systems use biomolecular condensates to concentrate, transiently sequester, and slow the diffusion of signaling proteins,” an emerging field expected to have significant biomedical importance.
Interested in learning how Double Helix Optics can enhance your research? Contact one of our engineers and discover how you can “explore what no one has seen before.”
[1] Read the original paper:
“Selective sequestration of signaling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus” Lasker, K., von Diezmann, L., Zhou, X. et al. Nat Microbiol 5, 418–429 (2020).