Selective sequestration of signalling proteins in a membraneless organelle reinforces the spatial regulation of asymmetry in Caulobacter crescentus

Abstract

Selective recruitment and concentration of signaling proteins within membraneless compartments is a ubiquitous mechanism for subcellular organization 1 – 3 . The dynamic flow of molecules into and out of these compartments occurs on faster timescales than for membrane-enclosed organelles, presenting a possible mechanism to control spatial patterning within cells. Here, we combined single-molecule tracking and super-resolution microscopy, light-induced subcellular localization, reaction-diffusion modeling, and a spatially-resolved promoter activation assay to study signal exchange in and out of the 200 nm cytoplasmic PopZ microdomain at the cell pole of the asymmetrically dividing bacterium Caulobacter crescentus 4 – 8 . Two phospho-signaling proteins, the transmembrane histidine kinase CckA and the cytoplasmic phosphotransferase ChpT, provide the only phosphate source for the cell fate-determining transcription factor CtrA ( Fig. 1a ) 9 – 18 . We found that all three proteins exhibit restricted rates of entry into and escape from the microdomain and enhanced phospho-signaling within, leading to a submicron gradient of activated CtrA~P 19 that is stable and sublinear. Entry into the microdomain is selective for cytosolic proteins and requires a binding pathway to PopZ. Our work demonstrates how nanoscale protein assemblies can modulate signal propagation with fine spatial-resolution, and that in Caulobacter , this modulation serves to reinforce asymmetry and differential cell fate of the two daughter cells.

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

“…Fortunately, single-molecule tracking has recently emerged as a powerful tool for examining LLPS in vivo (51,111,112). Indeed, the strongest evidence for bacterial condensates to date comes from single-molecule tracking of proteins in polar microdomains in Caulobacter (113). This study revealed that PopZ imposes a diffusion barrier to cytosolic proteins and restricts the mobility of signaling molecules at the cell poles.…”

The emergence of phase separation as an organizing principle in bacteria

“…The liquid-like behavior of condensates has been tested by fluorescence recovery after photobleaching (FRAP) and by observing droplet fusion events (17,21,24,26,28). Finally, (18,21,26,30) and measured the diffusive properties of their molecular components (23,26,31,32). As the methods improve and rigorous controls are implemented, single-molecule methods are becoming increasingly critical for understanding bacterial subcellular organization due to the very small size of the relevant features in these microscopic organisms.…”

Integrative and quantitative view of the CtrA regulatory network in a stalked budding bacterium

“…In C. crescentus, two hypotheses have been put forward to explain the condensation of CckA into a polar focus. First, CckA was shown to require a high enough local concentration to self-interact efficiently and become active as a kinase [26,27,77,78]. In addition, its Finally, there may be a pathway that acts in parallel to ChpT to transfer signals from CckA to CtrA (oval F, 29%).…”

“…accumulation at the flagellated pole may help to establish a gradient of CtrA~P with a maximum close to the pole-proximal replication origin, thereby blocking replication initiation in the (nascent) swarmer cell [77,79,80]. The absence of a polar CckA focus in the bud compartment of H. neptunium may suggest that CckA activity is not regulated at the level of protein concentration in this species, unless its activity is triggered at considerably lower threshold levels.…”

“…Another aspect that may contribute to gradient formation could be the specific and/or non-specific DNA-binding activity of CtrA(~P). A recent study in C. crescentus revealed that CtrA shows constrained diffusion, likely because of its interaction with the nucleoid [77]. Previous work showed that its affinity for specific DNA binding sites increases by two orders of magnitude upon phosphorylation [81][82][83], and it is conceivable that phosphorylation also increases, at least to some extent, its affinity for non-specific DNA.…”