Phospho-signaling couples polar asymmetry and proteolysis within a membraneless microdomain in C. crescentus

Abstract

Asymmetric cell division in bacteria is achieved through cell polarization, where regulatory proteins are directed to specific cell poles. Curiously, both poles contain a membraneless microdomain, established by the polar assembly hub PopZ, through most of the cell cycle, yet many PopZ clients are unipolar and transiently localized. We find that PopZ's interaction with the response regulator CpdR is controlled by phosphorylation, via the histidine kinase CckA. Phosphorylated CpdR does not interact with PopZ and is not localized to cell poles. At poles where CckA acts as a phosphatase, de-phosphorylated CpdR binds directly with PopZ and subsequently recruits ClpX, substrates, and other members of a protease complex to the cell pole. We also find that co-recruitment of protease components and substrates to polar microdomains enhances their coordinated activity. This study connects phosphosignaling with polar assembly and the activity of a protease that triggers cell cycle progression and cell differentiation.

Fig. 1|. CpdR Phosphorylation State Influences its Co-localization with PopZ.

a, CpdR phospho-signaling at stalked (left) and swarmer (right) cell poles, where CckA acts as a phosphatase or kinase, respectively. b, C. crescentus cells expressing CpdR-YFP were synchronized and, at indicated time points over a cell cycle time course, aliquots were removed for observation. The frequency of polar foci was plotted (n>300/time point). c, CpdR-YFP phosphorylation levels in lysates from b were observed by Phos-tag gel electrophoresis. d, Genetic modifications for controlling CpdR-YFP phosphorylation. Single-copy cpdR-yfp is expressed from the native promoter, multicopy cckA variants are expressed from P_xyl without xylose induction. e, Localization of CpdR-YFP or CpdR_D51A-YFP and mChy-PopZ in different CckA signaling contexts. Arrowheads mark polar localization. f, CpdR-YFP phosphorylation levels in lysates from e, observed using Phos-tag gel electrophoresis. For CckA variants, WT=wildtype; H+=hyperactive kinase; K−=kinase deficient. To equalize loading of unstable non-phosphorylated CpdR-YFP, 3-fold or 5-fold more material was loaded in K− and D51A lanes, respectively. g, Cells expressing CpdR-YFP or CpdR_D51A-YFP and mChy-PopZ were observed during cell division, using time-lapse microscopy at 15 minute intervals. Here, the fluorescence levels of individual panels were adjusted to aid visualization.

Fig. 2|. CpdR Phosphorylation State regulates CpdR-PopZ interactions.

a, Reconstitution of CpdR phosphorylation and PopZ interaction in E. coli. b, mChy-PopZ and CpdR-GFP were observed by microscopy and normalized fluorescence intensities were plotted against cell length (graphs). For CckA variants, WT=wildtype; H+=hyperactive kinase; K−=kinase deficient. c, CpdR-GFP phosphorylation levels in lysates from c, observed using Phos-tag gel electrophoresis. d, Images of CpdR-GFP interactions with PopZ condensates, with phase contrast and YFP fluorescence channels shown. GFP-only is included as a negative control. +AcP: after incubation with acetyl phosphate. e, CpdR-GFP phosphorylation levels in samples from d, observed using Phos-tag gel electrophoresis. Data shows composite images from two different gels. f, Fluorescence values from d were normalized to the maximum signal intensity and plotted against percentage of CpdR-YFP~P in 2e. g, FRAP and FLIP assay for CpdR-GFP in E. coli cells expressing PopZ. Recovery and loss of fluorescence were plotted against time in seconds.

Fig. 3|. CpdR Phosphorylation State Drives the Localization of Protease Components.

a, Diagram of CpdR-mediated substrate degradation by ClpXP. Depending on the substrate, an additional adaptor (green) could be RcdA and/or PopA. b, Genetic modifications for observing RcdA -GFP or ClpXP -GFP in different CpdR phosphorylation contexts, built in a ΔcpdR ; popZ::mChy-popZ C. crescentus strain background. c-d, Localization of RcdA-GFP or ClpX-GFP and mChy-PopZ in different CpdR phosphorylation contexts. Arrowheads mark polar localization.

Fig. 4|. Polar localization of CpdR substrates influences their rate of degradation.

a, Localization of YFP-tagged substrates in ΔcpdR; popZ::mChy-popZ and ΔpopZ C. crescentus strain backgrounds. b, Time-lapse images of YFP-tagged substrate localization in a WT C. crescentus background, at 4 minute intervals. Blue arrows mark frames with foci in stalked cell, orange arrows mark frames with foci in swarmer cell. Pink bar idicates the time of cell separation. After accounting for photobleaching and temporally alignging the cells with respect to the time of cell separation (n=20), average fluorescence intensities for stalked and swarmer cell bodies, normalized to maximum fluorescence intensity, were plotted against time (line graphs). The percentage of cell bodies ehxhibiting polar localization, normalized to the highest percentage, were plotted on the same time axis (solid curves). c, Degradation of HA-tagged proteolysis substrates following inducer wash-out, observed by western blotting with α-HA antibody. Average band intensities from three separate experiments were plotted against time (graphs, bar=standard deviation).

Fig. 5|. Conceptual models of substrate proteolysis in membraneless polar microdomains.

a-b, Three-dimensional reaction-diffusion simulations with two types of particles, colored red and yellow, that disappear after colliding. Parameters for cell size, particle diffusion rate in bulk cytoplasm, and the number of particles are based on physoligical values for C. crescentus. a, Cells with different sizes of membraneless polar microdomains, where particles become concentrated because they diffuse at 1/40^th the rate in bulk cytoplasm. PopZ microdomains occupy 0.5% of cell volume in C. crescentus. b, Cells with different concentrations of particles in polar microdomians. Slower diffusion rates in polar microdomains result in higher concentration. c, Asymmetric localizatin of CpdR and associated ClpXP complexes as a consequence of asymmetric CckA signaling activity. Inset panels show fluorescence images of a C. crescentus stalked cell, where mChy-PopZ is localized to both poles and CpdR-YFP is localized to only the stalked pole.