The material properties of a bacterial-derived biomolecular condensate tune biological function in natural and synthetic systems

Abstract

Intracellular phase separation is emerging as a universal principle for organizing biochemical reactions in time and space. It remains incompletely resolved how biological function is encoded in these assemblies and whether this depends on their material state. The conserved intrinsically disordered protein PopZ forms condensates at the poles of the bacterium Caulobacter crescentus, which in turn orchestrate cell-cycle regulating signaling cascades. Here we show that the material properties of these condensates are determined by a balance between attractive and repulsive forces mediated by a helical oligomerization domain and an expanded disordered region, respectively. A series of PopZ mutants disrupting this balance results in condensates that span the material properties spectrum, from liquid to solid. A narrow range of condensate material properties supports proper cell division, linking emergent properties to organismal fitness. We use these insights to repurpose PopZ as a modular platform for generating tunable synthetic condensates in human cells.

Fig. 1. PopZ phase separates in Caulobacter crescentus.

a PopZ self-assembles at the poles of wildtype Caulobacter cells. A fluorescent image of ΔpopZ Caulobacter cells expressing mCherry-PopZ (red) from the xylX promoter on a high copy plasmid overlaid on a corresponding phase-contrast image. Scale bar, 1 μm. b The PopZ microdomain excludes ribosomes and forms a sharp convex boundary. (left) Slice through a tomogram of a cryo-ET focused ion beam-thinned ΔpopZ Caulobacter cell overexpressing mCherry-PopZ. A dashed red line shows the boundaries of the PopZ region. (right) Segmentation of the tomogram in (left) showing the outer membrane (dark brown), inner membrane (light brown), and ribosomes (gold). Scale bar, 1 μm. c, d PopZ creates droplets in deformed Caulobacter cells. c A fluorescent image of Caulobacter cells bearing a mreB A325P mutant, expressing mCherry-PopZ (red) from the xylX promoter on a high copy plasmid overlaid on a corresponding phase-contrast image. Scale bar, 1 μm. d Fluorescent images show the PopZ microdomain (red) extending into the cell body, concurrent with the thinning of the polar region, producing a droplet that dynamically moves throughout the cell. Frames are 2 min apart. Scale bar, 1 μm. e PopZ dynamics are not affected by a release from the cell pole. Recovery following targeted photobleaching of a portion of an extended PopZ microdomain in wildtype and mreB A325P mutant cells. Cells expressing mCherry-PopZ from a high copy plasmid were imaged for 12 frames of laser scanning confocal microscopy following targeted photobleaching with high-intensity 561 nm laser light. Shown is the mean ± SEM of the normalized fraction of recovered signal in the bleached region; n equals 15 cells.

Fig. 2. PopZ phase separates in vitro and in human U2OS cells.

a The PopZ protein forms droplets in vitro in the presence of magnesium. Differential interference contrast microscopy images of PopZ at physiological concentration of 5 µM in 5 mM sodium phosphate at pH 6.0 with either 2 mM MgCl2 (left) or 5 mM MgCl2 (right). b Caulobacter PopZ expressed in human U2OS cells forms phase-separated condensates (black) in the cytoplasm but not the nucleus (N). c In vivo fusion and growth of PopZ condensates in human U2OS cells. 80 s time-lapse images of a small PopZ condensate (green) merging with a large PopZ condensate. Scale bar, 5 μm. d PopZ expressed in human U20S cells retains selectivity. (Top) EGFP-PopZ (green) and stress granule protein mCherry-G3BP1 (purple) form separate condensates. (Bottom) EGFP-PopZ (green) recruits the Caulobacter phosphotransfer protein mCherry-ChpT (magenta) when co-expressed in human U2OS cells. Scale bar, 10 μm.

Fig. 3. Domain organization of the PopZ condensate.

a Domain organization of the PopZ protein from Caulobacter crescentus. PopZ is composed of a short N-term region with a predicted helix, H1 (gray box), a 78 amino-acid intrinsically disordered region (IDR, blue curly line), and a C-term region with three predicted helices, H2, H3, H4 (gray boxes). b. Region deletion and its effect on PopZ condensation. (top) EGFP fused to five PopZ deletions (black) expressed in human U2OS cells. (bottom) mCherry fused to four PopZ deletions (Δ1–23, Δ24–101, Δ102–132, and Δ133–177) (red) expressed in ΔpopZ Caulobacter cells. Scale bar, 10 μm c. Region deletion and its effect on PopZ mobility. FRAP, shown as mobile fractions, the plateau of the FRAP curves for the wildtype (gray), for the five region deletions (blue, green, and brown). Also shown are the significances, calculated as Kruskall–Wallis tests with Dunn’s correction, of the difference in mobility between pairs of mutants. ns indicates no significant difference, two asterisks indicate p-value < 0.01, and four asterisks indicate p-value < 0.0001. n is between 15 and 20 granules per condition. Source data underlying graphs are provided in Source Data. d conservation of the PopZ protein regions. Graphical representation of a multiple alignment of 99 PopZ homologs within the Caulobacterales order. Each row corresponds to a PopZ homolog and each column to an alignment position. All homologs encode an N-terminal region (green), an IDR (blue), and a C-terminal helical region (brown). White regions indicate alignment gaps, and gray regions indicate predicted helices 1 to 4. Phylogeny tree of the corresponding species is shown, highlighting the four major genera in the Caulobacterales order: Asticcacaulis (pink), Brevundimonas (gray), Phenylobacterium (light purple), and Caulobacter (dark purple). Notably, all species within the Brevundimonas genus code for insertion between helix 2 and helix 3.

Fig. 4. Modular organization regulates the dynamics of the PopZ condensate.

a The predicted radius of gyration (R_G) for a half linker (IDR-40, 40 aa) (light blue), full wildtype linker (IDR-78, 78 aa) (gray), and a double linker (IDR-156, 156 aa) (dark blue). b PopZ linker expands beyond the denatured limit. The expected R_G of denatured proteins as a function of the number of amino acids is shown in black. Dimensions of PopZ-like linkers with varying lengths are shown in red, and dimensions of IDR-40, 78, and 156 are shown in shades of blue and gray. The red dashed line is an analytical fit with a scaling value of 0.80 with a prefactor value of 1.14. c Phase diagram of PopZ expressed in U2OS cells. (top) Three states of PopZ condensation: dilute phase (blue, left), two-phase (a diffused phase and condensed phase, red, middle), and a dense phase (gray, right). EGFP fluorescence intensity from blue (low) to white (high) and nucleus boundary as a white dotted line. Scale bar, 10 μm. (bottom) Phase diagrams of EGFP fused to either of the three PopZ variants. Each dot represents data from a single cell, positioned on the x-axis as a function of the cell mean cytoplasmic intensity. Dot color indicates phase. d Quantification of the partition coefficient of each of the three linkers. A higher partition coefficient indicates denser condensates. Two-sided student’s t-test; Four (two) asterisks indicate p-value < 0.0001 (0.01). n equals 30 granules per condition. Source data underlying graphs are provided in Source Data. e Schematics of the oligomerization domain (OD) of the wildtype PopZ (trivalent, left) and an OD with increased valency consisting of five helices, with a repeat of helices 3 and 4 (pentavalent, right). f The balance between condensation promoting and counteracting phase separation tunes condensate material properties. FRAP, shown as mobile fractions, for PopZ with a trivalent OD and a linker of three different lengths (gray and blue), and PopZ with a pentavalent OD with IDR-78 (light green) and IDR-156 (dark green). Two-sided student’s t-test; ****p-value < 0.0001. n equals 25 granules for each mutant. Source data underlying graphs are provided in Source Data.

Fig. 5. PopZ material properties are directly linked to Caulobacter viability and are modulated by conserved IDR properties.

a The sequence composition of the PopZ IDR is conserved across Caulobacterales. Histograms are calculated across 99 PopZ homologs within the Caulobacterales order and show a tight distribution for the following four parameters. (top, left) The mean fraction of acidic residues is 0.29 ± 0.004 (red). (top, right) The mean fraction of prolines is 0.23 ± 0.006 (purple). (bottom, left) Among the acidic residues within the IDR, the fraction of those found in the N-terminal half (light blue, 0.57 ± 0.011) and the C-terminal half of the IDR (dark blue, 0.43 ± 0.011). (bottom, right) Among the IDR prolines, the fraction of those found in the N-terminal half (light blue, 0.5 ± 0.015) and the C-terminal half of the IDR (dark blue, 0.5 ± 0.015). Source data underlying graphs are provided in Source Data. b Amino acid composition plays a role in PopZ mobility. FRAP, shown as mobile fractions, for PopZ with its wildtype IDR (light gray) and five mutants: Substituting either half or all of the acidic residues for asparagine (DE-N 50% in red and DE-N 100% in pink, respectively), substituting all prolines for glycines (P-G 100% in purple), and moving all acidic residues to either the N-terminal part or the C-terminal part of the linker (yellow and brown, respectively). n equals 20 granules per condition. c Growth is linked to PopZ’s material state. Growth, derived from serial dilution growth assay (Methods), as a function of FRAP mobility for ten mutants. These include from left to right: 100% DE-N (pink), Pentavalent (light green), 50% DE-N (red), IDR-156+pentavalent (green), C-acidity (orange), IDR-40 (light blue), wildtype (gray), 100% P-G (purple), N-acidity (yellow), and IDR-156 (blue). Examples of serial dilutions are shown for wildtype (gray box), 50% DE-N (red box), IDR-156 (blue box), and 100% DE-N (pink box). A polynomial fit with an R-square of 0.86 is shown in red. Three biological replicates, each with three technical replicates, were measured for each strain. a.u., arbitrary unit.

Fig. 6. PopZ material properties affect cytosol organization.

a PopZ IDR-156 condensates retain ribosome exclusion. (left) Slice through a tomogram of a cryo-focused ion beam-thinned ΔpopZ Caulobacter cell overexpressing mCherry-PopZ with IDR-156. (right) Segmentation of the tomogram in (left) showing annotated S-layer (orange), outer membrane (dark brown), inner membrane (light brown), and ribosomes (gold). Scale bar, 0.25 μm. b PopZ IDR-156 condensates retain DNA exclusion. PopZ IDR-156 condensates expressed in ΔPopZ cells dynamically moved in the cytosol and excluded DAPI-stained DNA (blue). Scale bar, 5 μm. c The CtrA activation network is sequestered to the PopZ condensate. The schematic shows the auto-kinase CckA phosphorylating the phospho-transfer protein, ChpT, which in turn phosphorylates the master transcription factor, CtrA. All three proteins are sequestered to the PopZ condensate^,,. Phosphorylation of CtrA occurs largely inside the condensate. CtrA~P leaves the DNA-free PopZ condensate and activates an array of asymmetry regulating genes, including sciP^, and pilA. d, e. PopZ material properties affect ChpT recruitment. d Representative cells are shown for pentavalent, IDR-40, and IDR-156. e The graph shows the partition coefficient of ChpT inside PopZ condensates as a function of condensate mobility. The coefficient was calculated as ChpT fluorescence intensity inside the PopZ condensates divided by the fluorescence intensity outside the condensates. A higher partitioning coefficient indicates stronger recruitment. Data shown for pentavalent (light green), pentavalent with IDR-156 (green), IDR-40 (light blue), wildtype (gray), and IDR-156 (blue). n equals 60 cells per strain. Two-sided student’s t-test; ns indicates no significant difference, two asterisks indicate p-value < 0.01, and four asterisks indicate p-value < 0.0001. Source data underlying graphs are provided in Source Data. f PopZ material properties affect the transcriptional program regulating asymmetry. Expression levels of CtrA activated genes sciP and pilA in cells expressing different PopZ mutants. Color code as in e. Three biological replicates (gray points), each with at least two technical replicates, were measured for each strain and each gene. Source data underlying graphs are provided in Source Data.

Fig. 7. A modular platform for generating synthetic condensates with tunable properties.

a Re-engineering PopZ as a modular platform for the generation of designer condensates. The oligomerization domain (PopTag) drives phase separation, the IDR (spacer) tunes material properties, and the n-terminal domain (actor) determines functionality. b Scheme highlighting setup of the PopTag system and formation of EGFP-PopTag condensates in U2OS cells. c Changing the linker length alters the FRAP dynamics and partitioning coefficient of PopTag condensates. Two-sided student’s t-test; ****p-value < 0.0001. For the FRAP dynamics plot, 15 condensates were analyzed per condition, and 30 condensates were analyzed per condition for the partitioning coefficient plot. Source data underlying graphs are provided in Source Data.

Fig. 8. NanoPop can inhibit nuclear import.

a, b The NanoPop system. a NanoPop is the fusion of the PopTag to a nanobody (nb), which allows the recruitment of clients into cytoplasmic condensates. In this example, PopTag is fused to a GFP nb, which allows the recruitment of EGFP-tagged protein. (b, top) Cells expressing EGFP and GFP nb fused to mCherry (GFPnb-mCherry) show diffused EGFP, diffused GFPnb-mCherry, and no correlation between them. (b, bottom) Cells expressing EGFP and GFPnb-mCherry-PopTag show GFPnb-mCherry-PopTag condensates with co-localized EGFP. c Schematics of the NanoPop system with EGFP fused to FUS. d–f NanoPop can inhibit nuclear import of FUS. N = nucleus, C = cytoplasm. d Co-expression of an EGFP-targeting nb does not impair the nuclear import of EGFP-FUS, whereas co-expression of NanoPop does. The strength of this effect is dependent on the NanoPop linker length (PopZ linker 40 versus PopZ linker 156). e Nuclear import is quantified by the nucleocytoplasmic ratios (N/C), the EGFP-FUS signal in the nucleus divided by the signal in the cytoplasm. Schematics of EGFP signals for low, medium, and high N/C are shown. f Quantification of EGFP-FUS N/C dependence on the material properties of its recruiting protein. Axes indicate average cellular mCherry and EGFP intensity for co-expression of EGFP-targeting nb alone (top), NanoPop-L40 (middle), and NanoPop-L156 (bottom). The color code indicates N/C, as illustrated in e. n equals 45, 39, and 31 cells for Nanobody, NanoPop-L40, and NanoPop-L156, respectively. a.u., arbitrary unit.