Daily Cycles of Reversible Protein Condensation in Cyanobacteria

Abstract

An emerging principle of cell biology is the regulated conversion of macromolecules between soluble and condensed states. To screen for such regulation of the cyanobacterial proteome, we use quantitative mass spectrometry to identify proteins that change solubility during the day-night cycle. We find a set of night-insoluble proteins that includes many enzymes in essential metabolic pathways. Using time-lapse microscopy and isotope labeling, we show that these proteins reversibly transition between punctate structures at night and a soluble state during the day without substantial degradation. We find that the cyanobacterial circadian clock regulates the kinetics of puncta formation during the night and that the appearance of puncta indicates the metabolic status of the cell. Reversible condensation of specific enzymes is thus a regulated response to the day-night cycle and may reflect a general bacterial strategy used in fluctuating growth conditions.

Keywords: circadian clock; cyanobacteria; metabolism; protein condensation.

Figure 1.. Protein Condensation in the Cyanobacterial Proteome During the Day-Night Cycle

(A) Schematic of experimental design. Wild-type cell cultures were synchronized by one light-dark cycle. Cells were harvested at the end of the day and 8 h into the dark (arrowheads). Cells were lysed and fractionated by ultracentrifugation into soluble and pellet fractions. Fractions were trypsin digested, labeled with TMT tags and analyzed by tandem mass spectrometry. (B) Scatter plot shows supernatant-pellet ratio changes in light vs dark samples for one biological replicate (gray dots, N =1777). The proportion of protein in each fraction was estimated from ratios of spectral counts. The shaded region indicates proteins with supernatant fraction >80% in the light and <45% in the dark (dark-demixing). Selected proteins marked in red. (C) Schematic showing steps catalyzed by dark-demixing enzymes (black rectangles) in central metabolism, including reactions from glycolysis and TCA cycle, pentose phosphate pathway, amino acid biosynthesis, lipid and cell wall synthesis.

Figure 2.. Live Cell Imaging of Reversible Protein Condensation

(A) Microscopy of dark-demixing proteins fused with EYFP. Cultures were grown under agar pads for live cell imaging during light-dark cycles. Micrograph filmstrips show the formation of EYFP puncta in dark and their disappearance following dawn (upper subpanels). Quantification of the total intensity of puncta during the experiment (lower subpanels). Black traces represent puncta fluorescence intensity from single cells. The red trace is the average of at least 10 cells. Pie charts show the percentage of cells with EYFP puncta. N = the number of cells analyzed. Scale bar = 3 μm. (B) Average time trace of fluorescence intensity in the cytosol (black line), puncta (red line) and total (blue dashed line) in EYFP-MetX (top) or EYFP-ASD (bottom) expressing cells before and after the end of night (shaded region). N=108 cells, bars show 95% CI. (C) Scatter plot showing change of cytosol fluorescence intensity (vertical axis) versus change in punctate fluorescence intensity (horizontal axis) over the 3 h window following lights-on. Dashed line shows linear regression with best-fit slope. (D) Pulse-chase labeling experiment. At end of the night, cultures were labeled with L-Leucine ¹³C₆ for 1 h before being transferred to light, harvested, and analyzed by tandem mass spectrometry. Normalized log-histogram in grey shows distribution of isotope labeling percentage in the soluble fraction after 4 h of light exposure for all detected proteins (gray, N = 864) and detected dark-demixing proteins (red, N=107).

Figure 3.. The Circadian Clock Regulates the Kinetics of Puncta Formation

(A) Imaging of dark-demixing proteins (MetX and ASD) follow transition to dark at either subjective dawn (upper) or subjective dusk (lower). (B-C) Imaging of MetX-EYFP and ASD-EYFP in circadian clock mutants. Micrograph filmstrips show the formation of EYFP puncta following the transfer of cells to darkness. Black bars indicates times when cells are not illuminated. (C-D) Puncta fluorescence intensity during a light-dark cycle in circadian clock mutants. Error bars show standard deviation from 2 independent experiments. All scale bars = 3 μm.

Figure 4.. Metabolic Limitation Is a Key Driver of Protein Condensation

(A) Live cell imaging of MetX-EYFP in the light (left) or after 1 h in darkness (right) in either wildtype (top) or in a glycogen breakdown-deficient mutant (ΔglgP). Scale bar: 3μm. (B) MetX-EYFP fluorescence in a strain expressing the GalP sugar transporter and capable of glucose-dependent growth in darkness. Images were captured after 12 h of dark treatment, with or without glucose. (C) MetX-EYFP fluorescence in cells incubated in light when ATP synthesis was blocked (DCCD, 15 μM). Control shows the effect of 0.1% methanol vehicle. Images were taken 5 h after treatment. (D) Representative micrographs of DNP-treated MetX-EYFP cells showing fluorescent puncta in light. The pH of the media was adjusted with Bis-Tris or Tris buffer. Scale bar is 3μm. Images were taken 5–8 h after treatment. Histograms show percentages of cells with foci, N = 50–100 cells.