Light-dependent modulation of protein localization and function in living bacteria cells

Abstract

Most bacteria lack membrane-enclosed organelles and rely on macromolecular scaffolds at different subcellular locations to recruit proteins for specific functions. Here, we demonstrate that the optogenetic CRY2-CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations with varying efficiencies in live Escherichia coli cells, including the nucleoid, the cell pole, the membrane, and the midcell division plane. Such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. We further show that CRY2-CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system. Finally, we test this optogenetic system in three additional bacterial species, Bacillus subtilis, Caulobacter crescentus, and Streptococcus pneumoniae, providing important considerations for this system's applicability in bacterial cell biology.

Fig. 1. Light-induced recruitment scheme using CRY2 and CIBN.

a CRY2 is targeted to different subcellular localizations in live E. coli cells: the chromosomal DNA, cell pole, inner membrane, and the midcell division plane. At each subcellular location, CIBN is fused to a bait protein at the target either at the C or N terminus, and CRY2 is fused to the N terminus of mCherry as a universal prey reporter for CIBN’s light (488 nm)-dependent association with CIBN-Target (inset box). The association is reversible in dark. b CIBN and CRY2 fusion proteins can be expressed co-transcriptionally (left) or independently (right) to suit different experimental needs.

Fig. 2. Rapid and reversible recruitment of cytoplasmic proteins to chromosomal DNA and the cell pole.

a Schematic depicting the relocalization of cytoplasmic CRY2-mCherry to TetR-CIBN bound tetO sites near OriC to from foci after activation with blue light. b CRY2-mCherry is uniformly distributed throughout the cell prior to blue light exposure (left panel). After blue light exposure CRY2-mCherry rapidly relocalizes to form foci (right panel). Images are representative of N = 3 independent experiments. Scale bar = 2 µm. c Single cell time course images demonstrating that the recruitment of CRY2-mCherry to DNA foci occurs only when blue light and the tetO array are both present (top row) but not in the absence of the tetO target (middle row) or blue light activation (bottom row). Images are representative of N = 3 independent experiments. Scale bar = 2 µm. d Averaged percent increase (black dots) of CRY2-mCherry signal at DNA foci (N = 3 independent experiments of 1359 cells in total) demonstrating that 90% recruitment is reached within 85 seconds. Shaded region indicated the s.e.m. e Example trace of the mean CRY2-mCherry signal at DNA foci (gray dots; N = 1 experiment of 89 cells) after activation with blue light (first blue section), relaxation in the dark (middle gray section), and after a second activation sequence (second blue region) demonstrating that CRY2/CIBN disassociation at DNA foci is reversible with a time constant of ~ 9 minutes. Error bars indicated s.e.m. f Schematic depicting the relocalization of cytoplasmic CRY2-mCherry to CIBN-GFP-PopZ foci at the cell pole after blue light activation. Scale bar = 2 µm. g CRY2-mCherry is uniformly distributed throughout the cell prior to blue light exposure (left panel). After blue light exposure, CRY2-mCherry rapidly relocalizes to the cell poles and form foci (right panel). Image is representative of N = 3 independent experiments. h Single cell time course images demonstrating that the recruitment of CRY2-mCherry to the cell pole occurs only when both blue light and the CIBN are present (top row) but not in the absence of the CIBN target (middle row) or blue light (bottom row). Images are representative of N = 3 independent experiments. Scale bar = 1 µm. i Averaged percent increase (black dots) of CRY2-mCherry signal at cell pole foci (N = 3 experiments with 315 cells in total) demonstrating that 90% recruitment is reached within ~8 seconds. Shaded region indicates the s.e.m.

Fig. 3. Recruitment of cytoplasmic protein to cell division plane and a Light-induced Inhibition of Cytokinesis (LInC) Assay.

a Schematic depicting the relocalization of cytoplasmic CRY2-mCherry to the ZapA-CIBN ring present at midcell after induction of CRY2/CIBN binding with blue light. b An example image of cells showing CRY2-mCherry’s diffusive cytoplasmic localization before blue light activation (left) and midcell localization after blue light activation (right). Image is representative of N = 3 independent experiments. Scale bar = 2 µm. c Single cell time course images demonstrating that CRY2-mCherry recruitment to midcell occurs only after activation with blue light. Scale bar = 1 µm. d Averaged percent increase (black dots) of CRY2-mCherry signal at midcell demonstrating that 90% recruitment is reached within 9 seconds (N = 3 experiments with 443 cells). Shaded region indicates the s.e.m. e Schematic depicting the LInC assay on both short and long time scales. Recruitment of CRY2-MinC to Z-rings via ZapA-CIBN by blue light activation results in instant destabilization of the Z-ring at a short time scale and cell division inhibition at a long time scale. In the absence of blue light cells grow and divide like WT cells. f Short timescale LInC Assay. Cells harboring the LInC system exhibited diffusive Z-rings (arrow heads) after blue light activation delivered every 10 s for 5 min. Scale bar = 2 µm. The projected fluorescence intensity of ZapA-mCherry (Z-ring) along the cell long-axis before (gray) and after a 5-minute blue light induction (blue) for the cell labeled with a white arrow in (f) is plotted in (g) to demonstrate the significant decondensation of the Z-ring (p = 0.032, determined by a two-sample KS-test). h Short timescale Z-ring destabilization by the LInC system is dependent on cell length. The percent reduction of ZapA-mCherry intensity (μ ± s.e.m, Supplementary Table 2) at midcell in cells expressing the LInC system before (N = 3 experiments with 1465 cells in total) and after (N = 3 experiments with 1658 cells in total) a 5-minute period of blue light activation (blue datapoints) or darkness (gray datapoints) plotted as a function of cell length. i Long timescale LInC assay. Time-lapse imaging shows that cells ectopically expressing CRY2-MinC only (Control, top row) grew and divided normally while cells expressing both CRY2-MinC and ZapA-CIBN (LInC, bottom row) filamented when both were exposed to 100 ms pulses of blue and green light every 5 minutes for a 12-h period. Cells harboring only CRY2-MinC (right panel, top) or both CRY2-MinC and ZapA-CIBN (right panel, bottom row) but were not exposed to blue light divided normally during the same period (right most panel). White arrow heads indicate the position of cells that are enlarged in the insets. Images are representative of N = 2 independent experiments. Scale bar = 2 µm. j Smoothed and averaged long axis projections of the Z-ring intensity measured by ZapA-mCherry at the 12-h timepoint after blue light activation demonstrated significant (p = 6.36 x 10⁻⁶, determined by a two-sample KS-test) widening of Z-ring in cells harboring the LInC system (FWHM = 221 nm, N = 2 experiments with 16 cells in total) compared with those in the Control cells (FWHM = 102 nm, N = 2 experiments with 33 cells in total).

Fig. 4. Green (561 nm) modulates the association of the CRY2-CIBN complex.

a Schematic of CRY2 photoactivation pathway highlighting the redox states of its flavin (FAD) cofactor along the path. Breifly, the fully oxidized FAD^ox is photoexcited by a blue light photon and accepts an electron from the nearby tryptophan (Trp397) to form a stable, semi-reduced neutral FADH^o. This formation induces a conformational change in CRY2, which allows it to bind to CIBN. In the absence of green light, the semi-reduced FADH^o slowly decays to a fully reduced state (FADH^-). As FADH^o is the only species in the cycle that can absorb green light, it is likely that green light speeds up the further reduction of FADH^o to FADH^-, which prevents CRY2 to associate or remain associated with CIBN. b The percent enrichment of cell pole-recruited CRY2-Halo shows a stepwise dependence on the intensity of the 100 ms blue (488 nm) light pulse used to trigger CRY2/CIBN complex formation. (N = 2 experiments with > 85 individual cells for each condition, error bars = s.d.) c The maximal fold enrichment of cell pole-recruited CRY2-Halo under varying green light intensities relative to that in the absence of green light showed a green light dependent reduction (left, N = 2 experiments with > 100 individual cells in total for each green light condition respectively, error bars = s.e.m. via bootstrapping), while the 90% recruitment time (τ_0.9) under varying green light intensities relative to that in the absence of green light was not significantly altered (right, N = 2 experiments, error bars = s.e.m. via bootstrapping) indicating that green light can modulate the levels of activated CRY2 available for complex formation with CIBN (p-values determined by a two-tailed t-test). d The change in the final fold reduction of cell pole-recruited CRY2-Halo dissociation under green light exposure relative to that in the absence of green light showed no change (left, N = 4 experiments with 91 cells in total, error bars = s.d.), while the reversion time (τ_rev) under green light exposure relative to that in the absence of green light showed a considerable but statistically insignificant reduction (right, N = 4 experiments with 62 cells in total, error bars = s.d., p = n.s., determined by a two-tailed t-test).

Fig. 5. Blue light induced midcell localization of CRY2-tdTomato in B. subtills cells.

a Schematic depicting the re-localization of cytoplasmic CRY2-tdTomato to ZapA-CIBN at midcell after activation with blue light. b Z-projections of cells undergoing blue light pulsing for the indicated number of frames. Consecutive 50 ms blue and 500 ms green light pulses were delivered every 3 seconds for a period of 8 minutes. The yellow triangles indicate midcell recruitment observed in a z-projection of the last 20 frames. Images are representative of N = 3 independent experiments. c Example showing blue light dependent localizing CRY2-tdTomato to midcell for a single cell over a 360 second time period. d Averaged percent increase (black dots) with s.e.m. (red) of CRY2-tdTomato signal at midcell demonstrating that light-dependent recruitment to midcell (N = 71 cells from three independent biological replicates). Scale bar = 1.6 µm.