Dynamics of the bacterial intermediate filament crescentin in vitro and in vivo

Abstract

Background: Crescentin, the recently discovered bacterial intermediate filament protein, organizes into an extended filamentous structure that spans the length of the bacterium Caulobacter crescentus and plays a critical role in defining its curvature. The mechanism by which crescentin mediates cell curvature and whether crescentin filamentous structures are dynamic and/or polar are not fully understood.

Methodology/principal findings: Using light microscopy, electron microscopy and quantitative rheology, we investigated the mechanics and dynamics of crescentin structures. Live-cell microscopy reveals that crescentin forms structures in vivo that undergo slow remodeling. The exchange of subunits between these structures and a pool of unassembled subunits is slow during the life cycle of the cell however; in vitro assembly and gelation of C. crescentus crescentin structures are rapid. Moreover, crescentin forms filamentous structures that are elastic, solid-like, and, like other intermediate filaments, can recover a significant portion of their network elasticity after shear. The assembly efficiency of crescentin is largely unaffected by monovalent cations (K(+), Na(+)), but is enhanced by divalent cations (Mg(2+), Ca(2+)), suggesting that the assembly kinetics and micromechanics of crescentin depend on the valence of the ions present in solution.

Conclusions/significance: These results indicate that crescentin forms filamentous structures that are elastic, labile, and stiff, and that their low dissociation rate from established structures controls the slow remodeling of crescentin in C. crescentus.

Figure 1. In vivo dynamics and FRAP/FLIP analysis of crescentin-GFP in C. crescentus.

A, crescentin-GFP FRAP kinetics. The half-life time for recovery is t_1/2 = 26±1.9 min (n = 12). The curve is a fit based on a FRAP kinetics model. B, Time-lapsed fluorescence microscopy images of crescentin-GFP following photobleaching in the indicated region of the cell. Columns show selected images before and after photobleaching. Arrow heads show fluorescence recovery in bleached region (white circle) of cells. Columns correspond to DIC, GFP fluorescence and overlay, respectively. C, Overlaid DIC and crescentin-GFP fluorescence micrographs in a cell that has been completely photobleached. The cell does not recover its fluorescence. D , Crescentin-GFP FLIP kinetics. A region in the cell was repeatedly photobleached for 120 min and the loss in fluorescence in the rest of the cell was measured. The rate of FLIP is 0.50±0.18 h⁻¹ (n = 12 cells) (mean ± standard error). The curve is a fit based on a FLIP kinetics model.

Figure 2. Gelation kinetics and steady-state mechanical properties of crescentin.

A, Time-dependent elastic modulus, G′, after the onset of crescentin assembly (dead time for specimen loading in the rheometer was 30 s). Crescentin concentrations: 0.25 mg/ml (filled squares), 0.5 mg/ml (open squares), 0.75 mg/ml (filled circles), and 1 mg/ml (open circles). B, Rate of gelation as a function of protein concentration measured as the inverse of the time required for the network elasticity to reach 90% of its plateau value. C, phase angle, δ, of crescentin structures as a function of concentration. A phase angle of 90° describes the rheological behavior of a liquid (e.g. glycerol); a phase angle of 0° describes the rheological behavior of an elastic solid (e.g. a stiff rubber). Phase angle was evaluated at a frequency of 1 rad/s and a strain amplitude of 1%. D, Crescentin filaments (0.2 mg/ml) visualized by negative staining and EM. Bar, 2 µm

Figure 3. Response of crescentin to mechanical deformation.

A, Frequency-dependent elastic modulus, G′(ω), of crescentin as different concentrations. B, Elasticity, G′, of crescentin as a function of strain amplitude, γ (see Methods section). Crescentin does not undergo strain-hardening, whereby G′ would increase with γ. Crescentin concentrations in panels A and B are 0.25 mg/ml (filled squares), 0.5 mg/ml (open squares), 0.75 mg/ml (filled circles), and 1 mg/ml (open circles). C, Recovery of the elasticity of crescentin following the application of a large short-lived shear deformation as a function of crescentin concentration. Percent recovery is defined as the ratio of the recovered elasticity after application of a couple of oscillatory shear deformations of 1000% and the initial elasticity. Elasticity was evaluated at a frequency of 1 rad/s and a strain amplitude of 1%.

Figure 4. EM of crescentin filaments assembled with monovalent cations.

Crescentin filaments (0.2 mg/ml) in the presence of A, 50 mM NaCl (scale bar, 2 µm) B, 100 mM NaCl (scale bar, 1 µm), and C, 150 mM KCl (scale bar, 1 µm) respectively, visualized by negative staining and EM.

Figure 5. Effect of divalent cations on mechanical properties of Crescentin.

A, Steady state elastic modulus, G′, of crescentin structures as a function of MgCl₂ (grey) and CaCl₂ (black) concentration. Elasticity was evaluated at a frequency of 1 rad/s and a strain amplitude of 1% after 2 h of gelation. B, Rate of gelation as a function of MgCl₂ (grey) and CaCl₂ (black) concentration measured as the inverse of the time required for the network elasticity to reach 90% of its plateau value. Crescentin filaments (0.2 mg/ml) in the presence of C, 5 mM MgCl₂ (scale bar, 2 µm) and D, 10 mM MgCl₂ (scale bar, 2 µm), respectively, visualized by negative staining and EM. Symbols correspond to control (closed squares), control with 10 mM CaCl₂ (open squares), and control with 10 mM MgCl₂ (closed circles).