Super-resolution imaging of the nucleoid-associated protein HU in Caulobacter crescentus

Abstract

Little is known about the structure and function of most nucleoid-associated proteins (NAPs) in bacteria. One reason for this is that the distribution and structure of the proteins is obfuscated by the diffraction limit in standard wide-field and confocal fluorescence imaging. In particular, the distribution of HU, which is the most abundant NAP, has received little attention. In this study, we investigate the distribution of HU in Caulobacter crescentus using a combination of super-resolution fluorescence imaging and spatial point statistics. By simply increasing the laser power, single molecules of the fluorescent protein fusion HU2-eYFP can be made to blink on and off to achieve super-resolution imaging with a single excitation source. Through quantification by Ripley's K-test and comparison with Monte Carlo simulations, we find the protein is slightly clustered within a mostly uniform distribution throughout the swarmer and stalked stages of the cell cycle but more highly clustered in predivisional cells. The methods presented in this letter should be of broad applicability in the future study of prokaryotic NAPs.

Figure 1

(Color) SR imaging of HU2-eYFP in fixed C. crescentus. (A) Frames of raw data (10 ms each) showing the blinking of eYFP achieved through the use of a single 514 nm laser. (B) A diffraction-limited (DL) image of HU2-eYFP with a white light transmission image in the inset. (C) SR image of HU2-eYFP. (D) Monte Carlo simulated image of a random distribution of molecules inside a model cell volume (inset).

Figure 2

(Color) Representative SR imaging of HU2-eYFP at different stages in the C. crescentus cell cycle. (A) SR image of a swarmer cell with corresponding L-test plots and DL epifluorescence image (inset). The blue line (upper) corresponds to the data and the red lines (center) to the CSR simulation (with upper and lower red lines corresponding to the 95% confidence intervals). (B) Same as panel A but for a stalked cell. In both swarmer and stalked, the distribution of HU2-eYFP is only slightly clustered compared to CSR. (C) Significant clustering of HU2-eYFP in predivisional cells can be observed both in the SR image and in the large deviation between the L function for the data compared to the simulation.