Cardiolipin microdomains localize to negatively curved regions of Escherichia coli membranes

Abstract

Many proteins reside at the cell poles in rod-shaped bacteria. Several hypotheses have drawn a connection between protein localization and the large cell-wall curvature at the poles. One hypothesis has centered on the formation of microdomains of the lipid cardiolipin (CL), its localization to regions of high membrane curvature, and its interaction with membrane-associated proteins. A lack of experimental techniques has left this hypothesis unanswered. This paper describes a microtechnology-based technique for manipulating bacterial membrane curvature and quantitatively measuring its effect on the localization of CL and proteins in cells. We confined Escherichia coli spheroplasts in microchambers with defined shapes that were embossed into a layer of polymer and observed that the shape of the membrane deformed predictably to accommodate the walls of the microchambers. Combining this technique with epifluorescence microscopy and quantitative image analyses, we characterized the localization of CL microdomains in response to E. coli membrane curvature. CL microdomains localized to regions of high intrinsic negative curvature imposed by microchambers. We expressed a chimera of yellow fluorescent protein fused to the N-terminal region of MinD--a spatial determinant of E. coli division plane assembly--in spheroplasts and observed its colocalization with CL to regions of large, negative membrane curvature. Interestingly, the distribution of MinD was similar in spheroplasts derived from a CL synthase knockout strain. These studies demonstrate the curvature dependence of CL in membranes and test whether these structures participate in the localization of MinD to regions of negative curvature in cells.

Fig. 1.

A schematic representation of the experimental procedure: E. coli cells are grown into filaments with a mean length of 50 μm. Removing the outer membrane and the peptidoglycan converts the filaments into spheroplasts. Spheroplasts are labeled with NAO (for CL, false-colored red) and DAPI (for DNA, false-colored blue), confined in individual microchambers with varying curvature, and imaged using phase-contrast and epifluorescence microscopy.

Fig. 2.

(A) Epifluorescence microscopy images of E. coli cells with labeled CL (NAO, red) and DNA (DAPI, blue). (B) Phase-contrast microscopy images of freestanding spheroplasts. (C) Epifluorescence microscopy images of freestanding spheroplasts with labeled CL (red) and DNA (blue). (Insets) White dashed lines indicate the perimeter of the cell (A) and spheroplast (C). White arrows (C) highlight CL microdomains.

Fig. 3.

(A) An analysis of the position of CL microdomains in spheroplasts confined in microchambers with different curvatures. CL was visualized by labeling spheroplasts with NAO. Each panel contains a representative image of CL localization in one confined spheroplast with the curvature indicated; the dashed line indicates the perimeter of the spheroplast. (B) Comparison of the CL microdomain distribution in spherical (curvature = 0.67 μm⁻¹, dark circles) and rod-shaped (curvature = 2.08 μm⁻¹, light circles) spheroplasts plotted against the normalized length of spheroplasts. We analyzed ≥400 spheroplasts for each microchamber curvature. (C) The predicted localization of CL microdomains in a spheroplast confined in a spherical microchamber.

Fig. 4.

The relative frequency of CL microdomains at the polar regions in spheroplasts plotted against the difference in the highest and lowest curvature regions of the membrane (Δcurvature). The data (open circles) were fitted to a sigmoidal function (dashed line) as a visual guide.

Fig. 5.

Plots depicting the relationship between the frequency of MinD localization and the normalized position in the confined spheroplast. Distributions of (A) MinD-YFP in CL synthase-positive (cls+) (MG1655 pFX40) and (B) CL synthase-negative (cls−) (JW1241 Δcls pFX40) E. coli strains. Dark circles represent data for spheroplasts confined in spherical microchambers (curvature = 0.67 μm⁻¹). Light circles represent data for rod-shaped microchambers (largest curvature = 2.08 μm⁻¹).

Fig. 6.

MinD and CL colocalization in E. coli MG1655 (+pFX40) spheroplasts confined in microchambers. (A) Microscopy images of spheroplasts confined in spherical microchambers. (B) Images of spheroplasts confined in rod-shaped microchambers. The microscopy images depict bright field (BF; phase contrast); CL labeled with NAO (CL); MinD-YFP (MinD); the overlay of the CL and MinD images (Merge); and DNA labeled with DAPI (DNA). Dashed circles indicate the perimeter of the microchambers and were added to the images for clarity.