Visualization of phospholipid domains in Escherichia coli by using the cardiolipin-specific fluorescent dye 10-N-nonyl acridine orange

Abstract

Cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange (NAO) was used to visualize CL distribution in Escherichia coli cells of different phospholipid compositions. In a filamentous mutant containing only anionic phospholipids, green fluorescent spots were observed along the filaments at approximately regular intervals. Three-dimensional image reconstruction obtained by optical sectioning and a deconvolution algorithm revealed NAO-binding domains in the plane of the cell membrane. Substantial red fluorescence emission of bound NAO supported labeling of CL-containing domains. These structures were not found in mutants deficient in CL biosynthesis. The domains were also observed mostly in the septal region and on the poles in cells of normal size with wild-type phospholipid composition.

FIG. 1-5

Fig. 1. Staining of living AD90 filamentous cells with NAO. Cells were grown in LB medium supplemented with 50 mM MgCl₂. (A) NAO was added to the cells in the exponential phase of growth, and pictures were taken after 1 h of incubation with the dye. (B) A part of filamentous AD90 cell at a higher magnification is shown. Bars, 2.5 μM. Exposure time, 0.25 s. Fig. 2. Deconvoluted images of an optical section of an AD90 cell. Cells were stained as described in the legend to Fig. 1. (A and B) Excitation was at 490 nm, and emission was at either 528 (A) or 617 (B) nm. Bar, 2.5 μM. Exposure time, 0.5 s. (C) Colocalization of green and red fluorescent domains. Fig. 3. Staining of living ADC/pDD72 (A), HDL11 (B), and E614 (C) cells with NAO. Cells were grown in LB medium supplemented with cephalexin and stained with NAO. Bars, 2.5 μM. Exposure times, 2 (A) and 1 (B and C) s. Fig. 4. Staining of living AD90/pDD72 cells with NAO. (A to F) Individual AD90/pDD72 cells. (G and H) Deconvoluted images of an optical section of an AD90/pDD72 cell. Excitation was at 490 nm and emission was at either 528 (G) or 617 (H) nm. (I) Three-dimensional picture of an AD90/pDD72 cell stained with NAO obtained by reconstruction of deconvolved optical sections. The same cell is shown at three different angles of rotation. Bars, 2.5 μM. Exposure time, 1 s. Fig. 5. AD90/pDD72 (A to C) and AD90 (D) cells stained with NAO and DAPI. Panels A and B show the same cell stained with NAO and DAPI, respectively. Panel C shows the overlay of the images in panels A and B. Panel D shows the overlay of NAO and DAPI staining. Stains, NAO (green) and DAPI (red). Bar, 2.5 μM.