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.2012 Jun 19;109(25):10036-41.
doi: 10.1073/pnas.1120979109. Epub 2012 Jun 4.

Wet-surface-enhanced ellipsometric contrast microscopy identifies slime as a major adhesion factor during bacterial surface motility

Affiliations

Wet-surface-enhanced ellipsometric contrast microscopy identifies slime as a major adhesion factor during bacterial surface motility

Adrien Ducret et al. Proc Natl Acad Sci U S A..

Abstract

In biology, the extracellular matrix (ECM) promotes both cell adhesion and specific recognition, which is essential for central developmental processes in both eukaryotes and prokaryotes. However, live studies of the dynamic interactions between cells and the ECM, for example during motility, have been greatly impaired by imaging limitations: mostly the ability to observe the ECM at high resolution in absence of specific staining by live microscopy. To solve this problem, we developed a unique technique, wet-surface enhanced ellipsometry contrast (Wet-SEEC), which magnifies the contrast of transparent organic materials deposited on a substrate (called Wet-surf) with exquisite sensitivity. We show that Wet-SEEC allows both the observation of unprocessed nanofilms as low as 0.2 nm thick and their accurate 3D topographic reconstructions, directly by standard light microscopy. We next used Wet-SEEC to image slime secretion, a poorly defined property of many prokaryotic and eukaryotic organisms that move across solid surfaces in absence of obvious extracellular appendages (gliding). Using combined Wet-SEEC and fluorescent-staining experiments, we observed slime deposition by gliding Myxococcus xanthus cells at unprecedented resolution. Altogether, the results revealed that in this bacterium, slime associates preferentially with the outermost components of the motility machinery and promotes its adhesion to the substrate on the ventral side of the cell. Strikingly, analogous roles have been proposed for the extracellular proteoglycans of gliding diatoms and apicomplexa, suggesting that slime deposition is a general means for gliding organisms to adhere and move over surfaces.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Wet-SEEC imaging and topographic reconstructions of nanometer-size transparent layers. (A) Contrast calculations for a film of optical indexn = 1.5 on a glass slide (red) and on a wet-surf (green) as a function of film thickness. (B) Wet-SEEC optical signal intensity as function of film thickness. Theoretical (solid line) and experimental (dots) normalized intensities on a wet-surf for thin resins films of optical indexn = 1.6. (C,E) Raw Wet-SEEC image (Left) and Wet-SEEC topographic reconstruction (Right) of (C) the calibrated resin steps, with thicknesses of 10, 18, and 40 nm (from left to right) were measured inB, and (E) a DOPC lipid bilayer of 4.5 nm thickness. (D) Phase contrast, and Wet-SEEC images of ECM deposition in the wake of a motile keratinocyte cell. Dark dashed line represents the cell contour. (Inset) Tubular structures at cell proximity. (Scale bar, 10 μm.)
Fig. 2.
Fig. 2.
Direct observation and topographic measurement of theMyxococcus slime trails by Wet-SEEC. (A) Phase contrast and Wet-SEEC images of slime deposition in the wake of a motile cell shown at different time points (for animation seeMovie S2). Pictures were taken every 30 s. Triangle arrow points to the slime trail. (Scale bar, 1 μm.) (B andC) Longitudinal (B) and lateral (C) profiles of the slime trail given in theInset ofB showing local measured thickness (nanometers) (for animation, seeMovie S3). (D) Schematic representation of the scale between the size of the cell body (500 nm of diameter) and the maximum slime thickness (5 nm).
Fig. 3.
Fig. 3.
Slime is deposited at constant rates underneath the cell body and does not mediate propulsion. (A) Slime thickness (green line) against cell velocity (blue line) or time residence at given locations (red line). Thickness, cell velocity, and residence were computed and normalized as discussed inMaterials and Methods. (B) Slime deposition by mutant nonmotile cells. Cells were allowed to settle on the substratum and flushed at different times. Slime deposition underneath the cell body was observed after flushing using Wet-SEEC. (Upper andLower) Two representative cells harboring different times of contact with the substratum. (Scale bar, 1 μm.)
Fig. 4.
Fig. 4.
Slime is deposited by the Agl/Glt motility complexes. (A) ConA staining of a moving cell and its resulting slime trail at different times. Note that the cell changes direction after t5 (2 min). Triangular arrows point to conspicuous bright dots that appear at the leading cell pole and remained fixed relative to the substratum before they eventually became deposited on the substratum (for animation, seeMovie S4). Fluorescent micrographs were taken every 30 s. (Scale bar, 1 μm.) (B) ConA clusters are transported down the cell body in immobile cells. Two ConA bright clusters (black triangles) are shown to move down the cell axis. Pictures were taken every 15 s. (Scale bar, 1 μm.) (C) Slime patches are deposited where the Agl/Glt machinery assembles. Time lapse of a cell expressing both AglZ-YFP and AglQ-mCherry is shown. Phase contrast and corresponding YFP and mCherry micrographs are shown. Slime was stained with ConA after the cell left the positions shown on theLeft. Triangular arrows point to fixed AglZ- and AglQ-bright motility complexes at positions where conspicuous slime patches were deposited. Fluorescent micrographs were taken every 15 s. (Scale bar, 1 μm.)
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