Journal: Nature Microbiology

Article Title: Cell wall synthesis and remodelling dynamics determine division site architecture and cell shape in Escherichia coli

doi: 10.1038/s41564-022-01210-z

Figure Lengend Snippet: a , Distribution of cell wall material in ∆envC cells was assessed by FDAA staining in 3 biological replicates. Images are sum-projections of a 1 µm spanning z -stack and were deconvolved. White arrowheads indicate double septa. b , Representative time-lapse series from 3 biological replicates of a ∆envC mutant expressing Pal-mCherry and ZipA-sfGFP as OM and IM markers, respectively. An example of double septum formation is shown. c , Examples of membrane blebbing (yellow arrowheads) and polar septa (blue arrowheads) formation are highlighted. d , Formation of double constrictions observed in cryo-electron tomograms of ∆envC ∆nlpD cells. Black arrowheads indicate constriction sites. e , The frequency of double septum formation was quantified from counting the number of Pal-mCherry doublets per cell. No Pal doublets were found in >10,000 cells for wild-type or ftsN-∆SPOR cells in 3 biological replicates (N.A., not applicable). Data are represented as median + 95% confidence interval. f , The distance between Pal doublets was measured manually using the line tool in Fiji. N = 91 ( ∆envC ), 46 ( ftsL* ∆envC ) Pal doublets measured. g , The frequency of polar septa per cell was measured for the indicated strains. No polar septa were observed in >10,000 wild-type or ftsN-∆SPOR cells. Data are represented as median + 95% confidence interval. h – j , Three-dimensional maximum intensity renderings showing Z-ring condensation based on ZipA-sfGFP localization ( h ). The degree of Z-ring condensation was quantified from averaged fluorescence intensity projections from summed 3D volumes ( i ) or from 5 time points (corresponding to 10 min) of a time-lapse series ( j ) (see Methods). Insets: FWHM of the fluorescence signal, with data represented as boxplots; line represents median, error bars depict minimum–maximum range. Inserts show average fluorescence intensity projection at the septum. Significance was tested against wild type by one-way ANOVA with Dunnett’s correction for multiple comparisons: * P < 0.05. N = 100 (wt, ∆envC , ftsL* ∆envC , ftsN-∆SPOR ) Z-rings from 3 biological replicates. Averaged Z-rings are shown and colour-coded according to graphs. Scale bars: a – c , 2 µm; d , 200 nm; h , 2 µm; i and j , 200 nm.

Article Snippet: Samples were imaged on a Nikon Ti2-E inverted widefield microscope equipped with a Lumencor Spectra III light engine, Semrock dichroics (LED-CFP/YFP/mCherry-3X-A-000, LED-DA/FI/TR/Cy5/Cy7-5X-A-000) and emission filters (FF01-432/36, FF01-515/30, FF01-544/24).

Techniques: Staining, Mutagenesis, Expressing, Membrane, Fluorescence

Journal: Nature Microbiology

Article Title: Cell wall synthesis and remodelling dynamics determine division site architecture and cell shape in Escherichia coli

doi: 10.1038/s41564-022-01210-z

Figure Lengend Snippet: ( a ) MreB-sw-mNeonGreen dynamics were followed by SIM-TRIF microscopy for 3 min at 3 s acquisitions per frame in indicated mutants. Time-lapse series was sum-projected and overlayed over a 3D-SIM Pal-mCherry and brightfield reference image. Larger fields of view are shown as compared to Fig. and are representative from three biological replicates. Bar = 1 µm ( b ) Slopes of MSD curves ( α ) were analyzed following log-log fit to l og[ MSD ] versus l og[ t ] using the MATLAB class msdanalyzer . Particles displaced by diffusive motion are characterized by a slope of their l og[ MSD ] = 1, while transported particles have slopes of 2 and constrained particles display slopes < 1. No significant difference (ns = non-significant, p = 0.0693; Kruskal Wallis test with Dunn’s correction for multiple comparisons) in the slopes of MSD curves were found indicating that MreB is displaced at a similar rate and manner in all strains. Box plot error bars displaying Min-Max range of values, blackline represents median. Tracks fit to l og[ MSD ] l og[ t ] with R 2 ≤ 0.95 ( c ) Cell length and ( d ) width was measured from three independent biological replicates for the indicated mutants using Morphometrics . Line represents median. Differences in significances were tested relative to wild-type using Kruskal-Wallis one-way ANOVA with Dunnett’s correction for multiple comparisons; * = p < 0.05, **** = p < 0.0001, ns = non-significant, p = 0.69. ( e ) MreB dynamics were imaged as in ( a ). Representative temporal SUM projections of MreB-mNeon trajectories were overlayed to brightfield images. Bar = 2 µm. ( f ) Directionally moving MreB tracks were filtered by MSD analysis (see Methods) and represented as boxplots (line indicating median; error bars depict Min-Max range) and normalized by cell area. Significance in each group was tested against non-filamented control (-Ara) by two-sided unpaired t-test; ** p < 0.01, ns = non-significant, wt p = 0.053, fstN-∆SPOR = 0.253. ( g ) Constriction curvature values of wild-type cells are plotted against division site width in 566 cells. Linear regression (R 2 = 0.135) indicates the negative correlation between cell width at the division site and constriction angle.

Article Snippet: Samples were imaged on a Nikon Ti2-E inverted widefield microscope equipped with a Lumencor Spectra III light engine, Semrock dichroics (LED-CFP/YFP/mCherry-3X-A-000, LED-DA/FI/TR/Cy5/Cy7-5X-A-000) and emission filters (FF01-432/36, FF01-515/30, FF01-544/24).

Techniques: Microscopy, Control