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( A ) Left, schematic of the <t>microfluidics</t> plasmolysis assay. GFP-expressing M. smegmatis were subjected to hyperosmotic shock (3 M sorbitol) in microfluidics device and imaged before, during, and after shock. Right, images before and after hyperosmotic shock. Cells were perfused with 7H9 medium for 5 min and then perfused with 7H9 + 3 M sorbitol for 5 min. Cells were imaged in phase (50 ms) and GFP (50 ms) every 30 s. An arrowhead indicates the septum while arrows indicate sites of plasmolysis. Scale bars, 2.5 μm. Images are representative of four independent experiments. ( B ) Cytoplasmic GFP enables visualization and quantitation of plasmolysis bays. Arrows indicate representative sites of plasmolysis. Scale bars, 2.5 μm. Images are representative of two independent experiments. ( C ) Analysis of the sites of plasmolysis (as defined in [ B] ) in wild-type (n = 85) and Δ ponA2 (n = 101) M. smegmatis from two biological replicates performed in duplicate. For this analysis, we removed cells that did not plasmolyse. Statistical significance was determined by the two-way ANOVA test, followed by Šídák’s multiple-comparisons test. ****p<0.0001, ns, p>0.99. Polar, subpolar, and midcell bays spatially correlate with the PM-CW, inner membrane domain (IMD), and a mixture of PM-CW and IMD, respectively ( ; ; ; ). ( D ) The proportion of cells that did not plasmolyse in ( C ) was calculated for wild-type (n = 90) and Δ ponA2 (n = 123) M. smegmatis . Statistical significance was determined by the Mann–Whitney U -test. **p=0.045. ( E ) Cell lengths were analyzed for wild-type (n = 84) and Δ ponA2 (n = 132) M. smegmatis from three biological replicates. Each color in the super plots represents an independent biological replicate. Smaller symbols are the lengths of individual cells, and larger symbols are the means of each replicate. Statistical significance was determined by the Mann–Whitney U -test. ns, p=0.4. Figure 5—source data 1. Raw values of the proportions of where plasmolysis bay were seen, population without plasmolysis, and cell length.
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( A ) Left, schematic of the <t>microfluidics</t> plasmolysis assay. GFP-expressing M. smegmatis were subjected to hyperosmotic shock (3 M sorbitol) in microfluidics device and imaged before, during, and after shock. Right, images before and after hyperosmotic shock. Cells were perfused with 7H9 medium for 5 min and then perfused with 7H9 + 3 M sorbitol for 5 min. Cells were imaged in phase (50 ms) and GFP (50 ms) every 30 s. An arrowhead indicates the septum while arrows indicate sites of plasmolysis. Scale bars, 2.5 μm. Images are representative of four independent experiments. ( B ) Cytoplasmic GFP enables visualization and quantitation of plasmolysis bays. Arrows indicate representative sites of plasmolysis. Scale bars, 2.5 μm. Images are representative of two independent experiments. ( C ) Analysis of the sites of plasmolysis (as defined in [ B] ) in wild-type (n = 85) and Δ ponA2 (n = 101) M. smegmatis from two biological replicates performed in duplicate. For this analysis, we removed cells that did not plasmolyse. Statistical significance was determined by the two-way ANOVA test, followed by Šídák’s multiple-comparisons test. ****p<0.0001, ns, p>0.99. Polar, subpolar, and midcell bays spatially correlate with the PM-CW, inner membrane domain (IMD), and a mixture of PM-CW and IMD, respectively ( ; ; ; ). ( D ) The proportion of cells that did not plasmolyse in ( C ) was calculated for wild-type (n = 90) and Δ ponA2 (n = 123) M. smegmatis . Statistical significance was determined by the Mann–Whitney U -test. **p=0.045. ( E ) Cell lengths were analyzed for wild-type (n = 84) and Δ ponA2 (n = 132) M. smegmatis from three biological replicates. Each color in the super plots represents an independent biological replicate. Smaller symbols are the lengths of individual cells, and larger symbols are the means of each replicate. Statistical significance was determined by the Mann–Whitney U -test. ns, p=0.4. Figure 5—source data 1. Raw values of the proportions of where plasmolysis bay were seen, population without plasmolysis, and cell length.
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( A ) Left, schematic of the microfluidics plasmolysis assay. GFP-expressing M. smegmatis were subjected to hyperosmotic shock (3 M sorbitol) in microfluidics device and imaged before, during, and after shock. Right, images before and after hyperosmotic shock. Cells were perfused with 7H9 medium for 5 min and then perfused with 7H9 + 3 M sorbitol for 5 min. Cells were imaged in phase (50 ms) and GFP (50 ms) every 30 s. An arrowhead indicates the septum while arrows indicate sites of plasmolysis. Scale bars, 2.5 μm. Images are representative of four independent experiments. ( B ) Cytoplasmic GFP enables visualization and quantitation of plasmolysis bays. Arrows indicate representative sites of plasmolysis. Scale bars, 2.5 μm. Images are representative of two independent experiments. ( C ) Analysis of the sites of plasmolysis (as defined in [ B] ) in wild-type (n = 85) and Δ ponA2 (n = 101) M. smegmatis from two biological replicates performed in duplicate. For this analysis, we removed cells that did not plasmolyse. Statistical significance was determined by the two-way ANOVA test, followed by Šídák’s multiple-comparisons test. ****p<0.0001, ns, p>0.99. Polar, subpolar, and midcell bays spatially correlate with the PM-CW, inner membrane domain (IMD), and a mixture of PM-CW and IMD, respectively ( ; ; ; ). ( D ) The proportion of cells that did not plasmolyse in ( C ) was calculated for wild-type (n = 90) and Δ ponA2 (n = 123) M. smegmatis . Statistical significance was determined by the Mann–Whitney U -test. **p=0.045. ( E ) Cell lengths were analyzed for wild-type (n = 84) and Δ ponA2 (n = 132) M. smegmatis from three biological replicates. Each color in the super plots represents an independent biological replicate. Smaller symbols are the lengths of individual cells, and larger symbols are the means of each replicate. Statistical significance was determined by the Mann–Whitney U -test. ns, p=0.4. Figure 5—source data 1. Raw values of the proportions of where plasmolysis bay were seen, population without plasmolysis, and cell length.

Journal: eLife

Article Title: A cell wall synthase accelerates plasma membrane partitioning in mycobacteria

doi: 10.7554/eLife.81924

Figure Lengend Snippet: ( A ) Left, schematic of the microfluidics plasmolysis assay. GFP-expressing M. smegmatis were subjected to hyperosmotic shock (3 M sorbitol) in microfluidics device and imaged before, during, and after shock. Right, images before and after hyperosmotic shock. Cells were perfused with 7H9 medium for 5 min and then perfused with 7H9 + 3 M sorbitol for 5 min. Cells were imaged in phase (50 ms) and GFP (50 ms) every 30 s. An arrowhead indicates the septum while arrows indicate sites of plasmolysis. Scale bars, 2.5 μm. Images are representative of four independent experiments. ( B ) Cytoplasmic GFP enables visualization and quantitation of plasmolysis bays. Arrows indicate representative sites of plasmolysis. Scale bars, 2.5 μm. Images are representative of two independent experiments. ( C ) Analysis of the sites of plasmolysis (as defined in [ B] ) in wild-type (n = 85) and Δ ponA2 (n = 101) M. smegmatis from two biological replicates performed in duplicate. For this analysis, we removed cells that did not plasmolyse. Statistical significance was determined by the two-way ANOVA test, followed by Šídák’s multiple-comparisons test. ****p<0.0001, ns, p>0.99. Polar, subpolar, and midcell bays spatially correlate with the PM-CW, inner membrane domain (IMD), and a mixture of PM-CW and IMD, respectively ( ; ; ; ). ( D ) The proportion of cells that did not plasmolyse in ( C ) was calculated for wild-type (n = 90) and Δ ponA2 (n = 123) M. smegmatis . Statistical significance was determined by the Mann–Whitney U -test. **p=0.045. ( E ) Cell lengths were analyzed for wild-type (n = 84) and Δ ponA2 (n = 132) M. smegmatis from three biological replicates. Each color in the super plots represents an independent biological replicate. Smaller symbols are the lengths of individual cells, and larger symbols are the means of each replicate. Statistical significance was determined by the Mann–Whitney U -test. ns, p=0.4. Figure 5—source data 1. Raw values of the proportions of where plasmolysis bay were seen, population without plasmolysis, and cell length.

Article Snippet: The cells were then back diluted and added to the B04A microfluidic perfusion plate (CellASIC) during exponential phase.

Techniques: Expressing, Quantitation Assay, Membrane, MANN-WHITNEY