Engineering Bacterial Shape Using Soft Matter Microchambers

Renner, Lars D.

doi:10.1002/cpch.59

Cited by 2 publications

(2 citation statements)

References 22 publications

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“…Cells were imaged directly afterward, so that the total time from taking cells out of culture and start of imaging was ~5 min. As we have done before for spheroplast formation (Renner and Weibel, 2011 ; Renner, 2019 ) and lysis (Wong and Amir, 2019 ), we treated cells with the β-lactam antibiotic cephalexin. Cephalexin hydrate (Sigma-Aldrich, St. Louis, MO) was dissolved in 1 M ammonium hydroxide stock solution.…”

Section: Methodsmentioning

confidence: 99%

Understanding Beta-Lactam-Induced Lysis at the Single-Cell Level

et al. 2021

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Mechanical rupture, or lysis, of the cytoplasmic membrane is a common cell death pathway in bacteria occurring in response to β-lactam antibiotics. A better understanding of the cellular design principles governing the susceptibility and response of individual cells to lysis could indicate methods of potentiating β-lactam antibiotics and clarify relevant aspects of cellular physiology. Here, we take a single-cell approach to bacterial cell lysis to examine three cellular features—turgor pressure, mechanosensitive channels, and cell shape changes—that are expected to modulate lysis. We develop a mechanical model of bacterial cell lysis and experimentally analyze the dynamics of lysis in hundreds of single Escherichia coli cells. We find that turgor pressure is the only factor, of these three cellular features, which robustly modulates lysis. We show that mechanosensitive channels do not modulate lysis due to insufficiently fast solute outflow, and that cell shape changes result in more severe cellular lesions but do not influence the dynamics of lysis. These results inform a single-cell view of bacterial cell lysis and underscore approaches of combatting antibiotic tolerance to β-lactams aimed at targeting cellular turgor.

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Section: Methodsmentioning

confidence: 99%

Understanding Beta-Lactam-Induced Lysis at the Single-Cell Level

et al. 2021

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“…We used a previously described approach [26,[49][50][51] to design squared microchambers to house T. brucei. The squared microchambers (more than 10,000 per array) were designed in AutoCAD (Autodesk, San Rafael, CA) and CleWin (Delta Mask).…”

Section: Microfabricationmentioning

confidence: 99%

Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging

et al. 2022

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Trypanosoma brucei, the causative agent of human African trypanosomiasis, is highly motile and must be able to move in all three dimensions for reliable cell division. These characteristics make long-term microscopic imaging of live T. brucei cells challenging, which has limited our understanding of important cellular events. To address this issue, we devised an imaging approach that confines cells in small volumes within cast agarose microwells that can be imaged continuously for up to 24 h. Individual T. brucei cells were imaged through multiple rounds of cell division with high spatial and temporal resolution. We developed a strategy that employs in-well “sentinel” cells to monitor potential imaging toxicity during loss-of-function experiments such as small-molecule inhibition and RNAi. Using our approach, we show that the asymmetric daughter cells produced during T. brucei division subsequently divide at different rates, with the old-flagellum daughter cell dividing first. The flagellar detachment phenotype that appears during inhibition of the Polo-like kinase homolog TbPLK occurs in a stepwise fashion, with the new flagellum initially linked by its tip to the old, attached flagellum. We probe the feasibility of a previously proposed “back-up” cytokinetic mechanism and show that cells that initiate this process do not appear to complete cell division. This live-cell imaging method will provide a novel avenue for studying a wide variety of cellular events in trypanosomatids that have previously been inaccessible.

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Engineering Bacterial Shape Using Soft Matter Microchambers

Cited by 2 publications

References 22 publications

Understanding Beta-Lactam-Induced Lysis at the Single-Cell Level

Understanding Beta-Lactam-Induced Lysis at the Single-Cell Level

Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging

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