iCellFusion

Santinha, João; Martins, Leonardo de Oliveira; Häkkinen, Antti; Lloyd-Price, Jason; Oliveira, Samuel M. D.; Gupta, Abhishekh; Annila, Teppo; Mora, André; Ribeiro, Alexandra B.; Fonseca, José

doi:10.4018/978-1-4666-8811-7.ch004

Cited by 3 publications

(2 citation statements)

References 59 publications

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“…Cell segmentation from the images was performed using the software 'iCellFusion' [32] and 'CellAging' [33], which perform automatic segmentation from phase contrast images (figure 1(A)) but allow manual corrections for increased accuracy. A 2D affine geometric transformation [34] was used to transform epifluorescence images (figure 1(B)) into the same resolution space as confocal images (figure 1(C)).…”

Section: Image Processing and Machine Learningmentioning

confidence: 99%

The precision of the symmetry in Z-ring placement inEscherichia coliis hampered at critical temperatures

et al. 2018

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Cell division in Escherichia coli is morphologically symmetric due to, among other things, the ability of these cells to place the Z-ring at midcell. Studies have reported that, at sub-optimal temperatures, this symmetry decreases at the single-cell level, but the causes remain unclear. Using fluorescence microscopy, we observe FtsZ-GFP and DAPI-stained nucleoids to assess the robustness of the symmetry of Z-ring formation and positioning in individual cells under sub-optimal and critical temperatures. We find the Z-ring formation and positioning to be robust at sub-optimal temperatures, as the Z-ring's mean width, density and displacement from midcell maintain similar levels of correlation to one another as at optimal temperatures. However, at critical temperatures, the Z-ring displacement from midcell is greatly increased. We present evidence showing that this is due to enhanced distance between the replicated nucleoids and, thus, reduced Z-ring density, which explains the weaker precision in setting a morphologically symmetric division site. This also occurs in rich media and is cumulative, i.e. combining richer media and critically high temperatures enhances the asymmetries in division, which is evidence that the causes are biophysical. To further support this, we show that the effects are reversible, i.e. shifting cells from optimal to critical, and then to optimal again, reduces and then enhances the symmetry in Z-ring positioning, respectively, as the width and density of the Z-ring return to normal values. Overall, our findings show that the Z-ring positioning in E. coli is a robust biophysical process under sub-optimal temperatures, and that critical temperatures cause significant asymmetries in division.

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Section: Image Processing and Machine Learningmentioning

confidence: 99%

The precision of the symmetry in Z-ring placement inEscherichia coliis hampered at critical temperatures

et al. 2018

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“…Many available tools [7,8] were developed for bacterial colonies and are not well adapted for the analysis of fly images. They lack the flexibility of switching between a specialized automatic mode, which is essential for efficient processing, to manual intervention mode, which are often required to maximize the amount of samples acquired from each time-consuming experiment.…”

Section: Introductionmentioning

confidence: 99%

LiveFly: A Toolbox for the Analysis of Transcription Dynamics in Live Drosophila Embryos

Tran¹,

Perez-Romero²,

Ferraro³

et al. 2018

Methods in Molecular Biology

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We present the LiveFly toolbox for quantitative analysis of transcription dynamics in live Drosophila embryos. The toolbox allows users to process two-color 3D confocal movies acquired using nucleilabeling and the fluorescent RNA-tagging system described in the previous chapter and export the nuclei's position as a function of time, their lineages and the intensity traces of the active loci. The toolbox, which is tailored for the context of Drosophila early development, is semi-automatic, and requires minimal user intervention. It also includes a tool to combine data from multiple movies and visualize several features of the intensity traces and the expression pattern.

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Chromosome and plasmid-borne PLacO3O1 promoters differ in sensitivity to critically low temperatures

Oliveira

Goncalves

Kandavalli

et al. 2019

Sci Rep

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Temperature shifts trigger genome-wide changes in Escherichia coli’s gene expression. We studied if chromosome integration impacts on a gene’s sensitivity to these shifts, by comparing the single-RNA production kinetics of a PLacO3O1 promoter, when chromosomally-integrated and when single-copy plasmid-borne. At suboptimal temperatures their induction range, fold change, and response to decreasing temperatures are similar. At critically low temperatures, the chromosome-integrated promoter becomes weaker and noisier. Dissection of its initiation kinetics reveals longer lasting states preceding open complex formation, suggesting enhanced supercoiling buildup. Measurements with Gyrase and Topoisomerase I inhibitors suggest hindrance to escape supercoiling buildup at low temperatures. Consistently, similar phenomena occur in energy-depleted cells by DNP at 30 °C. Transient, critically-low temperatures have no long-term consequences, as raising temperature quickly restores transcription rates. We conclude that the chromosomally-integrated PLacO3O1 has higher sensitivity to low temperatures, due to longer-lasting super-coiled states. A lesser active, chromosome-integrated native lac is shown to be insensitive to Gyrase overexpression, even at critically low temperatures, indicating that the rate of escaping positive supercoiling buildup is temperature and transcription rate dependent. A genome-wide analysis supports this, since cold-shock genes exhibit atypical supercoiling-sensitivities. This phenomenon might partially explain the temperature-sensitivity of some transcriptional programs of E. coli.

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iCellFusion

Cited by 3 publications

References 59 publications

The precision of the symmetry in Z-ring placement inEscherichia coliis hampered at critical temperatures

The precision of the symmetry in Z-ring placement inEscherichia coliis hampered at critical temperatures

LiveFly: A Toolbox for the Analysis of Transcription Dynamics in Live Drosophila Embryos

Chromosome and plasmid-borne PLacO3O1 promoters differ in sensitivity to critically low temperatures

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