High Resolution Scanning Electron Microscopy of Cells Using Dielectrophoresis

Tang, Shi‐Yang; Zhang, Wei; Soffe, Rebecca; Nahavandi, Sofia; Shukla, Ravi; Khoshmanesh, Khashayar

doi:10.1371/journal.pone.0104109

Cited by 29 publications

(28 citation statements)

References 50 publications

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“…SEM imaging of wild type and mutants ( sen1‐1 , sen1 ∆N and sen1‐K128E ) was performed by following a standard protocol was with slight modifications (Tang et al , ). In brief, 1 ml of overnight cultures were collected by centrifugation and washed twice with dH 2 O before prefixing the cells with 4% of Paraformaldehyde (PFA) for 5 minutes at RT.…”

Section: Methodsmentioning

confidence: 99%

Flocculation of Saccharomyces cerevisiae is dependent on activation of Slt2 and Rlm1 regulated by the cell wall integrity pathway

Sariki

Kumawat

Singh

et al. 2019

Molecular Microbiology

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Summary Flocculation is an essential characteristic of yeast cells required for survival under adverse conditions. The multicellular structure (flocs) of yeast provides a suitable microenvironment to enhance the chances of survival during stress conditions. Although the signaling events triggering flocculation have been studied earlier, molecular mechanisms remain elusive. In the present study, we used flocculating sen1 mutants to identify the mechanism of flocculation. Based on the abnormal cell surface morphology and constitutive phosphorylation of Slt2p in flocculating sen1 mutant cells, we hypothesized if flocculation was regulated by the cell wall integrity (CWI) pathway. Up‐regulation of FLO genes in wild‐type cells was observed upon the activation of CWI pathway either by chemical treatment or by deleting Slt2 phosphatase (Msg5). Our study with Slt2 mutants reveals that the active state of Slt2 is indispensable for flocculation. Deletion of either SLT2 or RLM1 leads to reduced flocculation. Furthermore, we observed overlapping binding sites for Rlm1 and Tup1 at the promoters of almost all the FLO genes. Finally, we show higher Rlm1 and lower Tup1 occupancy at the promoters of FLO1 and FLO5 in flocculating cells. Altogether we demonstrate that CWI MAPK (Slt2) pathway uses a non‐catalytic mechanism to activate the transcription of FLO genes.

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Section: Methodsmentioning

confidence: 99%

Flocculation of Saccharomyces cerevisiae is dependent on activation of Slt2 and Rlm1 regulated by the cell wall integrity pathway

Sariki

Kumawat

Singh

et al. 2019

Molecular Microbiology

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“…DEP-platforms have been used towards dynamic analysis of hematopoietic stem cells undergoing drug-induced apoptosis, using real-time fluorescent imaging 246 and environmental scanning electron microscopy (ESEM) 247 . This DEP-based ESEM platform has also been used to dynamically image the morphology of selected populations of DEP-immobilized cells 248 , for comparing the morphological changes of non-budding and budding yeast cells following drug treatment 249 . The application of such techniques to microbials, to investigate infectivity based on alterations in cell shape 73 , S-layer glycoprotein-induced cell wall structure 193 and lipid-induced cell membrane structure 250 can offer dynamic phenotypic information on antibiotic susceptibility.…”

Section: Challenges and Emerging Needsmentioning

confidence: 99%

Review: Microbial analysis in dielectrophoretic microfluidic systems

Fernandez

Rohani

Farmehini

et al. 2017

Analytica Chimica Acta

122

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Infections caused by various known and emerging pathogenic microorganisms, including antibiotic-resistant strains, are a major threat to global health and well-being. This highlights the urgent need for detection systems for microbial identification, quantification and characterization towards assessing infections, prescribing therapies and understanding the dynamic cellular modifications. Current state-of-the-art microbial detection systems exhibit a trade-off between sensitivity and assay time, which could be alleviated by selective and label-free microbial capture onto the sensor surface from dilute samples. AC electrokinetic methods, such as dielectrophoresis, enable frequency-selective capture of viable microbial cells and spores due to polarization based on their distinguishing size, shape and sub-cellular compositional characteristics, for downstream coupling to various detection modalities. Following elucidation of the polarization mechanisms that distinguish bacterial cells from each other, as well as from mammalian cells, this review compares the microfluidic platforms for dielectrophoretic manipulation of microbials and their coupling to various detection modalities, including immuno-capture, impedance measurement, Raman spectroscopy and nucleic acid amplification methods, as well as for phenotypic assessment of microbial viability and antibiotic susceptibility. Based on the urgent need within point-of-care diagnostics towards reducing assay times and enhancing capture of the target organism, as well as the emerging interest in isolating intact microbials based on their phenotype and subcellular features, we envision widespread adoption of these label-free and selective electrokinetic techniques.

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“…The SEM analysis of WT and mutants (sen1-1, sen1 ∆N, and sen1-K128E) by following a standard protocol was performed with slight modifications (TANG et al 2014). In brief, 1 ml of overnight cultures were collected by centrifugation and washed twice with dH 2 O before prefixing the cells with 4% Paraformaldehyde (PFA) for 5 min at RT.…”

Section: Scanning Electron Microscopy (Sem)mentioning

confidence: 99%

A novel link between Cell Wall Integrity pathway and Flocculation, regulated by yeast Sen1 dependent interplay between Rlm1 and Tup1

Sk¹,

Kumawat²,

Singh

et al. 2018

Preprint

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The budding yeast, Saccharomyces cerevisiae is one of the most studied organisms used for the synthesis of products, to explore the human diseases and eukaryotic gene expression mechanisms. The yeast cells with flocculation property are in high demand for industrial applications. However, the pathogenic yeast becomes drug-resistant due to flocculation/biofilm phenotype. The flocculation property of yeast depends on the expression of specific FLO genes. Genetic and epigenetic factors have been suggested to induce the expression of FLOs and flocculation, an evolutionarily conserved process. The present study was undertaken to identify a molecular link between stress caused by genetic and epigenetic factors and expression of FLOs. We utilized flocculating yeast strains to study the regulation of FLO genes and flocculation phenotype. We found rough surface morphology and constitutive activation of Slt2 in flocculating cells. The external cell wall stress factors as well as specific mutations in Sen1 and histone proteins strongly correlated with the induction of FLO genes whereas deletion of SLT2/RLM1, suppressed the expression and flocculation phenotype. We detected constitutive binding of Rlm1 and eviction of Tup1 from the promoters of FLO1 and FLO5 genes in flocculating cells. Thus we provide evidence for the CWI pathway dependent flocculation of yeast, regulated by Sen1 mediated interplay between Tup1 and Rlm1.

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High Resolution Scanning Electron Microscopy of Cells Using Dielectrophoresis

Cited by 29 publications

References 50 publications

Flocculation of Saccharomyces cerevisiae is dependent on activation of Slt2 and Rlm1 regulated by the cell wall integrity pathway

Flocculation of Saccharomyces cerevisiae is dependent on activation of Slt2 and Rlm1 regulated by the cell wall integrity pathway

Review: Microbial analysis in dielectrophoretic microfluidic systems

A novel link between Cell Wall Integrity pathway and Flocculation, regulated by yeast Sen1 dependent interplay between Rlm1 and Tup1

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