scholarly journals Spatiotemporal mapping of bacterial membrane potential responses to extracellular electron transfer

2020 ◽  
Vol 117 (33) ◽  
pp. 20171-20179 ◽  
Author(s):  
Sahand Pirbadian ◽  
Marko S. Chavez ◽  
Mohamed Y. El-Naggar
Keyword(s):  
Electron Transfer ◽  
Membrane Potential ◽  
Single Cell ◽  
Electrochemical Techniques ◽  
Model Organisms ◽  
Extracellular Electron Transfer ◽  
Single Cell Level ◽  
Cell Level ◽  
Membrane Hyperpolarization ◽  
Wide Range

Extracellular electron transfer (EET) allows microorganisms to gain energy by linking intracellular reactions to external surfaces ranging from natural minerals to the electrodes of bioelectrochemical renewable energy technologies. In the past two decades, electrochemical techniques have been used to investigate EET in a wide range of microbes, with emphasis on dissimilatory metal-reducing bacteria, such asShewanella oneidensisMR-1, as model organisms. However, due to the typically bulk nature of these techniques, they are unable to reveal the subpopulation variation in EET or link the observed electrochemical currents to energy gain by individual cells, thus overlooking the potentially complex spatial patterns of activity in bioelectrochemical systems. Here, to address these limitations, we use the cell membrane potential as a bioenergetic indicator of EET byS. oneidensisMR-1 cells. Using a fluorescent membrane potential indicator during in vivo single-cell-level fluorescence microscopy in a bioelectrochemical reactor, we demonstrate that membrane potential strongly correlates with EET. Increasing electrode potential and associated EET current leads to more negative membrane potential. This EET-induced membrane hyperpolarization is spatially limited to cells in contact with the electrode and within a near-electrode zone (<30 μm) where the hyperpolarization decays with increasing cell-electrode distance. The high spatial and temporal resolution of the reported technique can be used to study the single-cell-level dynamics of EET not only on electrode surfaces, but also during respiration of other solid-phase electron acceptors.

scholarly journals Single-Cell Electrophysiological Measurements Reveal Bacterial Membrane Potential Dynamics during Extracellular Electron Transfer

2020 ◽  
Author(s):  
Sahand Pirbadian ◽  
Marko S. Chavez ◽  
Mohamed Y. El-Naggar
Keyword(s):  
Electron Transfer ◽  
Membrane Potential ◽  
Single Cell ◽  
Solid Phase ◽  
Electrochemical Techniques ◽  
Model Organisms ◽  
Extracellular Electron Transfer ◽  
Single Cell Level ◽  
Cell Level ◽  
Wide Range

AbstractExtracellular electron transfer (EET) allows microorganisms to gain energy by linking intracellular reactions to external surfaces ranging from natural minerals to the electrodes of bioelectrochemical renewable energy technologies. In the past two decades, electrochemical techniques have been used to investigate EET in a wide range of microbes, with emphasis on dissimilatory metal-reducing bacteria, such as Shewanella oneidensis MR-1, as model organisms. However, due to the typically bulk nature of these techniques, they are unable to reveal the subpopulation variation in EET or link the observed electrochemical currents to energy gain by individual cells, thus overlooking the potentially complex spatial patterns of activity in bioelectrochemical systems. Here, to address these limitations, we use the cell membrane potential as a bioenergetic indicator of EET by S. oneidensis MR-1 cells. Using a fluorescent membrane potential indicator during in vivo single-cell level fluorescence microscopy in a bioelectrochemical reactor, we demonstrate that membrane potential strongly correlates with the electrode potential, produced current, and position of cells relative to the electrodes. The high spatial and temporal resolution of the reported technique can be used to study the single-cell level dynamics of EET not only on electrode surfaces, but also during respiration of other solid-phase electron acceptors.

scholarly journals Mito Hacker: a set of tools to enable high-throughput analysis of mitochondrial network morphology

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Ali Rohani ◽  
Jennifer A. Kashatus ◽  
Dane T. Sessions ◽  
Salma Sharmin ◽  
David F. Kashatus
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Data Science ◽  
Structural Information ◽  
Morphological Changes ◽  
Mitochondrial Morphology ◽  
Single Cell Level ◽  
Mitochondrial Network ◽  
Cell Level ◽  
Wide Range

Abstract Mitochondria are highly dynamic organelles that can exhibit a wide range of morphologies. Mitochondrial morphology can differ significantly across cell types, reflecting different physiological needs, but can also change rapidly in response to stress or the activation of signaling pathways. Understanding both the cause and consequences of these morphological changes is critical to fully understanding how mitochondrial function contributes to both normal and pathological physiology. However, while robust and quantitative analysis of mitochondrial morphology has become increasingly accessible, there is a need for new tools to generate and analyze large data sets of mitochondrial images in high throughput. The generation of such datasets is critical to fully benefit from rapidly evolving methods in data science, such as neural networks, that have shown tremendous value in extracting novel biological insights and generating new hypotheses. Here we describe a set of three computational tools, Cell Catcher, Mito Catcher and MiA, that we have developed to extract extensive mitochondrial network data on a single-cell level from multi-cell fluorescence images. Cell Catcher automatically separates and isolates individual cells from multi-cell images; Mito Catcher uses the statistical distribution of pixel intensities across the mitochondrial network to detect and remove background noise from the cell and segment the mitochondrial network; MiA uses the binarized mitochondrial network to perform more than 100 mitochondria-level and cell-level morphometric measurements. To validate the utility of this set of tools, we generated a database of morphological features for 630 individual cells that encode 0, 1 or 2 alleles of the mitochondrial fission GTPase Drp1 and demonstrate that these mitochondrial data could be used to predict Drp1 genotype with 87% accuracy. Together, this suite of tools enables the high-throughput and automated collection of detailed and quantitative mitochondrial structural information at a single-cell level. Furthermore, the data generated with these tools, when combined with advanced data science approaches, can be used to generate novel biological insights.

scholarly journals Methane-Linked Mechanisms of Electron Uptake from Cathodes byMethanosarcina barkeri

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Annette R. Rowe ◽  
Shuai Xu ◽  
Emily Gardel ◽  
Arpita Bose ◽  
Peter Girguis ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Methane Production ◽  
Extracellular Enzymes ◽  
Solid Phase ◽  
Electrochemical Techniques ◽  
Methanosarcina Barkeri ◽  
Extracellular Electron Transfer ◽  
Hydrogen Electrode ◽  
Content Type ◽  
Wide Range

ABSTRACTTheMethanosarcinales, a lineage of cytochrome-containing methanogens, have recently been proposed to participate in direct extracellular electron transfer interactions within syntrophic communities. To shed light on this phenomenon, we applied electrochemical techniques to measure electron uptake from cathodes byMethanosarcina barkeri, which is an important model organism that is genetically tractable and utilizes a wide range of substrates for methanogenesis. Here, we confirm the ability ofM. barkerito perform electron uptake from cathodes and show that this cathodic current is linked to quantitative increases in methane production. The underlying mechanisms we identified include, but are not limited to, a recently proposed association between cathodes and methanogen-derived extracellular enzymes (e.g., hydrogenases) that can facilitate current generation through the formation of reduced and diffusible methanogenic substrates (e.g., hydrogen). However, after minimizing the contributions of such extracellular enzymes and using a mutant lacking hydrogenases, we observe a lower-potential hydrogen-independent pathway that facilitates cathodic activity coupled to methane production inM. barkeri. Our electrochemical measurements of wild-type and mutant strains point to a novel and hydrogenase-free mode of electron uptake with a potential near −484 mV versus standard hydrogen electrode (SHE) (over 100 mV more reduced than the observed hydrogenase midpoint potential under these conditions). These results suggest thatM. barkerican perform multiple modes (hydrogenase-mediated and free extracellular enzyme-independent modes) of electrode interactions on cathodes, including a mechanism pointing to a direct interaction, which has significant applied and ecological implications.IMPORTANCEMethanogenic archaea are of fundamental applied and environmental relevance. This is largely due to their activities in a wide range of anaerobic environments, generating gaseous reduced carbon that can be utilized as a fuel source. While the bioenergetics of a wide variety of methanogens have been well studied with respect to soluble substrates, a mechanistic understanding of their interaction with solid-phase redox-active compounds is limited. This work provides insight into solid-phase redox interactions inMethanosarcinaspp. using electrochemical methods. We highlight a previously undescribed mode of electron uptake from cathodes that is potentially informative of direct interspecies electron transfer interactions in theMethanosarcinales.

scholarly journals Visualization and Modeling of Inhibition of IL-1β and TNFα mRNA Transcription at the Single-Cell Level

2020 ◽  
Author(s):  
Daniel Kalb ◽  
Huy D. Vo ◽  
Samantha Adikari ◽  
Elizabeth Hong-Geller ◽  
Brian Munsky ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Single Cell ◽  
Inflammatory Responses ◽  
Leukemia Cell ◽  
26S Proteasome ◽  
Leukemia Cell Line ◽  
Single Cell Level ◽  
Cell Level ◽  
Wide Range

AbstractIL-1β and TNFα are canonical immune response mediators that play key regulatory roles in a wide range of inflammatory responses to both chronic and acute conditions. Here we employ an automated microscopy platform for the analysis of messenger RNA (mRNA) expression of IL-1β and TNFα at the single-cell level. The amount of IL-1β and TNFα mRNA expressed in a human monocytic leukemia cell line (THP-1) is visualized and counted using single-molecule fluorescent in-situ hybridization (smFISH) following exposure of the cells to lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria. We show that the small molecule inhibitors MG132 (a 26S proteasome inhibitor used to block NF-κB signaling) and U0126 (a MAPK Kinase inhibitor used to block CCAAT-enhancer-binding proteins C/EBP) successfully block IL-1β and TNFα mRNA expression. Based upon this single-cell mRNA expression data, mathematical models of gene expression indicate that the drugs U0126 and MG132 affect gene activation/deactivation rates between the basal and highly activated states. Models for which the parameters were informed by the action of each drug independently were able to predict the effects of the combined drug treatment. From our data and models, we postulate that IL-1β is activated by both NF-κB and C/EBP, while TNFα is predominantly activated by NF-κB. Our combined single-cell experimental modeling efforts shows the interconnection between these two genes and demonstrates how the single-cell responses, including the distribution shapes, mean expression, and kinetics of gene expression, change with inhibition.

scholarly journals Visualization and modeling of inhibition of IL-1β and TNF-α mRNA transcription at the single-cell level

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Daniel Kalb ◽  
Huy D. Vo ◽  
Samantha Adikari ◽  
Elizabeth Hong-Geller ◽  
Brian Munsky ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mrna Expression ◽  
Single Cell ◽  
Leukemia Cell ◽  
26S Proteasome ◽  
Leukemia Cell Line ◽  
Single Cell Level ◽  
Cell Level ◽  
Tnf Α ◽  
Wide Range

AbstractIL-1β and TNF-α are canonical immune response mediators that play key regulatory roles in a wide range of inflammatory responses to both chronic and acute conditions. Here we employ an automated microscopy platform for the analysis of messenger RNA (mRNA) expression of IL-1β and TNF-α at the single-cell level. The amount of IL-1β and TNF-α mRNA expressed in a human monocytic leukemia cell line (THP-1) is visualized and counted using single-molecule fluorescent in-situ hybridization (smFISH) following exposure of the cells to lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria. We show that the small molecule inhibitors MG132 (a 26S proteasome inhibitor used to block NF-κB signaling) and U0126 (a MAPK Kinase inhibitor used to block CCAAT-enhancer-binding proteins C/EBP) successfully block IL-1β and TNF-α mRNA expression. Based upon this single-cell mRNA expression data, we screened 36 different mathematical models of gene expression, and found two similar models that capture the effects by which the drugs U0126 and MG132 affect the rates at which the genes transition into highly activated states. When their parameters were informed by the action of each drug independently, both models were able to predict the effects of the combined drug treatment. From our data and models, we postulate that IL-1β is activated by both NF-κB and C/EBP, while TNF-α is predominantly activated by NF-κB. Our combined single-cell experimental and modeling efforts show the interconnection between these two genes and demonstrates how the single-cell responses, including the distribution shapes, mean expression, and kinetics of gene expression, change with inhibition.

scholarly journals Visualizing Metabolic Processes at the Single-Cell Level - Using Genetically Encoded Biosensor and Biomarker

2021 ◽  
pp. 276-314
Author(s):  
Elena Locci ◽  
Silvia Raymond
Keyword(s):  
Artificial Intelligence ◽  
Endothelial Cells ◽  
Blood Vessel ◽  
Single Cell ◽  
Single Cell Level ◽  
Cell Level ◽  
Wide Range ◽  
Keywords Cancer ◽  
Glycolysis Process ◽  
Genetically Encoded Biosensor

Understanding cellular metabolism (how cells use energy) can be key in treating a wide range of diseases, including vascular disease and cancer. Although many techniques can measure these processes in tens of thousands of cells, researchers have not been able to measure them at the single-cell level. Researchers have used a genetically encoded biosensor with artificial intelligence to measure glycolysis. (Process of converting glucose to energy, single endothelial cells, blood vessel cells). Keywords: Cancer; Cells; Tissues, Tumors; Prevention, Prognosis; Diagnosis; Imaging; Screening; Treatment; Management

scholarly journals Decoding mitochondrial heterogeneity in single muscle fibres by imaging mass cytometry

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Charlotte Warren ◽  
David McDonald ◽  
Roderick Capaldi ◽  
David Deehan ◽  
Robert W. Taylor ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Oxidative Phosphorylation ◽  
Single Cell ◽  
Mitochondrial Disease ◽  
Muscle Fibres ◽  
Single Muscle ◽  
Single Cell Level ◽  
Mass Cytometry ◽  
Cell Level ◽  
Wide Range

Abstract The study of skeletal muscle continues to support the accurate diagnosis of mitochondrial disease and remains important in delineating molecular disease mechanisms. The heterogeneous expression of oxidative phosphorylation proteins and resulting respiratory deficiency are both characteristic findings in mitochondrial disease, hence the rigorous assessment of these at a single cell level is incredibly powerful. Currently, the number of proteins that can be assessed in individual fibres from a single section by immunohistochemistry is limited but imaging mass cytometry (IMC) enables the quantification of further, discrete proteins in individual cells. We have developed a novel workflow and bespoke analysis for applying IMC in skeletal muscle biopsies from patients with genetically-characterised mitochondrial disease, investigating the distribution of nine mitochondrial proteins in thousands of single muscle fibres. Using a semi-automated analysis pipeline, we demonstrate the accurate quantification of protein levels using IMC, providing an accurate measure of oxidative phosphorylation deficiency for complexes I–V at the single cell level. We demonstrate signatures of oxidative phosphorylation deficiency for common mtDNA variants and nuclear-encoded complex I variants and a compensatory upregulation of unaffected oxidative phosphorylation components. This technique can now be universally applied to evaluate a wide range of skeletal muscle disorders and protein targets.

scholarly journals Methane-linked mechanisms of electron uptake from cathodes byMethanosarcina barkeri

2018 ◽  
Author(s):  
Annette R. Rowe ◽  
Shuai Xu ◽  
Emily Gardel ◽  
Arpita Bose ◽  
Peter Girguis ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Methane Production ◽  
Extracellular Enzymes ◽  
Solid Phase ◽  
Electrochemical Techniques ◽  
Extracellular Enzyme ◽  
Methanosarcina Barkeri ◽  
Extracellular Electron Transfer ◽  
Cathodic Current ◽  
Wide Range

AbstractTheMethanosarcinales, a lineage of cytochrome-containing methanogens, have recently been proposed to participate in direct extracellular electron transfer interactions within syntrophic communities. To shed light on this phenomenon, we applied electrochemical techniques to measure electron uptake from cathodes byMethanosarcina barkeri, which is an important model organism that is genetically tractable and utilizes a wide range of substrates for methanogenesis. Here we confirm the ability ofM. barkerito perform electron uptake from cathodes and show that this cathodic current is linked to quantitative increases in methane production. The underlying mechanisms we identified include, but are not limited to, a recently proposed association between cathodes and methanogen-derived extracellular enzymes (e.g. hydrogenases) that can facilitate current generation through the formation of reduced and diffusible methanogenic substrates (e.g. hydrogen). However, after minimizing the contributions of such extracellular enzymes and using a mutant lacking hydrogenases, we observe a lower potential hydrogen-independent pathway that facilitates cathodic activity coupled to methane production inM. barkeri. Our electrochemical measurements of wild-type and mutant strains point to a novel and extracellular-enzyme-free mode of electron uptake able to take up electrons at potentials lower than - 498 mV vs. SHE (over 100 mV more reduced than the observed hydrogenase midpoint potential under these conditions). These results suggest thatM. barkerican perform multiple modes (hydrogenase-mediated and free extracellular enzyme-independent) of electrode interactions on cathodes including a mechanism pointing to a direct interaction, which has significant applied and ecological implications.ImportanceMethanogenic Archaea are of fundamental applied and environmental relevance. This is largely due to their activities in a wide range of anaerobic environments, generating gaseous reduced carbon that can be utilized as a fuel source. While the bioenergetics of a wide variety of methanogens has been well studied with respect to soluble substrates, mechanistic understanding of their interaction with solid phase redox active compounds is limited. This work provides insight into solid phase redox interactions inMethanosarcinausing electrochemical methods. We highlight a previously undescribed mode of electron uptake from cathodes, that is potentially informative of direct interspecies electron transfer interactions in theMethanosarcinales.

Cytoplasmic dynein

2011 ◽  
Vol 39 (5) ◽  
pp. 1169-1178 ◽  
Author(s):  
Victoria J. Allan
Keyword(s):  
Single Cell ◽  
Recent Progress ◽  
Motor Proteins ◽  
Molecular Motor ◽  
Cytoplasmic Dynein ◽  
Eukaryotic Cells ◽  
Single Cell Level ◽  
Cell Level ◽  
Wide Range ◽  
And Function

The organization and function of eukaryotic cells rely on the action of many different molecular motor proteins. Cytoplasmic dynein drives the movement of a wide range of cargoes towards the minus ends of microtubules, and these events are needed, not just at the single-cell level, but are vital for correct development. In the present paper, I review recent progress on understanding dynein's mechanochemistry, how it is regulated and how it binds to such a plethora of cargoes. The importance of a number of accessory factors in these processes is discussed.

Sign in / Sign up

Export Citation Format

Share Document