scholarly journals A novel single-cell screening platform reveals proteome plasticity during yeast stress responses

2013 ◽  
Vol 201 (2) ◽  
pp. 353-353
Author(s):  
Michal Breker ◽  
Melissa Gymrek ◽  
Maya Schuldiner
Keyword(s):  
Single Cell ◽  
Stress Responses ◽  
Screening Platform

scholarly journals A novel single-cell screening platform reveals proteome plasticity during yeast stress responses

2013 ◽  
Vol 200 (6) ◽  
pp. 839-850 ◽  
Author(s):  
Michal Breker ◽  
Melissa Gymrek ◽  
Maya Schuldiner
Keyword(s):  
Single Cell ◽  
Stress Responses ◽  
Nitrogen Starvation ◽  
Bet Hedging ◽  
Transcriptional Responses ◽  
Cellular Physiology ◽  
Protein Levels ◽  
Hedging Strategies ◽  
Screening Platform ◽  
External Stimuli

Uncovering the mechanisms underlying robust responses of cells to stress is crucial for our understanding of cellular physiology. Indeed, vast amounts of data have been collected on transcriptional responses in Saccharomyces cerevisiae. However, only a handful of pioneering studies describe the dynamics of proteins in response to external stimuli, despite the fact that regulation of protein levels and localization is an essential part of such responses. Here we characterized unprecedented proteome plasticity by systematically tracking the localization and abundance of 5,330 yeast proteins at single-cell resolution under three different stress conditions (DTT, H2O2, and nitrogen starvation) using the GFP-tagged yeast library. We uncovered a unique “fingerprint” of changes for each stress and elucidated a new response arsenal for adapting to radical environments. These include bet-hedging strategies, organelle rearrangement, and redistribution of protein localizations. All data are available for download through our online database, LOQATE (localization and quantitation atlas of yeast proteome).

scholarly journals Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses

2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Ciara H. O’Flanagan ◽  
◽  
Kieran R. Campbell ◽  
Allen W. Zhang ◽  
Farhia Kabeer ◽  
...  
Keyword(s):  
Stress Response ◽  
Single Cell ◽  
Solid Tumor ◽  
Stress Responses ◽  
Immune Recognition ◽  
Cell Type ◽  
Collagenase Digestion ◽  
Tumor Tissues ◽  
Cold Active ◽  
Active Protease

Abstract Background Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumor tissues and patient-derived mouse xenografts for scRNA-seq are not well understood. Results We use low temperature (6 °C) protease and collagenase (37 °C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNA-seq dataset comprising 155,165 cells from patient cancer tissues, patient-derived breast cancer xenografts, and cancer cell lines. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. From the contrast between tissue protease dissociation at 37 °C or 6 °C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 °C), which are minimized by dissociation with a cold active protease (6 °C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. Conclusions The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. Interpretation of stress pathway expression differences in cancer single-cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments.

scholarly journals Novel microfluidic platform accelerates processing of tissue into single cells for molecular analysis and primary culture models

2020 ◽  
Author(s):  
Jeremy Lombardo ◽  
Marzieh Aliaghaei ◽  
Quy Nguyen ◽  
Kai Kessenbrock ◽  
Jered Haun
Keyword(s):  
Single Cell ◽  
Stress Responses ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cell Types ◽  
Tissue Preparation ◽  
Cell Analysis ◽  
Processing Technologies ◽  
Microfluidic Platform ◽  
Wide Range

Abstract Tissues are composed of highly heterogeneous mixtures of cell subtypes, and this diversity is increasingly being characterized using high-throughput single cell analysis methods. However, these efforts are hindered by the fact that tissues must first be dissociated into single cell suspensions that are viable and still accurately represent phenotypes from the original tissue. Current methods for breaking down tissues are inefficient, labor-intensive, subject to high variability, and potentially biased towards cell subtypes that are easier to release. Here, we present a microfluidic platform consisting of three different tissue processing technologies that can perform the complete tissue to single cell workflow, including digestion, disaggregation, and filtration. First, we developed a new microfluidic digestion device that can be loaded with minced tissue specimens quickly and easily, and then use the combination of proteolytic enzyme activity and fluid shear forces to accelerate tissue breakdown. Next, we integrated dissociation and filter technologies into a single device, which enhanced single cell numbers and fully prepared the sample for single cell analysis. The final multi-device platform was then evaluated using a diverse array of tissue types that exhibited a wide range of properties. For murine kidney and mammary tumor, we found that microfluidic processing produced 2.5-fold more single, viable cells. Single cell RNA sequencing (scRNA-seq) further revealed that device processing enriched for endothelial cells, fibroblasts, and basal epithelium, and did not increase stress responses. For murine liver and heart, which are softer tissues containing fragile cell types, processing time could be reduced to 15 min, and even as short as 1 min. We also demonstrated that periodic recovery at defined time intervals produced substantially more hepatocytes and cardiomyocytes than continuous operation, most likely by preventing damage to fragile cell types. In future work, we will seek to integrate additional operations such as upstream tissue preparation and downstream microfluidic cell sorting and detection to create powerful point-of-care single cell diagnostic platforms.

scholarly journals Characterization of hormone-producing cell types in the teleost pituitary gland using single-cell RNA-seq

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Khadeeja Siddique ◽  
Eirill Ager-Wick ◽  
Romain Fontaine ◽  
Finn-Arne Weltzien ◽  
Christiaan V. Henkel
Keyword(s):  
Single Cell ◽  
Stress Responses ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Peptide Hormone ◽  
Cell Types ◽  
Specific Cell ◽  
Endocrine Gland ◽  
Hormone Production ◽  
Male Adult

AbstractThe pituitary is the vertebrate endocrine gland responsible for the production and secretion of several essential peptide hormones. These, in turn, control many aspects of an animal’s physiology and development, including growth, reproduction, homeostasis, metabolism, and stress responses. In teleost fish, each hormone is presumably produced by a specific cell type. However, key details on the regulation of, and communication between these cell types remain to be resolved. We have therefore used single-cell sequencing to generate gene expression profiles for 2592 and 3804 individual cells from the pituitaries of female and male adult medaka (Oryzias latipes), respectively. Based on expression profile clustering, we define 15 and 16 distinct cell types in the female and male pituitary, respectively, of which ten are involved in the production of a single peptide hormone. Collectively, our data provide a high-quality reference for studies on pituitary biology and the regulation of hormone production, both in fish and in vertebrates in general.

Pulses and delays, anticipation and memory: seeing bacterial stress responses from a single-cell perspective

2020 ◽  
Vol 44 (5) ◽  
pp. 565-571
Author(s):  
Valentine Lagage ◽  
Stephan Uphoff
Keyword(s):  
Stress Response ◽  
Single Cell ◽  
Single Molecule ◽  
Stress Responses ◽  
Phenotypic Diversity ◽  
Single Cells ◽  
Super Resolution ◽  
Population Level ◽  
Protective Mechanisms ◽  
Single Cell Imaging

ABSTRACT Stress responses are crucial for bacteria to survive harmful conditions that they encounter in the environment. Although gene regulatory mechanisms underlying stress responses in bacteria have been thoroughly characterised for decades, recent advances in imaging technologies helped to uncover previously hidden dynamics and heterogeneity that become visible at the single-cell level. Despite the diversity of stress response mechanisms, certain dynamic regulatory features are frequently seen in single cells, such as pulses, delays, stress anticipation and memory effects. Often, these dynamics are highly variable across cells. While any individual cell may not achieve an optimal stress response, phenotypic diversity can provide a benefit at the population level. In this review, we highlight microscopy studies that offer novel insights into how bacteria sense stress, regulate protective mechanisms, cope with response delays and prepare for future environmental challenges. These studies showcase developments in the single-cell imaging toolbox including gene expression reporters, FRET, super-resolution microscopy and single-molecule tracking, as well as microfluidic techniques to manipulate cells and create defined stress conditions.

scholarly journals Single-cell microscopy of suspension cultures using a microfluidics-assisted cell screening platform

2017 ◽  
Vol 13 (1) ◽  
pp. 170-194 ◽  
Author(s):  
Burak Okumus ◽  
Charles J Baker ◽  
Juan Carlos Arias-Castro ◽  
Ghee Chuan Lai ◽  
Emanuele Leoncini ◽  
...  
Keyword(s):  
Single Cell ◽  
Suspension Cultures ◽  
Screening Platform ◽  
Cell Microscopy
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