scholarly journals Metabolic activity affects response of single cells to a nutrient switch in structured populations

2019 ◽  
Author(s):  
Alma Dal Co ◽  
Martin Ackermann ◽  
Simon van Vliet
Keyword(s):  
Metabolic Activity ◽  
Phenotypic Variation ◽  
Single Cells ◽  
Structured Populations ◽  
Cell Level ◽  
Changing Environments ◽  
Spatially Structured Populations ◽  
Fluctuating Environments ◽  
Structured Groups ◽  
Microfluidic Chambers

AbstractMicrobes live in ever-changing environments where they need to adapt their metabolism to different nutrient conditions. Many studies have characterized the response of genetically identical cells to nutrient switches in homogenous cultures, however in nature microbes often live in spatially structured groups such as biofilms where cells can create metabolic gradients by consuming and releasing nutrients. Consequently, cells experience different local microenvironments and vary in their phenotype. How does this phenotypic variation affect the ability of cells to cope with nutrient switches? Here we address this question by growing dense populations of Escherichia coli in microfluidic chambers and studying a switch from glucose to acetate at the single cell level. Before the switch, cells vary in their metabolic activity: some grow on glucose while others cross-feed on acetate. After the switch, only few cells can resume growth after a period of lag. The probability to resume growth depends on a cells’ phenotype prior to the switch: it is highest for cells crossfeeding on acetate, while it depends in a non-monotonic way on growth rate for cells growing on glucose. Our results suggest that the strong phenotypic variation in spatially structured populations might enhance their ability to cope with fluctuating environments.

scholarly journals Metabolic activity affects the response of single cells to a nutrient switch in structured populations

2019 ◽  
Vol 16 (156) ◽  
pp. 20190182 ◽  
Author(s):  
Alma Dal Co ◽  
Martin Ackermann ◽  
Simon van Vliet
Keyword(s):  
Metabolic Activity ◽  
Phenotypic Variation ◽  
Single Cells ◽  
Structured Populations ◽  
Cell Level ◽  
Changing Environments ◽  
Spatially Structured Populations ◽  
Fluctuating Environments ◽  
Structured Groups ◽  
Microfluidic Chambers

Microbes live in ever-changing environments where they need to adapt their metabolism to different nutrient conditions. Many studies have characterized the response of genetically identical cells to nutrient switches in homogeneous cultures; however, in nature, microbes often live in spatially structured groups such as biofilms where cells can create metabolic gradients by consuming and releasing nutrients. Consequently, cells experience different local microenvironments and vary in their phenotype. How does this phenotypic variation affect the ability of cells to cope with nutrient switches? Here, we address this question by growing dense populations of Escherichia coli in microfluidic chambers and studying a switch from glucose to acetate at the single-cell level. Before the switch, cells vary in their metabolic activity: some grow on glucose, while others cross-feed on acetate. After the switch, only few cells can resume growth after a period of lag. The probability to resume growth depends on a cells' phenotype prior to the switch: it is highest for cells cross-feeding on acetate, while it depends in a non-monotonic way on the growth rate for cells growing on glucose. Our results suggest that the strong phenotypic variation in spatially structured populations might enhance their ability to cope with fluctuating environments.

scholarly journals A yeast FRET biosensor enlightens cAMP signalling

2019 ◽  
Author(s):  
Dennis Botman ◽  
Tom G. O’Toole ◽  
Joachim Goedhart ◽  
Frank J. Bruggeman ◽  
Johan H. van Heerden ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cells ◽  
Peak Height ◽  
Metabolic Adaptation ◽  
Single Cell Level ◽  
Cell Level ◽  
Dynamic Changes ◽  
Changing Environments ◽  
Intracellular Metabolism ◽  
Signalling Cascade

AbstractThe cAMP-PKA signalling cascade in budding yeast regulates adaptation to changing environments. We developed yEPAC, a FRET-based biosensor for cAMP measurements in yeast. We used this sensor with flow cytometry for high-throughput single cell-level quantification during dynamic changes in response to sudden nutrient transitions. We found that the characteristic cAMP peak differentiates between different carbon source transitions, and is rather homogenous among single-cells, especially for transitions to glucose. The peaks are mediated by a combination of extracellular sensing and intracellular metabolism. Moreover, the cAMP peak follows Weber’s law; its height scales with the relative, and not the absolute, change in glucose. Lastly, our results suggest that the cAMP peak height conveys information about prospective growth rates. In conclusion, our yEPAC-sensor makes possible new avenues for understanding yeast physiology, signalling and metabolic adaptation.

scholarly journals Identifying extracellular vesicle populations from single cells

2021 ◽  
Vol 118 (38) ◽  
pp. e2106630118 ◽  
Author(s):  
Jonas M. Nikoloff ◽  
Mario A. Saucedo-Espinosa ◽  
André Kling ◽  
Petra S. Dittrich
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Single Cells ◽  
Extracellular Vesicle ◽  
Single Cell Level ◽  
Cell Level ◽  
Highly Sensitive ◽  
High Resolution Images ◽  
Microfluidic Chambers

Extracellular vesicles (EVs) are constantly secreted from both eukaryotic and prokaryotic cells. EVs, including those referred to as exosomes, may have an impact on cell signaling and an incidence in diseased cells. In this manuscript, a platform to capture, quantify, and phenotypically classify the EVs secreted from single cells is introduced. Microfluidic chambers of about 300 pL are employed to trap and isolate individual cells. The EVs secreted within these chambers are then captured by surface-immobilized monoclonal antibodies (mAbs), irrespective of their intracellular origin. Immunostaining against both plasma membrane and cytosolic proteins was combined with highly sensitive, multicolor total internal reflection fluorescence microscopy to characterize the immobilized vesicles. The data analysis of high-resolution images allowed the assignment of each detected EV to one of 15 unique populations and demonstrated the presence of highly heterogeneous phenotypes even at the single-cell level. The analysis also revealed that each mAb isolates phenotypically different EVs and that more vesicles were effectively immobilized when CD63 was targeted instead of CD81. Finally, we demonstrate how a heterogeneous suppression in the secreted vesicles is obtained when the enzyme neutral sphingomyelinase is inhibited.

scholarly journals A yeast FRET biosensor enlightens cAMP signalling

2021 ◽  
pp. mbc.E20-05-0319
Author(s):  
Dennis Botman ◽  
Tom G. O'Toole ◽  
Joachim Goedhart ◽  
Frank J. Bruggeman ◽  
Johan H. van Heerden ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cells ◽  
Peak Height ◽  
Metabolic Adaptation ◽  
Single Cell Level ◽  
Cell Level ◽  
Dynamic Changes ◽  
Changing Environments ◽  
Intracellular Metabolism ◽  
Signalling Cascade

The cAMP-PKA signalling cascade in budding yeast regulates adaptation to changing environments. We developed yEPAC, a FRET-based biosensor for cAMP measurements in yeast. We used this sensor with flow cytometry for high-throughput single cell-level quantification during dynamic changes in response to sudden nutrient transitions. We found that the characteristic cAMP peak differentiates between different carbon source transitions, and is rather homogenous among single-cells, especially for transitions to glucose. The peaks are mediated by a combination of extracellular sensing and intracellular metabolism. Moreover, the cAMP peak follows the Weber-Fechner law; its height scales with the relative, and not the absolute, change in glucose. Lastly, our results suggest that the cAMP peak height conveys information about prospective growth rates. In conclusion, our yEPAC-sensor makes possible new avenues for understanding yeast physiology, signalling and metabolic adaptation. [Media: see text]

scholarly journals SUGAR-seq enables simultaneous detection of glycans, epitopes, and the transcriptome in single cells

2021 ◽  
Vol 7 (8) ◽  
pp. eabe3610
Author(s):  
Conor J. Kearney ◽  
Stephin J. Vervoort ◽  
Kelly M. Ramsbottom ◽  
Izabela Todorovski ◽  
Emily J. Lelliott ◽  
...  
Keyword(s):  
Single Cell ◽  
Posttranslational Modification ◽  
Cellular Differentiation ◽  
Single Cells ◽  
Simultaneous Detection ◽  
Surface Protein ◽  
Rna Seq ◽  
Cell Level ◽  
Simultaneous Integration ◽  
Unique Surface

Multimodal single-cell RNA sequencing enables the precise mapping of transcriptional and phenotypic features of cellular differentiation states but does not allow for simultaneous integration of critical posttranslational modification data. Here, we describe SUrface-protein Glycan And RNA-seq (SUGAR-seq), a method that enables detection and analysis of N-linked glycosylation, extracellular epitopes, and the transcriptome at the single-cell level. Integrated SUGAR-seq and glycoproteome analysis identified tumor-infiltrating T cells with unique surface glycan properties that report their epigenetic and functional state.

scholarly journals Vector-borne disease risk indexes in spatially structured populations

2018 ◽  
Vol 12 (2) ◽  
pp. e0006234 ◽  
Author(s):  
Jorge Velázquez-Castro ◽  
Andrés Anzo-Hernández ◽  
Beatriz Bonilla-Capilla ◽  
Moisés Soto-Bajo ◽  
Andrés Fraguela-Collar
Keyword(s):  
Disease Risk ◽  
Structured Populations ◽  
Vector Borne Disease ◽  
Spatially Structured Populations ◽  
Vector Borne

scholarly journals High Throughput Immunophenotyping and Expression Profiling at Single Cell Level Reveal BCR-ABL1 Dependent Surface Markers of Chronic Myeloid Leukemia Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2920-2920
Author(s):  
Marianna Romzova ◽  
Dagmar Smitalova ◽  
Peter Taus ◽  
Jiri Mayer ◽  
Martin Culen
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Expression Profiling ◽  
Myeloid Leukemia ◽  
Single Cells ◽  
Transcript Level ◽  
Surface Expression ◽  
Surface Markers ◽  
Cell Level ◽  
Tki Treatment

BACKGROUND: Bcr-abl1 oncogene targeted treatment with tyrosine kinase inhibitors (TKI) showed an impressive efficacy against proliferating chronic myeloid leukemia (CML) cells. However, rapid relapses in more than half of CML patients after discontinuation of the treatment suggest a presence of quiescent leukemic stem cells inherently resistant to BCR-ABL1 inhibition. Understanding the heterogeneity of CML stem cell compartment is crucial for preventing the treatment failure. Specificity of already established leukemic stem cell (LSC) markers has been tested mainly in bulk CD34+CD38- populations at diagnosis. Phenotypes and molecular signatures of therapy resistant BCR ABL1 positive stem cells is however yet to be established. AIMS: Identification of BCR-ABL1 dependent LSC markers at single cell level by direct comparison their surface and transcript expression with the levels and the presence of BCR-ABL1 transcript at diagnosis and after administration of TKI treatment. METHODS: Total number of 375 cells were obtained from bone marrow and peripheral blood of 4 chronic phase CML patients. Cells were collected prior any treatment and three months after TKI treatment initiation. Normal bone marrow cells and BCR-ABL1 positive K562 cell line were used as controls. Indexed immuno-phenotyping and sorting of CD34+CD38- single cells was performed using a panel of 11 specific surface markers. Collected single cells were lysed and cDNA was enriched for 11 targets using 22 cycle pre-amplification. Expression profiling was carried on SmartChip real-time PCR system (Takara Bio) detecting following genes: BCR-ABL1, CD26, CD25, IL1-Rap, CD56, CD90, CD93, CD69, KI67, and control genes GUS and HPRT. Unsupervised clustering was performed using principal component analysis (PCA). Correlations were measured by Spearman rank method. RESULTS: At diagnosis, majority of BCR-ABL1+ C34+CD38- stem cells co-express IL1-Rap, CD26, and CD69 on their surface (88%, 82%, 78% overlap). Only 56% of BCR-ABL1+ cells positive for aforementioned markers co-express CD25, 28% CD93 and 16% CD56. The expression of these markers could also be detected in 4-11% of BCR-ABL1- cell, although this could be technical inaccuracy caused by the single cell profiling. CD90 marker did not show any correlation with BCR-ABL1 expression. At transcript level the expression of IL-1Rap, CD26, CD25 and CD56 was observed in 62%, 52% 45% and 16% BCR-ABL1+ cells, and up to 7% of BCR-ABL1- cells. CD69 expression was observed in 90% of BCR-ABL+ cells at transcript level, but also in 71% BCR-ABL- cells. BCR-ABL1 independent expression was observed for cKIT. (60% vs. 76 % in positive vs negative). Finally proliferation marker KI67 was expressed only in 6% of the BCR-ABL1+ cells. PCA analysis divided cells into several distinct clusters with BCR-ABL1 as the main contributor, and cKIT, CD69 and CD26, IL-1RAP as other significant factors. Interestingly BCR-ABL1+ cells collected during TKI treatment showed persistent surface expression of IL-1Rap and CD26, while CD56, CD69 and CD93 were only on part of the BCR-ABL1+ cells. CD25 was significantly deregulated during TKI treatment. CONCLUSION: At diagnosis up to 80% of LSC co-express 3 specific surface markers - IL-1RAP, CD26 and CD69. Variable portion of LSC co-express additional markers such are CD25, CD56 and CD93. During TKI treatment the surface expression of majority of markers is decreased, where the best correlated LSC marker is IL-1Rap, followed by CD26 and CD69. CD56 marker seems to persist in the same proportion of cells while CD25 disappears. cKIT is highly expressed in normal BM and HSC from CML patients, but also in some LSC. CD34+CD38- cells show non-proliferating phenotype. Disclosures Mayer: AOP Orphan Pharmaceuticals AG: Research Funding.

scholarly journals Estado Atual de Conhecimento de Dinâmica de Populações

2019 ◽  
Vol 23 (3) ◽  
pp. 256
Author(s):  
Anna Clara Balbina Silva ◽  
Afonso Pelli
Keyword(s):  
Population Dynamics ◽  
Full Text ◽  
Mortality Rates ◽  
Structured Populations ◽  
Exclusion Criteria ◽  
Spatially Structured Populations ◽  
The Subject ◽  
Population Interactions ◽  
Dynamic Population ◽  
Critical Challenge

Compreender os mecanismos que regulam a dinâmica das populações espacialmente estruturadas é um desafio crítico para os ecólogos e gestores de conservação. A dinâmica de populações é um ramo da ecologia que estuda as populações como sistema em atividades, relacionando as influências ambientais com a distribuição e abundância dos indivíduos e suas interações com o ambiente. O presente artigo é uma revisão bibliográfica, com o objetivo de identificar produções científicas relevantes sobre dinâmica populacional. Para isso, foram utilizados periódicos revisados por pares, na base de Periódicos Capes. A pesquisa foi realizada em junho de 2019, utilizando-se as palavras-chave para título contendo: "population dynamics" e no assunto “ecology”, a partir de 2014, quando o texto completo estava disponível. Foram considerados como critérios de exclusão os artigos publicados antes de 2014. Após a leitura dos títulos dos artigos, foram selecionados 34 artigos que foram lidos na íntegra. Em livros disponíveis no acervo da biblioteca da Universidade Federal do Triângulo Mineiro, foram selecionados quatro livros no tema dinâmica populacional. O referencial teórico aborda os aspectos da dinâmica de populações, tabela de vida, formas de crescimento e interações populacionais. Ressalta-se a necessidade de novos estudos que ainda possuem lacunas, que venha complementar e contribuir para o conhecimento de organismos que faltam ou ainda não possuem registros de estudos. Palavras-chave: Taxas de Natalidade e Mortalidade. Atributos Populacionais. Dispersão. AbstractUnderstanding the mechanisms that regulate the dynamics of spatially structured populations is a critical challenge for ecologists and conservation managers. Population dynamics is a branch of ecology that studies populations as a system in activities, relating environmental influences to the individuals’ distribution and abundance and their interactions with the environment. This article is a bibliographic review, aiming to identify relevant scientific productions about population dynamics. Thus. peer-reviewed journals were used in the Capes Periodicals base, the research was conducted in June 2019, using the keywords for title containing "population dynamics" and in the subject "ecology", from 2014, when the full text was available. Exclusion criteria were: articles published before 2014, after reading the article titles, 34 articles were selected that met the initially proposed criteria and were read in full. In books available in the library collection of the Federal University of Triângulo Mineiro, with a search for the dynamic population theme, 4 books were used. The theoretical framework addresses the aspects of population dynamics, life table, forms of growth and population interactions. It is emphasized  the need for further studies that still have gaps, which will complement and contribute to the knowledge of organisms that are missing or do not have study records. Keywords: Birth and Mortality Rates. Population Attributes. Dispersion.

scholarly journals Microfluidic Platforms Designed for Morphological and Photosynthetic Investigations of Chlamydomonas reinhardtii on a Single-Cell Level

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 285
Author(s):  
Eszter Széles ◽  
Krisztina Nagy ◽  
Ágnes Ábrahám ◽  
Sándor Kovács ◽  
Anna Podmaniczki ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Single Cell ◽  
Single Cells ◽  
Fluorescence Induction ◽  
Photochemical Quenching ◽  
Support Surface ◽  
Single Cell Level ◽  
Microfluidic Platforms ◽  
Cell Level ◽  
Non Photochemical Quenching

Chlamydomonas reinhardtii is a model organism of increasing biotechnological importance, yet, the evaluation of its life cycle processes and photosynthesis on a single-cell level is largely unresolved. To facilitate the study of the relationship between morphology and photochemistry, we established microfluidics in combination with chlorophyll a fluorescence induction measurements. We developed two types of microfluidic platforms for single-cell investigations: (i) The traps of the “Tulip” device are suitable for capturing and immobilizing single cells, enabling the assessment of their photosynthesis for several hours without binding to a solid support surface. Using this “Tulip” platform, we performed high-quality non-photochemical quenching measurements and confirmed our earlier results on bulk cultures that non-photochemical quenching is higher in ascorbate-deficient mutants (Crvtc2-1) than in the wild-type. (ii) The traps of the “Pot” device were designed for capturing single cells and allowing the growth of the daughter cells within the traps. Using our most performant “Pot” device, we could demonstrate that the FV/FM parameter, an indicator of photosynthetic efficiency, varies considerably during the cell cycle. Our microfluidic devices, therefore, represent versatile platforms for the simultaneous morphological and photosynthetic investigations of C. reinhardtii on a single-cell level.

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