Illuminating stem cell transcription factor dynamics: long-term single-cell imaging of fluorescent protein fusions

2017 ◽  
Vol 49 ◽  
pp. 77-83 ◽  
Author(s):  
Martin Etzrodt ◽  
Timm Schroeder
Keyword(s):  
Transcription Factor ◽  
Stem Cell ◽  
Single Cell ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Single Cell Imaging

scholarly journals SunRISE: long-term imaging of individual mRNA molecules in living cells

2020 ◽  
Author(s):  
Yue Guo ◽  
Robin E. C. Lee
Keyword(s):  
Phase Separation ◽  
Single Cell ◽  
Cell Imaging ◽  
Living Cells ◽  
Mrna Detection ◽  
Central Dogma ◽  
Single Cell Imaging ◽  
Cell Fixation ◽  
Limited Sensitivity

AbstractSingle-cell imaging of individual mRNAs has revealed core mechanisms of the central dogma. However, most approaches require cell fixation or have limited sensitivity for live-cell applications. Here, we describe SunRISE (SunTag-based Reporter for Imaging Signal Enriched mRNA), a computationally and experimentally optimized approach for unambiguous single-mRNA detection in living cells. We demonstrate SunRISE with long-term epifluorescence imaging, using translational stress to track mRNA phase separation and recovery from cytosolic droplets.

Long-term single-cell imaging of mammalian stem cells

2011 ◽  
Vol 8 (S4) ◽  
pp. S30-S35 ◽  
Author(s):  
Timm Schroeder
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Cell Imaging ◽  
Single Cell Imaging

scholarly journals High-throughput Automated Single Cell Imaging Analysis Reveals Dynamics Of Glioblastoma Stem Cell Population During State Transition

2018 ◽  
Author(s):  
Anastasia P. Chumakova ◽  
Masahiro Hitomi ◽  
Erik P. Sulman ◽  
Justin D. Lathia
Keyword(s):  
Stem Cell ◽  
Protein Expression ◽  
Single Cell ◽  
High Throughput ◽  
Cell Imaging ◽  
State Transition ◽  
Imaging Analysis ◽  
Sox2 Expression ◽  
Single Cell Imaging ◽  
Stem Cell State

ABSTRACTCancer stem cells (CSCs) are a heterogeneous and dynamic population that stands at the top of tumor cellular hierarchy and is responsible for maintenance of the tumor microenvironment. As methods of CSC isolation and functional interrogation advance, there is a need for a reliable and accessible quantitative approach to assess heterogeneity and state transition dynamics in CSCs. We developed a High-throughput Automated Single Cell Imaging Analysis (HASCIA) approach for quantitative assessment of protein expression with single cell resolution and applied the method to investigate spatiotemporal factors that influence CSC state transition using glioblastoma (GBM) CSC as a model system. We were able to validate the quantitative nature of this approach through comparison of the protein expression levels determined by HASCIA to those determined by immunoblotting. A virtue of HASCIA was exemplified by detection of a subpopulation of SOX2-low cells, which expanded in fraction size during state transition. HASCIA also revealed that CSCs were committed to loose stem cell state at an earlier time point than the average SOX2 level decreased. Functional assessment of stem cell frequency in combination with quantification of SOX2 expression by HASCIA defined a stable cut-off of SOX2 expression level for stem cell state. We also developed an approach to assess local cell density and found that denser monolayer areas possess higher average levels of SOX2, higher cell diversity and a presence of a sub-population of slowly proliferating SOX2-low CSCs. HASCIA is an open source software that facilitates understanding the dynamics of heterogeneous cell population such as that of CSCs and their progeny. It is a powerful and easy-to-use image analysis and statistical analysis tool available athttps://hascia.lerner.ccf.org.

Microglia turnover with aging and in an Alzheimer's model via long-term in vivo single-cell imaging

2017 ◽  
Vol 20 (10) ◽  
pp. 1371-1376 ◽  
Author(s):  
Petra Füger ◽  
Jasmin K Hefendehl ◽  
Karthik Veeraraghavalu ◽  
Ann-Christin Wendeln ◽  
Christine Schlosser ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Imaging ◽  
Single Cell Imaging

scholarly journals Understanding cell fate control by continuous single-cell quantification

Blood ◽  
2019 ◽  
Vol 133 (13) ◽  
pp. 1406-1414 ◽  
Author(s):  
Dirk Loeffler ◽  
Timm Schroeder
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Cell Imaging ◽  
Population Based ◽  
Molecular Processes ◽  
Cell Fate Decisions ◽  
Single Cell Imaging

Abstract Cells and the molecular processes underlying their behavior are highly dynamic. Understanding these dynamic biological processes requires noninvasive continuous quantitative single-cell observations, instead of population-based average or single-cell snapshot analysis. Ideally, single-cell dynamics are measured long-term in vivo; however, despite progress in recent years, technical limitations still prevent such studies. On the other hand, in vitro studies have proven to be useful for answering long-standing questions. Although technically still demanding, long-term single-cell imaging and tracking in vitro have become valuable tools to elucidate dynamic molecular processes and mechanisms, especially in rare and heterogeneous populations. Here, we review how continuous quantitative single-cell imaging of hematopoietic cells has been used to solve decades-long controversies. Because aberrant cell fate decisions are at the heart of tissue degeneration and disease, we argue that studying their molecular dynamics using quantitative single-cell imaging will also improve our understanding of these processes and lead to new strategies for therapies.

ERK phosphorylation and nuclear accumulation: insights from single-cell imaging

2012 ◽  
Vol 40 (1) ◽  
pp. 224-229 ◽  
Author(s):  
Christopher J. Caunt ◽  
Craig A. McArdle
Keyword(s):  
Single Cell ◽  
Nuclear Localization ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Mitogen Activated Protein Kinase ◽  
Nuclear Accumulation ◽  
Nuclear Targeting ◽  
Single Cell Imaging ◽  
Erk Phosphorylation

Many stimuli mediate activation and nuclear translocation of ERK (extracellular-signal-regulated kinase) by phosphorylation on the TEY (Thr-Glu-Tyr) motif. This is necessary to initiate transcriptional programmes controlling cellular responses, but the mechanisms that govern ERK nuclear targeting are unclear. Single-cell imaging approaches have done much to increase our understanding of input–output relationships in the ERK cascade, but few studies have addressed how the range of ERK phosphorylation responses observed in cell populations influences subcellular localization. Using automated microscopy to explore ERK regulation in single adherent cells, we find that nuclear localization responses increase in proportion to stimulus level, but not the level of TEY phosphorylation. This phosphorylation-unattributable nuclear localization response occurs in the presence of tyrosine phosphatase and protein synthesis inhibitors. It is also seen with a catalytically inactive ERK2–GFP (green fluorescent protein) mutant, and with a mutant incapable of binding the DEF (docking site for ERK, F/Y-X-F/Y-P) domains found in many ERK-binding partners. It is, however, reduced by MEK (mitogen-activated protein kinase/ERK kinase) inhibition and by mutations preventing TEY phosphorylation or in the ERK common docking region. We therefore show that TEY phosphorylation of ERK is necessary, but not sufficient, for the full nuclear accumulation response and that this ‘phosphorylation-unattributable’ component of stimulus-mediated ERK nuclear localization requires association with partner proteins via the common docking motif.

Long-term Live Single Cell Quantification of Transcription Factor Dynamics

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-4-SCI-4
Author(s):  
Timm Schroeder
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Cell Fate ◽  
Fluorescent Protein ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Molecular Control ◽  
Endogenous Gene ◽  
Stem And Progenitor Cells

Abstract Hematopoiesis is highly complex and dynamic, and consist of large numbers of different cells expressing many molecules. Despite intensive research, many long-standing questions in hematopoiesis research remain disputed. One major reason is the fact that we usually only analyze populations of cells - rather than individual cells - at very few time points of an experiment. Tracking of individual cells would be an extremely powerful approach to improve our understanding of molecular cell fate control. We are therefore developing imaging systems to follow the fate of single cells over many generations. We program new software to help recording and displaying the divisional history, position, properties, interaction, etc. of all individual cells over many generations. In addition, novel microfluidics devices are designed and produced to allow improved observation and manipulation of cells. Our technologies allow continuous long-term quantification of protein expression or activity in living cells. Among other approaches, we generate knock in models expressing transcription factor to fluorescent protein fusions from endogenous gene loci. This enables non-invasive long-term live quantification of transcription factor protein dynamics in single stem and progenitor cells throughout their differentiation. The resulting novel kind of continuous quantitative single cell data is used for the generation and falsification of models describing the molecular control of hematopoietic cell fates. Disclosures No relevant conflicts of interest to declare.

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