scholarly journals Real-time single-cell characterization of the eukaryotic transcription cycle reveals correlations between RNA initiation, elongation, and cleavage

2021 ◽  
Vol 17 (5) ◽  
pp. e1008999
Author(s):  
Jonathan Liu ◽  
Donald Hansen ◽  
Elizabeth Eck ◽  
Yang Joon Kim ◽  
Meghan Turner ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Cell ◽  
Real Time ◽  
Fruit Fly ◽  
Effective Parameters ◽  
Spatiotemporal Resolution ◽  
Eukaryotic Transcription ◽  
Nascent Rna ◽  
Transcription Cycle

The eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel application of Bayesian inference techniques to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters as well as single-cell correlations between these parameters. These measurements, combined with theoretical modeling, suggest a substantial variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

scholarly journals Single-cell characterization of the eukaryotic transcription cycle using live imaging and statistical inference

2020 ◽  
Author(s):  
Jonathan Liu ◽  
Donald Hansen ◽  
Elizabeth Eck ◽  
Yang Joon Kim ◽  
Meghan Turner ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Single Cell ◽  
Fruit Fly ◽  
Computational Technique ◽  
Effective Parameters ◽  
Spatiotemporal Resolution ◽  
Eukaryotic Transcription ◽  
Nascent Rna ◽  
Transcription Cycle

AbstractThe eukaryotic transcription cycle consists of three main steps: initiation, elongation, and cleavage of the nascent RNA transcript. Although each of these steps can be regulated as well as coupled with each other, their in vivo dissection has remained challenging because available experimental readouts lack sufficient spatiotemporal resolution to separate the contributions from each of these steps. Here, we describe a novel computational technique to simultaneously infer the effective parameters of the transcription cycle in real time and at the single-cell level using a two-color MS2/PP7 reporter gene and the developing fruit fly embryo as a case study. Our method enables detailed investigations into cell-to-cell variability in transcription-cycle parameters with high precision. These measurements, combined with theoretical modeling, reveal a significant variability in the elongation rate of individual RNA polymerase molecules. We further illustrate the power of this technique by uncovering a novel mechanistic connection between RNA polymerase density and nascent RNA cleavage efficiency. Thus, our approach makes it possible to shed light on the regulatory mechanisms in play during each step of the transcription cycle in individual, living cells at high spatiotemporal resolution.

scholarly journals Differential Responses of Transplanted Stem Cells to Diseased Environment Unveiled by a Molecular NIR-II Cell Tracker

Research ◽  
2021 ◽  
Vol 2021 ◽  
pp. 1-16
Author(s):  
Hao Chen ◽  
Huaxiao Yang ◽  
Chen Zhang ◽  
Si Chen ◽  
Xin Zhao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Therapy ◽  
Single Cell ◽  
Real Time ◽  
Stem Cell Therapy ◽  
Cellular Migration ◽  
Cell Cluster ◽  
Spatiotemporal Resolution

Stem cell therapy holds high promises in regenerative medicine. The major challenge of clinical translation is to precisely and quantitatively evaluate the in vivo cell distribution, migration, and engraftment, which cannot be easily achieved by current techniques. To address this issue, for the first time, we have developed a molecular cell tracker with a strong fluorescence signal in the second near-infrared (NIR-II) window (1,000-1,700 nm) for real-time monitoring of in vivo cell behaviors in both healthy and diseased animal models. The NIR-II tracker (CelTrac1000) has shown complete cell labeling with low cytotoxicity and profound long-term tracking ability for 30 days in high spatiotemporal resolution for semiquantification of the biodistribution of transplanted stem cells. Taking advantage of the unique merits of CelTrac1000, the responses of transplanted stem cells to different diseased environments have been discriminated and unveiled. Furthermore, we also demonstrate CelTrac1000 as a universal and effective technique for ultrafast real-time tracking of the cellular migration and distribution in a 100 μm single-cell cluster spatial resolution, along with the lung contraction and heart beating. As such, this NIR-II tracker will shift the optical cell tracking into a single-cell cluster and millisecond temporal resolution for better evaluating and understanding stem cell therapy, affording optimal doses and efficacy.

scholarly journals Multi-Modal Multi-Spectral Intravital Microscopic Imaging of Signaling Dynamics in Real-Time during Tumor–Immune Interactions

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 499
Author(s):  
Tracy W. Liu ◽  
Seth T. Gammon ◽  
David Piwnica-Worms
Keyword(s):  
Molecular Imaging ◽  
Single Cell ◽  
Real Time ◽  
Bioluminescence Imaging ◽  
Emission Spectra ◽  
Ccd Camera ◽  
Microscopic Imaging ◽  
Signaling Dynamics ◽  
Window Chamber

Intravital microscopic imaging (IVM) allows for the study of interactions between immune cells and tumor cells in a dynamic, physiologically relevant system in vivo. Current IVM strategies primarily use fluorescence imaging; however, with the advances in bioluminescence imaging and the development of new bioluminescent reporters with expanded emission spectra, the applications for bioluminescence are extending to single cell imaging. Herein, we describe a molecular imaging window chamber platform that uniquely combines both bioluminescent and fluorescent genetically encoded reporters, as well as exogenous reporters, providing a powerful multi-plex strategy to study molecular and cellular processes in real-time in intact living systems at single cell resolution all in one system. We demonstrate that our molecular imaging window chamber platform is capable of imaging signaling dynamics in real-time at cellular resolution during tumor progression. Importantly, we expand the utility of IVM by modifying an off-the-shelf commercial system with the addition of bioluminescence imaging achieved by the addition of a CCD camera and demonstrate high quality imaging within the reaches of any biology laboratory.

Transcriptional Pausing in Vivo: A Nascent RNA Hairpin Restricts Lateral Movements of RNA Polymerase in Both Forward and Reverse Directions

2005 ◽  
Vol 351 (1) ◽  
pp. 39-51 ◽  
Author(s):  
Francine Toulmé ◽  
Christine Mosrin-Huaman ◽  
Irina Artsimovitch ◽  
A. Rachid Rahmouni
Keyword(s):  
Rna Polymerase ◽  
Transcriptional Pausing ◽  
Nascent Rna ◽  
Rna Hairpin

scholarly journals Visualizing ATP Dynamics in Live Mice

2020 ◽  
Author(s):  
Norimichi Koitabashi ◽  
Riki Ogasawara ◽  
Ryuto Yasui ◽  
Yuki Sugiura ◽  
Hinako Matsuda ◽  
...  
Keyword(s):  
Mouse Model ◽  
Real Time ◽  
Biological Activities ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spatiotemporal Resolution ◽  
Physiological Mechanisms ◽  
Multiple Organs ◽  
Atp Levels

ABSTRACTAnalysis of the dynamics of adenosine triphosphate (ATP) is vital to quantitatively define the actual roles of ATP in biological activities. Here, we applied a genetically encoded Förster resonance energy transfer biosensor “GO-ATeam” and created a transgenic mouse model that allows systemic ATP levels to be quantitatively, sensitively, noninvasively, and spatiotemporally measured under physiological and pathological conditions. We used this model to readily conduct intravital imaging of ATP dynamics under three different conditions: during exercise, in all organs and cells; during myocardial infarction progression; and in response to the application of cardiotoxic drugs. These findings provide compelling evidence that the GO-ATeam mouse model is a powerful tool to investigate the multifarious functions of cellular ATP in vivo with unprecedented spatiotemporal resolution in real-time. This will inform predictions of molecular and morphological responses to perturbations of ATP levels, as well as the elucidation of physiological mechanisms that control ATP homeostasis.One Sentence SummaryIntravital real-time imaging of ATP dynamics in multiple organs using GO-ATeam mice, can be used to quantitatively, sensitively, noninvasively, and spatiotemporally measure systemic ATP levels and provide a platform for preclinical pharmacological studies.

scholarly journals Genetically-encoded fluorescent biosensor for rapid detection of protein expression

2020 ◽  
Author(s):  
Matthew G Eason ◽  
Antonia T Pandelieva ◽  
Marc M Mayer ◽  
Safwat T Khan ◽  
Hernan G Garcia ◽  
...  
Keyword(s):  
Protein Expression ◽  
Real Time ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
E Coli ◽  
Fluorescent Biosensor ◽  
Green Fluorescent

Fluorescent proteins are widely used as fusion tags to detect protein expression in vivo. To become fluorescent, these proteins must undergo chromophore maturation, a slow process with a half-time of 5 to >30 min, which causes delays in real-time detection of protein expression. Here, we engineer a genetically-encoded fluorescent biosensor to enable detection of protein expression within seconds in live cells. This sensor for transiently-expressed proteins (STEP) is based on a fully matured but dim green fluorescent protein in which pre-existing fluorescence increases 11-fold in vivo following the specific and rapid binding of a protein tag (Kd 120 nM, kon 1.7 x 10^5 M-1s-1). In live E. coli cells, our STEP biosensor enables detection of protein expression twice as fast as the use of standard fluorescent protein fusions. Our biosensor opens the door to the real-time study of short-timescale processes in research model animals with high spatiotemporal resolution.

scholarly journals Transcription of fractionated mammalian chromatin by mammalian ribonucleic acid polymerase. Demonstration of temperature-dependent rifampicin-resistant initiation sites in euchromatin deoxyribonucleic acid

1974 ◽  
Vol 143 (1) ◽  
pp. 73-81 ◽  
Author(s):  
C. James Chesterton ◽  
Barbara E. H. Coupar ◽  
Peter H. W. Butterworth
Keyword(s):  
Rna Polymerase ◽  
Rat Liver ◽  
Deoxyribonucleic Acid ◽  
Temperature Dependent ◽  
Fractionation Method ◽  
Liver Nuclei ◽  
Nascent Rna ◽  
Chromatin Fractionation

The chromatin fractionation method of Frenster et al. (1963) as modified by Leake et al. (1972) was used to prepare fragments of euchromatin from rat liver nuclei. These remain soluble in 5mm-MgCl2, and contain DNA of maximum mol.wt. 1×106–2×106. The fragments were separated from condensable chromatin on a sucrose gradient. Euchromatin contains endogenous DNA-dependent RNA polymerase, and most of the nascent RNA labelled in vivo or in vitro. Euchromatin fragments allow initiation of transcription by added purified rat liver form-B RNA polymerase and contain temperature-dependent rifampicin-resistant initiation sites for the form-B enzyme. These findings indicate that transcription of the euchromatin regions of interphase chromosomes is not initiated in condensed chromatin, but is initiated within the euchromatin stretches. Condensable chromatin also contains most of these activities, but is not associated with nascent RNA.

scholarly journals MATRIEX imaging: multiarea two-photon real-time in vivo explorer

2019 ◽  
Vol 8 (1) ◽  
Author(s):  
Mengke Yang ◽  
Zhenqiao Zhou ◽  
Jianxiong Zhang ◽  
Shanshan Jia ◽  
Tong Li ◽  
...  
Keyword(s):  
Single Cell ◽  
Real Time ◽  
Neuronal Activity ◽  
Laser Scanning ◽  
Primary Motor Cortex ◽  
Laser Scanning Microscopy ◽  
Functional Region ◽  
Two Photon ◽  
Optical Module

AbstractTwo-photon laser scanning microscopy has been extensively applied to study in vivo neuronal activity at cellular and subcellular resolutions in mammalian brains. However, the extent of such studies is typically confined to a single functional region of the brain. Here, we demonstrate a novel technique, termed the multiarea two-photon real-time in vivo explorer (MATRIEX), that allows the user to target multiple functional brain regions distributed within a zone of up to 12 mm in diameter, each with a field of view (FOV) of ~200 μm in diameter, thus performing two-photon Ca2+ imaging with single-cell resolution in all of the regions simultaneously. For example, we demonstrate real-time functional imaging of single-neuron activities in the primary visual cortex, primary motor cortex and hippocampal CA1 region of mice in both anesthetized and awake states. A unique advantage of the MATRIEX technique is the configuration of multiple microscopic FOVs that are distributed in three-dimensional space over macroscopic distances (>1 mm) both laterally and axially but that are imaged by a single conventional laser scanning device. In particular, the MATRIEX technique can be effectively implemented as an add-on optical module for an existing conventional single-beam-scanning two-photon microscope without requiring any modification to the microscope itself. Thus, the MATRIEX technique can be readily applied to substantially facilitate the exploration of multiarea neuronal activity in vivo for studies of brain-wide neural circuit function with single-cell resolution.

scholarly journals Role for the Ssu72 C-Terminal Domain Phosphatase in RNA Polymerase II Transcription Elongation

2006 ◽  
Vol 27 (3) ◽  
pp. 926-936 ◽  
Author(s):  
Mariela Reyes-Reyes ◽  
Michael Hampsey
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Elongation Factor ◽  
Elongation Complex ◽  
Terminal Domain ◽  
Rnap Ii ◽  
Transcription Cycle

ABSTRACT The RNA polymerase II (RNAP II) transcription cycle is accompanied by changes in the phosphorylation status of the C-terminal domain (CTD), a reiterated heptapeptide sequence (Y1S2P3T4S5P6S7) present at the C terminus of the largest RNAP II subunit. One of the enzymes involved in this process is Ssu72, a CTD phosphatase with specificity for serine-5-P. Here we report that the ssu72-2-encoded Ssu72-R129A protein is catalytically impaired in vitro and that the ssu72-2 mutant accumulates the serine-5-P form of RNAP II in vivo. An in vitro transcription system derived from the ssu72-2 mutant exhibits impaired elongation efficiency. Mutations in RPB1 and RPB2, the genes encoding the two largest subunits of RNAP II, were identified as suppressors of ssu72-2. The rpb1-1001 suppressor encodes an R1281A replacement, whereas rpb2-1001 encodes an R983G replacement. This information led us to identify the previously defined rpb2-4 and rpb2-10 alleles, which encode catalytically slow forms of RNAP II, as additional suppressors of ssu72-2. Furthermore, deletion of SPT4, which encodes a subunit of the Spt4-Spt5 early elongation complex, also suppresses ssu72-2, whereas the spt5-242 allele is suppressed by rpb2-1001. These results define Ssu72 as a transcription elongation factor. We propose a model in which Ssu72 catalyzes serine-5-P dephosphorylation subsequent to addition of the 7-methylguanosine cap on pre-mRNA in a manner that facilitates the RNAP II transition into the elongation stage of the transcription cycle.

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