scholarly journals Detection and quantification of single mRNA dynamics with the Riboglow fluorescent RNA tag

2019 ◽  
Author(s):  
Esther Braselmann ◽  
Timothy J. Stasevich ◽  
Kenneth Lyon ◽  
Robert T. Batey ◽  
Amy E. Palmer
Keyword(s):  
Single Molecule ◽  
Fluorescent Probes ◽  
Mammalian Cells ◽  
Fluorescent Antibody ◽  
Living Cells ◽  
Rna Molecules ◽  
Single Molecule Level ◽  
Rna Biology ◽  
Tagging System ◽  
Detection And Quantification

AbstractLabeling and tracking biomolecules with fluorescent probes on the single molecule level enables quantitative insights into their dynamics in living cells. We previously developed Riboglow, a platform to label RNAs in live mammalian cells, consisting of a short RNA tag and a small organic probe that increases fluorescence upon binding RNA. Here, we demonstrate that Riboglow is capable of detecting and tracking single RNA molecules. We benchmark RNA tracking by comparing results with the established MS2 RNA tagging system. To demonstrate versatility of Riboglow, we assay translation on the single molecule level, where the translated mRNA is tagged with Riboglow and the nascent polypeptide is labeled with a fluorescent antibody. The growing effort to investigate RNA biology on the single molecule level requires sophisticated and diverse fluorescent probes for multiplexed, multi-color labeling of biomolecules of interest, and we present Riboglow as a new member in this toolbox.

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew N. Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Computational Models ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

scholarly journals An Optical Setup for the Study of Mechanotransduction in Living Cells at the Single Molecule Level

2017 ◽  
Vol 112 (3) ◽  
pp. 588a
Author(s):  
Marios Sergides ◽  
Tommaso Galgani ◽  
Claudia Arbore ◽  
Francesco S. Pavone ◽  
Marco Capitanio
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Single Molecule Level ◽  
Optical Setup

Nanomechanochemical Delivery of Nanoparticles for Nanomechanics Inside Living Cells

2010 ◽  
Author(s):  
Kyungsuk Yum ◽  
Sungsoo Na ◽  
Yang Xiang ◽  
Ning Wang ◽  
Min-Feng Yu
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Endocytic Pathway ◽  
Great Promise ◽  
Biological Processes ◽  
Temporal Precision ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Cellular Environment ◽  
Biological Studies

Studying biological processes and mechanics in living cells is challenging but highly rewarding. Recent advances in experimental techniques have provided numerous ways to investigate cellular processes and mechanics of living cells. However, most of existing techniques for biomechanics are limited to experiments outside or on the membrane of cells, due to the difficulties in physically accessing the interior of living cells. On the other hand, nanomaterials, such as fluorescent quantum dots (QDs) and magnetic nanoparticles, have shown great promise to overcome such limitations due to their small sizes and excellent functionalities, including bright and stable fluorescence and remote manipulability. However, except a few systems, the use of nanoparticles has been limited to the study of biological studies on cell membranes or related to endocytosis, because of the difficulty of delivering dispersed and single nanoparticles into living cells. Various strategies have been explored, but delivered nanoparticles are often trapped in the endocytic pathway or form aggregates in the cytoplasm, limiting their further use. Here we show a nanoscale direct delivery method, named nanomechanochemical delivery, where we manipulate a nanotube-based nanoneedle, carrying “cargo” (QDs in this study), to mechanically penetrate the cell membrane, access specific areas inside cells, and release the cargo [1]. We selectively delivered well-dispersed QDs into either the cytoplasm or the nucleus of living cells. We quantified the dynamics of the delivered QDs by single-molecule tracking and demonstrated the applicability of the QDs as a nanoscale probe for studying nanomechanics inside living cells (by using the biomicrorhology method), revealing the biomechanical heterogeneity of the cellular environment. This method may allow new strategies for studying biological processes and mechanics in living cells with spatial and temporal precision, potentially at the single-molecule level.

scholarly journals A dual role for H2A.Z.1 in modulating the dynamics of RNA Polymerase II initiation and elongation

2020 ◽  
Author(s):  
Constantine Mylonas ◽  
Alexander L. Auld ◽  
Choongman Lee ◽  
Ibrahim I. Cisse ◽  
Laurie A. Boyer
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Mammalian Cells ◽  
Embryonic Stem ◽  
Super Resolution ◽  
Fine Tuning ◽  
Transcriptional Initiation ◽  
Single Molecule Level

AbstractRNAPII pausing immediately downstream of the transcription start site (TSS) is a critical rate limiting step at most metazoan genes that allows fine-tuning of gene expression in response to diverse signals1–5. During pause-release, RNA Polymerase II (RNAPII) encounters an H2A.Z.1 nucleosome6–8, yet how this variant contributes to transcription is poorly understood. Here, we use high resolution genomic approaches2,9 (NET-seq and ChIP-nexus) along with live cell super-resolution microscopy (tcPALM)10 to investigate the role of H2A.Z.1 on RNAPII dynamics in embryonic stem cells (ESCs). Using a rapid, inducible protein degron system11 combined with transcriptional initiation and elongation inhibitors, our quantitative analysis shows that H2A.Z.1 slows the release of RNAPII, impacting both RNAPII and NELF dynamics at a single molecule level. We also find that H2A.Z.1 loss has a dramatic impact on nascent transcription at stably paused, signal-dependent genes. Furthermore, we demonstrate that H2A.Z.1 inhibits re-assembly and re-initiation of the PIC to reinforce the paused state and acts as a strong additional pause signal at stably paused genes. Together, our study suggests that H2A.Z.1 fine-tunes gene expression by regulating RNAPII kinetics in mammalian cells.

scholarly journals Blinking fluorescent probes for tubulin nanoscopy in living and fixed cells

2021 ◽  
Author(s):  
Ruta Gerasimaite ◽  
Jonas Bucevicius ◽  
Kamila A. Kiszka ◽  
Georgij Kostiuk ◽  
Tanja Koenen ◽  
...  
Keyword(s):  
Small Molecule ◽  
Single Molecule ◽  
Fluorescent Probes ◽  
Living Cells ◽  
Stimulated Emission ◽  
Sted Microscopy ◽  
Linker Length ◽  
Localisation Microscopy ◽  
Red Dye ◽  
Small Molecule Probe

Here we report a small molecule probe for single molecule localisation microscopy (SMLM) of tubulin in living and fixed cells. We explored a series of constructs composed of taxanes and spontaneously blinking far-red dye hydroxymethyl silicon-rhodamine (HMSiR). We found that the linker length profoundly affects the probe permeability and off-targeting. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and 6'-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameters of <50 nm can be routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin also performs well in 3D stimulated emission depletion (STED) microscopy, allowing a complementary use of both nanoscopy methods for investigating microtubule functions in living cells.

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2020 ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell ◽  
Highly Efficient

The carboxyl-terminal domain of RNA polymerase II is dynamically phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to unphosphorylated and phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as clusters of Serine 5 phosphorylated RNAP2 that form around active genes and are separated in both space and time from nascent mRNA synthesis. Computational models fit to our data determine that 5 to 40 RNAP2 cluster around the promoter of a gene during typical transcriptional bursts. Nearly all RNAP2 either arrive with Serine 5 phosphorylation or acquire the modification within a minute. Transcription from the cluster appears to be highly efficient, with nearly half of the clustered RNAP2 ultimately escaping the promoter in a minute or so to elongate a full-length mRNA in approximately five minutes. The highly dynamic and spatially organized concentrations of RNAP2 we observe support the notion of highly efficient transcription clusters that form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

Fluorescent Probes for Chemical Transformations on the Single-Molecule Level

2008 ◽  
Vol 1130 (1) ◽  
pp. 131-137 ◽  
Author(s):  
Gregor Jung ◽  
Alexander Schmitt ◽  
Michaela Jacob ◽  
Babette Hinkeldey
Keyword(s):  
Single Molecule ◽  
Fluorescent Probes ◽  
Chemical Transformations ◽  
Single Molecule Level

scholarly journals Fast 3D imaging of giant unilamellar vesicles using reflected light-sheet microscopy with single molecule sensitivity

2020 ◽  
Author(s):  
Sven A. Szilagyi ◽  
Moritz Burmeister ◽  
Q. Tyrell Davis ◽  
Gero L. Hermsdorf ◽  
Suman De ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Numerical Aperture ◽  
Light Sheet ◽  
Living Cells ◽  
High Signal ◽  
Active Optics ◽  
Single Molecule Level ◽  
Light Sheet Microscopy ◽  
Reflected Light

AbstractObservation of highly dynamic processes inside living cells at the single molecule level is key for a quantitative understanding of biological systems. However, imaging of single molecules in living cells usually is limited by the spatial and temporal resolution, photobleaching and the signal-to-background ratio. To overcome these limitations, light-sheet microscopes with thin selective plane illumination have recently been developed. For example, a reflected light-sheet design combines the illumination by a thin light-sheet with a high numerical aperture objective for single-molecule detection. Here, we developed a reflected light-sheet microscope with active optics for fast, high contrast, two-color acquisition of z-stacks. We demonstrate fast volume scanning by imaging a two-color giant unilamellar vesicle (GUV) hemisphere. In addition, the high signal-to-noise ratio enabled the imaging and tracking of single lipids in the cap of a GUV. In the long term, the enhanced reflected scanning light sheet microscope enables fast 3D scanning of artificial membrane systems and cells with single-molecule sensitivity and thereby will provide quantitative and molecular insight into the operation of cells.

scholarly journals Central dogma at the single-molecule level in living cells

Nature ◽  
2011 ◽  
Vol 475 (7356) ◽  
pp. 308-315 ◽  
Author(s):  
Gene-Wei Li ◽  
X. Sunney Xie
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Central Dogma ◽  
Single Molecule Level
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