scholarly journals Kinetics of HTLV-1 reactivation from latency quantified by single-molecule RNA FISH and stochastic modelling

2019 ◽  
Vol 15 (11) ◽  
pp. e1008164 ◽  
Author(s):  
Michi Miura ◽  
Supravat Dey ◽  
Saumya Ramanayake ◽  
Abhyudai Singh ◽  
David S. Rueda ◽  
...  
Keyword(s):  
Single Molecule ◽  
Stochastic Modelling ◽  
Rna Fish ◽  
Kinetics Of

scholarly journals Kinetics of HTLV-1 reactivation from latency quantified by single-molecule RNA FISH and stochastic modelling

2019 ◽  
Author(s):  
Michi Miura ◽  
Supravat Dey ◽  
Saumya Ramanayake ◽  
Abhyudai Singh ◽  
David Rueda ◽  
...  
Keyword(s):  
T Cell ◽  
Single Molecule ◽  
Cell Clone ◽  
New Host ◽  
Rna Fish ◽  
Human T Cell ◽  
T Cell Clone ◽  
Sense Strand ◽  
Infected Cells ◽  
Kinetics Of

AbstractThe human T cell leukemia virus HTLV-1 establishes a persistent infectionin vivoin which the viral sense-strand transcription is usually silent at a given time in each cell. However, cellular stress responses trigger the reactivation of HTLV-1, enabling the virus to transmit to a new host cell. Using single-molecule RNA FISH, we measured the kinetics of the HTLV-1 transcriptional reactivation in peripheral blood mononuclear cells (PBMCs) isolated from HTLV-1+individuals. The abundance of the HTLV-1 sense and antisense transcripts was quantified hourly during incubation of the HTLV-1-infected PBMCsex vivo. We found that, in each cell, the sense-strand transcription occurs in two distinct phases: the initial low-rate transcription is followed by a phase of rapid transcription. The onset of transcription peaked between 1 and 3 hours after the start ofin vitroincubation. The variance in the transcription intensity was similar in polyclonal HTLV-1+PBMCs (with tens of thousands of distinct provirus insertion sites), and in samples with a single dominant HTLV-1+clone. A stochastic simulation model was developed to estimate the parameters of HTLV-1 proviral transcription kinetics. In PBMCs from a leukemic subject with one dominant T-cell clone, the model indicated that the average duration of HTLV-1 sense-strand activation by Tax (i.e. the rapid transcription) was less than one hour. HTLV-1 antisense transcription was stable during reactivation of the sense-strand. The antisense transcriptHBZwas produced at an average rate of x~0.1 molecules per hour per HTLV-1+cell; however, between 20% and 70% of HTLV-1-infected cells wereHBZ-negative at a given time, the percentage depending on the individual subject. HTLV-1-infected cells are exposed to a range of stresses when they are drawn from the host, which initiate the viral reactivation. We conclude that whereas antisense-strand transcription is stable throughout the stress response, the HTLV-1 sense-strand reactivation is highly heterogeneous and occurs in short, self-terminating bursts.Author summaryHuman retroviruses such as HIV-1 and HTLV-1 (human T cell leukemia virus) can establish a latent infection in the host cell. However, these viruses need to be able to produce viral genome to propagate in a new host. HTLV-1-infected cells are transmitted through breastfeeding, blood transfusion and sexual contact, and HTLV-1 restores transcription once the infected cells are drawn from infected individuals. We measured the kinetics of the HTLV-1 transcriptional reactivation in blood cells isolated from HTLV-1+individuals by single-molecule RNA FISH. Viral transcripts were visualised as diffraction-limited spots and their abundance was quantified at one-hour intervals. The onset of the virus transcription peaked after one to three hours of incubation. In each cell, a short period of slow HTLV-1 transcription was followed by a phase of rapid transcription. Computer simulation, based on experimental data on PBMCs from a leukemic patient with a single dominant HTLV-1-infected T cell clone, indicated that this rapid transcription from the HTLV-1 sense-strand promoter activated by Tax was terminated in less than an hour. The HTLV-1 antisense transcriptHBZwas constantly produced at a low level, and 50% ± 20% of HTLV-1+cells were negative forHBZat a given time. These results demonstrate how rapidly HTLV-1 is reactivated and potentially becomes infectious, once HTLV-1+cells are transmitted into a new host.

Single-Molecule Force-Clamp Experiments Reveal Kinetics of Mechanically Activated Silyl Ester Hydrolysis

ACS Nano ◽  
2012 ◽  
Vol 6 (2) ◽  
pp. 1314-1321 ◽  
Author(s):  
Sebastian W. Schmidt ◽  
Pavel Filippov ◽  
Alfred Kersch ◽  
Martin K. Beyer ◽  
Hauke Clausen-Schaumann
Keyword(s):  
Single Molecule ◽  
Ester Hydrolysis ◽  
Silyl Ester ◽  
Force Clamp ◽  
Kinetics Of

scholarly journals Increasing the time resolution of single-molecule experiments with Bayesian inference

2017 ◽  
Author(s):  
Colin D. Kinz-Thompson ◽  
Ruben L. Gonzalez
Keyword(s):  
Bayesian Inference ◽  
Single Molecule ◽  
Time Resolution ◽  
Rate Constants ◽  
Computational Approach ◽  
Biomolecular Systems ◽  
Time Resolved ◽  
The Given ◽  
Kinetics Of ◽  
Resolution Data

AbstractMany time-resolved, single-molecule biophysics experiments seek to characterize the kinetics of biomolecular systems exhibiting dynamics that challenge the time resolution of the given technique. Here we present a general, computational approach to this problem that employs Bayesian inference to learn the underlying dynamics of such systems, even when they are much faster than the time resolution of the experimental technique being used. By accurately and precisely inferring rate constants, our Bayesian Inference for the Analysis of Sub-temporal-resolution Data (BIASD) approach effectively enables the experimenter to super-resolve the poorly resolved dynamics that are present in their data.

scholarly journals Transcription factor residence time dominates over concentration in transcription activation

2020 ◽  
Author(s):  
Achim P. Popp ◽  
Johannes Hettich ◽  
J. Christof M. Gebhardt
Keyword(s):  
Transcription Factor ◽  
Residence Time ◽  
Single Molecule ◽  
Search Time ◽  
Mrna Synthesis ◽  
Burst Frequency ◽  
Quantitative Measurements ◽  
Rna Fish ◽  
Transition Times ◽  
Basic Course

Transcription is a vital process activated by transcription factor (TF) binding. The active gene releases a burst of transcripts before turning inactive again. While the basic course of transcription is well understood, it is unclear how binding of a TF affects the frequency, duration and size of a transcriptional burst. We systematically varied the residence time and concentration of a synthetic TF and characterized the transcription of a reporter gene by combining single molecule imaging, single molecule RNA-FISH, live transcript visualisation and analysis with a novel algorithm, Burst Inference from mRNA Distributions (BIRD). For this well-defined system, we found that TF binding solely affected burst frequency and variations in TF residence time had a stronger influence than variations in concentration. This enabled us to device a model of gene transcription, in which TF binding triggers multiple successive steps before the gene transits to the active state and actual mRNA synthesis is decoupled from TF presence. We quantified all transition times of the TF and the gene, including the TF search time and the delay between TF binding and the onset of transcription. Our quantitative measurements and analysis revealed detailed kinetic insight, which may serve as basis for a bottom-up understanding of gene regulation.

scholarly journals Dynamics of Tpm1.8 domains on actin filaments with single molecule resolution

2020 ◽  
Author(s):  
Ilina Bareja ◽  
Hugo Wioland ◽  
Miro Janco ◽  
Philip R. Nicovich ◽  
Antoine Jégou ◽  
...  
Keyword(s):  
Actin Filament ◽  
Single Molecule ◽  
Actin Filaments ◽  
High Probability ◽  
Actin Binding ◽  
Single Molecule Level ◽  
Filament Dynamics ◽  
Kinetics Of ◽  
Insight Into

ABSTRACTTropomyosins regulate dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of fluorescently labelled tropomyosin isoform Tpm1.8 to unlabelled actin filaments in real time. This approach in conjunction with mathematical modeling enabled us to quantify the nucleation, assembly and disassembly kinetics of Tpm1.8 on single filaments and at the single molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilised by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented towards the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

Dynamic epistasis analysis reveals how chromatin remodeling regulates transcriptional bursting

2021 ◽  
Author(s):  
Ineke Brouwer ◽  
Emma Kerklingh ◽  
Fred van Leeuwen ◽  
Tineke L Lenstra
Keyword(s):  
Transcription Factor ◽  
Chromatin Remodeling ◽  
Single Molecule ◽  
Binding Sites ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Nucleosome Remodeling ◽  
Epistasis Analysis ◽  
Transcriptional Bursting ◽  
Kinetics Of

Transcriptional bursting has been linked to the stochastic positioning of nucleosomes. However, how bursting is regulated by remodeling of promoter nucleosomes is unknown. Here, we use single-molecule live-cell imaging of GAL10 transcription in budding yeast to measure how transcriptional bursting changes upon single and double perturbations of chromatin remodeling factors, the transcription factor Gal4 and preinitiation complex (PIC) components. Using dynamic epistasis analysis, we reveal how remodeling of different nucleosomes regulates individual transcriptional bursting parameters. At the nucleosome covering the Gal4 binding sites, RSC acts synergistically with Gal4 binding to facilitate each burst. Conversely, nucleosome remodeling at the TATA box controls only the first burst upon galactose induction. In the absence of remodelers, nucleosomes at canonical TATA boxes are displaced by TBP binding to allow for transcription activation. Overall, our results reveal how promoter nucleosome remodeling, together with transcription factor and PIC binding regulates the kinetics of transcriptional bursting.

Single-Molecule Kinetics of Styrene Hydrogenation on Silica-Supported Vanadium: The Role of Disorder for Single-Atom Catalysts

2021 ◽  
Vol 125 (37) ◽  
pp. 20286-20300
Author(s):  
Robert H. Wells ◽  
Suming An ◽  
Prajay Patel ◽  
Cong Liu ◽  
Rex T. Skodje
Keyword(s):  
Single Molecule ◽  
Single Atom ◽  
Styrene Hydrogenation ◽  
Kinetics Of

Single Molecule Detection Approach to Muscle Study: Kinetics of a Single Cross-Bridge During Contraction of Muscle

2012 ◽  
pp. 311-334 ◽  
Author(s):  
Julian Borejdo ◽  
Danuta Szczesna-Cordary ◽  
Priya Muthu ◽  
Prasad Metticolla ◽  
Rafal Luchowski ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Molecule Detection ◽  
Cross Bridge ◽  
Detection Approach ◽  
Single Cross ◽  
Kinetics Of
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