scholarly journals Insights into chromatin fibre structure by in vitro and in silico single-molecule stretching experiments

2013 ◽  
Vol 41 (2) ◽  
pp. 494-500 ◽  
Author(s):  
Rosana Collepardo-Guevara ◽  
Tamar Schlick
Keyword(s):  
Gene Regulation ◽  
Single Molecule ◽  
Fibre Structure ◽  
Detailed Structure ◽  
Single Molecule Force Spectroscopy ◽  
Linker Histones ◽  
Chromatin Fibre ◽  
Structure And Dynamics ◽  
Experimental Findings

The detailed structure and dynamics of the chromatin fibre and their relation to gene regulation represent important open biological questions. Recent advances in single-molecule force spectroscopy experiments have addressed these questions by directly measuring the forces that stabilize and alter the folded states of chromatin, and by investigating the mechanisms of fibre unfolding. We present examples that demonstrate how complementary modelling approaches have helped not only to interpret the experimental findings, but also to advance our knowledge of force-induced events such as unfolding of chromatin with dynamically bound linker histones and nucleosome unwrapping.

scholarly journals Fluorescence-free quantification of protein/nucleic-acid binding through single-molecule kinetic locking

2020 ◽  
Author(s):  
Martin Rieu ◽  
Jessica Valle-Orero ◽  
Bertrand Ducos ◽  
Jean-François Allemand ◽  
Vincent Croquette
Keyword(s):  
Single Molecule ◽  
Functional Characterization ◽  
Nucleic Acid Binding ◽  
Single Molecule Force Spectroscopy ◽  
E Coli ◽  
Natural Function ◽  
Protein Nucleic Acid ◽  
Free Quantification

ABSTRACTFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a new method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5,6,7 bases) and use it to measure the dynamical interactions of E. coli RecQ helicase with its DNA substrate.

Role of linker histones in extended chromatin fibre structure

1994 ◽  
Vol 1 (11) ◽  
pp. 761-763 ◽  
Author(s):  
Guoliang Yang ◽  
Sanford H. Leuba ◽  
Carlos Bustamante ◽  
Jordanka Zlatanove ◽  
Kensal van Holde
Keyword(s):  
Fibre Structure ◽  
Linker Histones ◽  
Chromatin Fibre

Single-molecule tracking technologies for quantifying the dynamics of gene regulation in cells, tissue and embryos

Development ◽  
2021 ◽  
Vol 148 (18) ◽  
Author(s):  
Alan P. Boka ◽  
Apratim Mukherjee ◽  
Mustafa Mir
Keyword(s):  
Gene Regulation ◽  
Single Molecule ◽  
Fluorescent Labelling ◽  
Single Molecule Tracking ◽  
Trade Offs ◽  
Molecular Scale ◽  
Key Technologies ◽  
Single Molecule Studies ◽  
Localization And Tracking

ABSTRACT For decades, we have relied on population and time-averaged snapshots of dynamic molecular scale events to understand how genes are regulated during development and beyond. The advent of techniques to observe single-molecule kinetics in increasingly endogenous contexts, progressing from in vitro studies to living embryos, has revealed how much we have missed. Here, we provide an accessible overview of the rapidly expanding family of technologies for single-molecule tracking (SMT), with the goal of enabling the reader to critically analyse single-molecule studies, as well as to inspire the application of SMT to their own work. We start by overviewing the basics of and motivation for SMT experiments, and the trade-offs involved when optimizing parameters. We then cover key technologies, including fluorescent labelling, excitation and detection optics, localization and tracking algorithms, and data analysis. Finally, we provide a summary of selected recent applications of SMT to study the dynamics of gene regulation.

scholarly journals Single-molecule kinetic locking allows fluorescence-free quantification of protein/nucleic-acid binding

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Martin Rieu ◽  
Jessica Valle-Orero ◽  
Bertrand Ducos ◽  
Jean-François Allemand ◽  
Vincent Croquette
Keyword(s):  
Single Molecule ◽  
Functional Characterization ◽  
Nucleic Acid Binding ◽  
Single Molecule Force Spectroscopy ◽  
E Coli ◽  
Natural Function ◽  
Protein Nucleic Acid ◽  
Free Quantification

AbstractFluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5, 6, 7 bases) and use it to measure the dynamical interactions of Escherichia coli/E. coli RecQ helicase with its DNA substrate.

scholarly journals A mechanism with severing near barbed ends and annealing explains structure and dynamics of dendritic actin networks

2021 ◽  
Author(s):  
Danielle Holz ◽  
Aaron R Hall ◽  
Eiji Usukura ◽  
Sawako Yamashiro ◽  
Naoki Watanabe ◽  
...  
Keyword(s):  
Single Molecule ◽  
Leading Edge ◽  
Lifetime Distribution ◽  
Diffusion Annealing ◽  
Actin Networks ◽  
Structure And Dynamics ◽  
In Vitro Measurements ◽  
Filament Network ◽  
Orientation Pattern

Single molecule imaging has shown that part of actin disassembles within a few seconds after incorporation into the dendritic filament network in lamellipodia, suggestive of frequent destabilization near barbed ends. To investigate the mechanisms behind network remodeling, we created a stochastic model with polymerization, depolymerization, branching, capping, uncapping, severing, oligomer diffusion, annealing, and debranching. We find that filament severing, enhanced near barbed ends, can explain the single molecule actin lifetime distribution, if oligomer fragments reanneal to free ends with rate constants comparable to in vitro measurements. The same mechanism leads to actin networks consistent with measured filament, end, and branch concentrations. These networks undergo structural remodeling, leading to longer filaments away from the leading edge, at the +/- 35$^o$ orientation pattern. Imaging of actin speckle lifetimes at sub-second resolution verifies frequent disassembly of newly-assembled actin. We thus propose a unified mechanism that fits a diverse set of basic lamellipodia phenomenology.

A Combined In Vitro and In Silico Approach to Estimate the Molecular Arrangement Within a Fibronectin Fiber

2011 ◽  
Author(s):  
Mark J. Bradshaw ◽  
Michael L. Smith
Keyword(s):  
Single Molecule ◽  
Strain State ◽  
Cell Adhesion Molecule ◽  
Tertiary Structure ◽  
Cell Behavior ◽  
Single Molecule Force Spectroscopy ◽  
Binding Partner ◽  
Molecular Arrangement ◽  
Cellular Mechanotransduction

It has become apparent that the extracellular matrix (ECM) is a powerful modulator of cell behavior. Fibronectin (Fn) is of particular interest because it is a requisite cell adhesion molecule for development and wound healing and it is a promiscuous binding partner for many soluble signaling molecules. It was recently shown that the binding affinity of some molecules is dependent on the strain state of the Fn [2,3], reinvigorating our interest in the molecular mechanism of Fn fiber extension. The tertiary structure of the approximately 30 Fn type III domains of the protein has been shown to be capable of unfolding in single molecule force spectroscopy experiments, although evidence that unfolding occurs in Fn fibers has been indirect and has not been quantified. Nevertheless, unfolding of Fn molecules predicts a possible mechanism of strain dependant binding in Fn matrix and commensurate strain feedback to attached cells, contributing to the cellular mechanotransduction toolbox [3].

scholarly journals Chromatin fiber breaks into clutches under tension and crowding

2021 ◽  
Author(s):  
Shuming Liu ◽  
Xingcheng Lin ◽  
Bin Zhang
Keyword(s):  
Single Molecule ◽  
Sol Gel ◽  
In Vivo Studies ◽  
Binding Modes ◽  
Cell Nuclei ◽  
Force Extension ◽  
Fiber Breaks ◽  
Experimental Findings

The arrangement of nucleosomes inside chromatin is of extensive interest. While in vitro experiments have revealed the formation of 30 nm fibers, most in vivo studies have failed to confirm their presence in cell nuclei. To reconcile the diverging experimental findings, we characterized chromatin organization using a near atomistic model. The computed force-extension curve matches well with measurements from single-molecule experiments. Notably, we found that a dodeca-nucleosome in the two-helix zigzag conformation breaks into structures with nucleosome clutches and a mix of trimers and tetramers under tension. Such unfolded configurations can also be stabilized through trans interactions with other chromatin chains. Our study supports a hypothesis that disordered, in vivo chromatin configurations arise as folding intermediates from regular fibril structures. We further revealed that chromatin segments with fibril or clutch structures engaged in distinct binding modes and discussed the implications of these inter-chain interactions for a potential sol-gel phase transition.

In Vitro Studies on the Mechanism of the Antifibrino-lytic Action of E-Aminocaproic Acid (EACA)

1968 ◽  
Vol 19 (03/04) ◽  
pp. 584-592 ◽  
Author(s):  
Hanna Lukasiewicz ◽  
S Niewiarowski
Keyword(s):  
Aminocaproic Acid ◽  
Test Tube ◽  
In Vitro Studies ◽  
Lytic Action ◽  
Human Plasminogen ◽  
Experimental Findings ◽  
Fibrin Clots

Summary and Conclusion1. It has been found that EACA does not inhibit activation of human plasminogen into plasmin by SK and UK in a concentration of 5 × 10–2 M. The activation of bovine plasminogen by SK and UK is inhibited by this concentration of EACA but not by a lower one.2. EACA in concentrations of 1,5 × 10–1 – 10–4 M does not inhibit casein proteolysis by plasmin. The proteolysis of fibrinogen and fibrin measured by the release of TCA soluble tyrosine is inhibited by EACA in concentrations of 1,5 × 10–1 – 10–2 M.3. The lysis of non-stabilized clots by plasmin measured in a test tube was inhibited by an EACA concentration of 5 × 10–3 – 5 × 10–4 M. The lysis of stabilized clots by plasmin was inhibited by an EACA concentration of 10–5 M.4. On the basis of experimental findings and data given in literature the authors postulate that the mechanism of the antifibrinolytic effects of EACA consists mainly in a modification of plasmin action on fibrin. These effects are dependent on the structure of the fibrin clots.

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