scholarly journals Super-resolved visualization of single DNA-based tension sensors in cell adhesion

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Thomas Schlichthaerle ◽  
Caroline Lindner ◽  
Ralf Jungmann
Keyword(s):  
Receptor Binding ◽  
Single Molecule ◽  
Super Resolution ◽  
Integrin Receptors ◽  
Single Molecule Level ◽  
Cell Matrix ◽  
Ligand Interactions ◽  
Tension Sensor ◽  
Macromolecular Protein ◽  
Cell Matrix Interactions

AbstractCell-extracellular matrix sensing plays a crucial role in cellular behavior and leads to the formation of a macromolecular protein complex called the focal adhesion. Despite their importance in cellular decision making, relatively little is known about cell-matrix interactions and the intracellular transduction of an initial ligand-receptor binding event on the single-molecule level. Here, we combine cRGD-ligand-decorated DNA tension sensors with DNA-PAINT super-resolution microscopy to study the mechanical engagement of single integrin receptors and the downstream influence on actin bundling. We uncover that integrin receptor clustering is governed by a non-random organization with complexes spaced at 20–30 nm distances. The DNA-based tension sensor and analysis framework provide powerful tools to study a multitude of receptor-ligand interactions where forces are involved in ligand-receptor binding.

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 FluoSim: simulator of single molecule dynamics for fluorescence live-cell and super-resolution imaging of membrane proteins

2020 ◽  
Author(s):  
Matthieu Lagardère ◽  
Ingrid Chamma ◽  
Emmanuel Bouilhol ◽  
Macha Nikolski ◽  
Olivier Thoumine
Keyword(s):  
Real Time ◽  
Protein Dynamics ◽  
Single Molecule ◽  
Imaging Techniques ◽  
Simulated Data ◽  
Super Resolution ◽  
Molecular Complexes ◽  
Live Cell ◽  
Single Molecule Level ◽  
Resolution Imaging

AbstractFluorescence live-cell and super-resolution microscopy methods have considerably advanced our understanding of the dynamics and mesoscale organization of macro-molecular complexes that drive cellular functions. However, different imaging techniques can provide quite disparate information about protein motion and organization, owing to their respective experimental ranges and limitations. To address these limitations, we present here a unified computer program that allows one to model and predict membrane protein dynamics at the ensemble and single molecule level, so as to reconcile imaging paradigms and quantitatively characterize protein behavior in complex cellular environments. FluoSim is an interactive real-time simulator of protein dynamics for live-cell imaging methods including SPT, FRAP, PAF, and FCS, and super-resolution imaging techniques such as PALM, dSTORM, and uPAINT. The software, thoroughly validated against experimental data on the canonical neurexin-neuroligin adhesion complex, integrates diffusion coefficients, binding rates, and fluorophore photo-physics to calculate in real time the distribution of thousands of independent molecules in 2D cellular geometries, providing simulated data of protein dynamics and localization directly comparable to actual experiments.

scholarly journals Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy

2018 ◽  
Author(s):  
Gerti Beliu ◽  
Andreas Kurz ◽  
Alexander Kuhlemann ◽  
Lisa Behringer-Pliess ◽  
Natalia Wolf ◽  
...  
Keyword(s):  
Single Molecule ◽  
Signal To Noise Ratio ◽  
Super Resolution ◽  
Alder Reaction ◽  
Cell Permeability ◽  
High Signal ◽  
Noncanonical Amino Acids ◽  
Single Molecule Level ◽  
Genetic Code Expansion ◽  
Super Resolution Microscopy

Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine as the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.

Potential role of RGD-binding integrins in mammalian lung branching morphogenesis

Development ◽  
1991 ◽  
Vol 112 (2) ◽  
pp. 551-558 ◽  
Author(s):  
J. Roman ◽  
C.W. Little ◽  
J.A. McDonald
Keyword(s):  
Lung Development ◽  
Branching Morphogenesis ◽  
Integrin Receptors ◽  
Murine Lung ◽  
Cell Matrix ◽  
Matrix Interactions ◽  
Mammalian Lung ◽  
Cell Matrix Interactions ◽  
Lung Branching

Cell-matrix interactions are generally considered critical for normal lung development. This is particularly likely to be true during the glandular stage, when the primitive airways are formed through a process termed branching morphogenesis. Integrins, transmembrane receptors that bind to extracellular matrices, are likely to mediate important interactions between embryonic cells and their matrices during branching morphogenesis. In this report, we examine the role of integrin receptors in this process. Immunohistochemical studies revealed that the integrins VLA 3, VLA 5 and integrin receptors to vitronectin are expressed in the epithelium and/or mesenchyme during the glandular stage of murine lung development. To correlate expression with function, an in vitro model of murine lung branching morphogenesis was utilized to examine branching in the presence of inhibitors of ligand binding to integrin receptors. One such reagent, a hexapeptide containing the RGD (Arg-Gly-Asp) sequence, diminished branching and resulted in an abnormal morphology, whereas a control peptide RGESP (Arg-Gly-Glu-Ser-Pro) had no effect. These findings suggest a critical role for cell-matrix interactions mediated via integrin receptors in early stages of mammalian lung development.

scholarly journals Dynamic single-molecule counting for the quantification and optimization of nanoparticle functionalization protocols

Nanoscale ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 4128-4136 ◽  
Author(s):  
Matěj Horáček ◽  
Dion J. Engels ◽  
Peter Zijlstra
Keyword(s):  
Single Molecule ◽  
Super Resolution ◽  
Medical Applications ◽  
Single Molecule Level ◽  
Microscopy Method ◽  
Super Resolution Microscopy ◽  
Direct Quantification

We provide a super-resolution microscopy method to characterize the chemical interface of nanoparticles at the single-molecule level. This provides a direct quantification and optimization of functionalization protocols for bio-medical applications.

scholarly journals RGD-Dependent Epithelial Cell-Matrix Interactions in the Human Intestinal Crypt

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Yannick D. Benoit ◽  
Jean-François Groulx ◽  
David Gagné ◽  
Jean-François Beaulieu
Keyword(s):  
Cell Function ◽  
Intestinal Epithelial ◽  
Integrin Receptors ◽  
Cell Microenvironment ◽  
Cell Matrix ◽  
Epithelial Homeostasis ◽  
Intracellular Signals ◽  
Matrix Interactions ◽  
Key Players ◽  
Cell Matrix Interactions

Interactions between the extracellular matrix (ECM) and integrin receptors trigger structural and functional bonds between the cell microenvironment and the cytoskeleton. Such connections are essential for adhesion structure integrity and are key players in regulating transduction of specific intracellular signals, which in turn regulate the organization of the cell microenvironment and, consequently, cell function. The RGD peptide-dependent integrins represent a key subgroup of ECM receptors involved in the maintenance of epithelial homeostasis. Here we review recent findings on RGD-dependent ECM-integrin interactions and their roles in human intestinal epithelial crypt cells.

Protein clustering and spatial organization in T-cells

2015 ◽  
Vol 43 (3) ◽  
pp. 315-321 ◽  
Author(s):  
Michael J. Shannon ◽  
Garth Burn ◽  
Andrew Cope ◽  
Georgina Cornish ◽  
Dylan M. Owen
Keyword(s):  
T Cell ◽  
Single Molecule ◽  
Spatial Organization ◽  
Super Resolution ◽  
Cell Protein ◽  
Single Molecule Level ◽  
Composition And Structure ◽  
Resolution Analysis ◽  
And Function ◽  
Super Resolution Microscopy

T-cell protein microclusters have until recently been investigable only as microscale entities with their composition and structure being discerned by biochemistry or diffraction-limited light microscopy. With the advent of super resolution microscopy comes the ability to interrogate the structure and function of these clusters at the single molecule level by producing highly accurate pointillist maps of single molecule locations at ~20nm resolution. Analysis tools have also been developed to provide rich descriptors of the pointillist data, allowing us to pose questions about the nanoscale organization which governs the local and cell wide responses required of a migratory T-cell.

Competitive multicomponent anion exchange adsorption of proteins at the single molecule level

The Analyst ◽  
2017 ◽  
Vol 142 (17) ◽  
pp. 3127-3131 ◽  
Author(s):  
Lydia Kisley ◽  
Ujwal Patil ◽  
Sagar Dhamane ◽  
Katerina Kourentzi ◽  
Lawrence J. Tauzin ◽  
...  
Keyword(s):  
Anion Exchange ◽  
Single Molecule ◽  
Adsorption Kinetics ◽  
Competitive Adsorption ◽  
Super Resolution ◽  
Single Molecule Level ◽  
Exchange Adsorption ◽  
Resolution Imaging

Super-resolution imaging of multicomponent, competitive adsorption demonstrates that competitors block certain ligands from the analyte without changing analyte adsorption kinetics.

scholarly journals Monitoring HIV-1 Assembly in Living Cells: Insight from Dynamic and Single Molecule Microscopy

2018 ◽  
Author(s):  
Kaushik Inamdar ◽  
Charlotte Floderer ◽  
Cyril Favard ◽  
Delphine Muriaux
Keyword(s):  
Host Cell ◽  
Single Molecule ◽  
High Speed ◽  
Time Course ◽  
Super Resolution ◽  
Living Cells ◽  
Single Molecule Level ◽  
Host Cell Factors ◽  
Immature Virus ◽  
Hiv 1

HIV-1 assembly is a complex mechanism taking place at the plasma membrane of the host cell. It requires nice spatial and temporal coordination to end up with a full immature virus. Researchers have extensively studied HIV-1 assembly molecular mechanism during the past decades, in order to dissect the respective roles of viral proteins, viral genome and host cell factors. Nevertheless, the time course of the process has been observed in living cells only a decade ago. The very recent revolution of optical microscopy, combining high speed and high spatial resolution now permit to study assemblies and their consequences at the single molecule level within (living) cells. In this review, after a short description of these new approaches, we will show how HIV-1 assembly in cells has been revisited using these advanced super resolution microscopy techniques and how much it could make a bridge in studying assembly from the single molecule to the host cell.

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