scholarly journals Fluorescent Single Molecule Orientation Imaging for Exploring 3D Architectural Dynamics of Cytoskeletal Protein Assembly in Living Cells

2013 ◽  
Vol 104 (2) ◽  
pp. 340a
Author(s):  
Tomomi Tani ◽  
Shalin Mehta ◽  
Bradley S. DeMay ◽  
Patricia Occhipinti ◽  
Rudolf Oldenbourg ◽  
...  
Keyword(s):  
Single Molecule ◽  
Protein Assembly ◽  
Living Cells ◽  
Cytoskeletal Protein ◽  
Orientation Imaging

A Molecular Logic Gate Enables Super-Resolved Imaging of Intracellular Lipid Droplets

2019 ◽  
Author(s):  
Adam Eördögh ◽  
Carolina Paganini ◽  
Dorothea Pinotsi ◽  
Paolo Arosio ◽  
Pablo Rivera-Fuentes
Keyword(s):  
Single Molecule ◽  
Lipid Droplets ◽  
Neutral Lipids ◽  
Logic Gate ◽  
Living Cells ◽  
Three Dimensions ◽  
Single Molecule Imaging ◽  
Molecular Logic Gate ◽  
Molecular Logic ◽  
Cellular Compartments

<div>Photoactivatable dyes enable single-molecule imaging in biology. Despite progress in the development of new fluorophores and labeling strategies, many cellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid droplets, which are organelles that contain mostly neutral lipids, have eluded single-molecule imaging. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid droplets with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment and a competent nucleophile to produce a fluorescent product. The combination of these requirements results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolutions beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipids within and between droplets in living cells.</div>

scholarly journals A binding protein regulates myosin-7a dimerization and actin bundle assembly

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rong Liu ◽  
Neil Billington ◽  
Yi Yang ◽  
Charles Bond ◽  
Amy Hong ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Actin Filament ◽  
Single Molecule ◽  
Binding Protein ◽  
Living Cells ◽  
Actin Bundle ◽  
Actin Bundles ◽  
Cytoskeletal Remodeling ◽  
Actin Protrusions

AbstractMyosin-7a, despite being monomeric in isolation, plays roles in organizing actin-based cell protrusions such as filopodia, microvilli and stereocilia, as well as transporting cargoes within them. Here, we identify a binding protein for Drosophila myosin-7a termed M7BP, and describe how M7BP assembles myosin-7a into a motile complex that enables cargo translocation and actin cytoskeletal remodeling. M7BP binds to the autoinhibitory tail of myosin-7a, extending the molecule and activating its ATPase activity. Single-molecule reconstitution show that M7BP enables robust motility by complexing with myosin-7a as 2:2 translocation dimers in an actin-regulated manner. Meanwhile, M7BP tethers actin, enhancing complex’s processivity and driving actin-filament alignment during processive runs. Finally, we show that myosin-7a-M7BP complex assembles actin bundles and filopodia-like protrusions while migrating along them in living cells. Together, these findings provide insights into the mechanisms by which myosin-7a functions in actin protrusions.

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.

Single-molecule dynamics of site-specific labeled transforming growth factor type II receptors on living cells

2014 ◽  
Vol 50 (94) ◽  
pp. 14724-14727 ◽  
Author(s):  
Ming Cheng ◽  
Wei Zhang ◽  
Jinghe Yuan ◽  
Wangxi Luo ◽  
Nan Li ◽  
...  
Keyword(s):  
Growth Factor ◽  
Single Molecule ◽  
Transforming Growth Factor ◽  
Living Cells ◽  
Unnatural Amino Acid ◽  
Type Ii ◽  
Site Specific ◽  
Single Molecule Dynamics ◽  
Factor Type ◽  
Molecule Dynamics

Single-molecule dynamics of the transforming growth factor type II receptor (TβRII) labeled by an unnatural amino acid.

scholarly journals Single-Molecule Analysis of Human Immunodeficiency Virus Type 1 gp120-Receptor Interactions in Living Cells

2005 ◽  
Vol 79 (23) ◽  
pp. 14748-14755 ◽  
Author(s):  
Melissa I. Chang ◽  
Porntula Panorchan ◽  
Terrence M. Dobrowsky ◽  
Yiider Tseng ◽  
Denis Wirtz
Keyword(s):  
Human Immunodeficiency Virus ◽  
Host Cell ◽  
Single Molecule ◽  
Quantitative Description ◽  
Living Cells ◽  
Viral Envelope ◽  
Binding Interactions ◽  
Immunodeficiency Virus ◽  
Single Molecule Analysis

ABSTRACT A quantitative description of the binding interactions between human immunodeficiency virus (HIV) type 1 envelope glycoproteins and their host cell surface receptors remains incomplete. Here, we introduce a single-molecule analysis that directly probes the binding interactions between an individual viral subunit gp120 and a single receptor CD4 and/or chemokine coreceptor CCR5 in living cells. This analysis differentiates single-molecule binding from multimolecule avidity and shows that, while the presence of CD4 is required for gp120 binding to CCR5, the force required to rupture a single gp120-coreceptor bond is significantly higher and its lifetime is much longer than those of a single gp120-receptor bond. The lifetimes of these bonds are themselves shorter than those of the P-selectin/PSGL-1 bond involved in leukocyte attachment to the endothelium bonds during an inflammation response. These results suggest an amended model of HIV entry in which, immediately after the association of gp120 to its receptor, gp120 seeks its coreceptor to rapidly form a new bond. This “bond transfer” occurs only if CCR5 is in close proximity to CD4 and CD4 is still attached to gp120. The analysis presented here may serve as a general framework to study mechanisms of receptor-mediated interactions between viral envelope proteins and host cell receptors at the single-molecule level in living cells.

scholarly journals Single-Molecule Kinetics in Living Cells

2019 ◽  
Vol 88 (1) ◽  
pp. 635-659 ◽  
Author(s):  
Johan Elf ◽  
Irmeli Barkefors
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
The Past

In the past decades, advances in microscopy have made it possible to study the dynamics of individual biomolecules in vitro and resolve intramolecular kinetics that would otherwise be hidden in ensemble averages. More recently, single-molecule methods have been used to image, localize, and track individually labeled macromolecules in the cytoplasm of living cells, allowing investigations of intermolecular kinetics under physiologically relevant conditions. In this review, we illuminate the particular advantages of single-molecule techniques when studying kinetics in living cells and discuss solutions to specific challenges associated with these methods.

Single-molecule imaging of EGFR signalling on the surface of living cells

2000 ◽  
Vol 2 (3) ◽  
pp. 168-172 ◽  
Author(s):  
Yasushi Sako ◽  
Shigeru Minoghchi ◽  
Toshio Yanagida
Keyword(s):  
Single Molecule ◽  
Living Cells ◽  
Single Molecule Imaging
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