scholarly journals Live-cell single-molecule labeling and analysis of myosin motors with quantum dots

2017 ◽  
Vol 28 (1) ◽  
pp. 173-181 ◽  
Author(s):  
Hiroyasu Hatakeyama ◽  
Yoshihito Nakahata ◽  
Hirokazu Yarimizu ◽  
Makoto Kanzaki
Keyword(s):  
Quantum Dots ◽  
Single Molecule ◽  
Intracellular Trafficking ◽  
Glucose Transporter ◽  
Insulin Treatment ◽  
Single Particle Tracking ◽  
Single Molecule Level ◽  
Cellular Processes ◽  
Myosin Motors ◽  
Glucose Transporter Glut4

Quantum dots (QDs) are a powerful tool for quantitatively analyzing dynamic cellular processes by single-particle tracking. However, tracking of intracellular molecules with QDs is limited by their inability to penetrate the plasma membrane and bind to specific molecules of interest. Although several techniques for overcoming these problems have been proposed, they are either complicated or inconvenient. To address this issue, in this study, we developed a simple, convenient, and nontoxic method for labeling intracellular molecules in cells using HaloTag technology and electroporation. We labeled intracellular myosin motors with this approach and tracked their movement within cells. By simultaneously imaging myosin movement and F-actin architecture, we observed that F-actin serves not only as a rail but also as a barrier for myosin movement. We analyzed the effect of insulin on the movement of several myosin motors, which have been suggested to regulate intracellular trafficking of the insulin-responsive glucose transporter GLUT4, but found no significant enhancement in myosin motor motility as a result of insulin treatment. Our approach expands the repertoire of proteins for which intracellular dynamics can be analyzed at the single-molecule level.

scholarly journals Single-molecule diffusometry reveals no catalysis-induced diffusion enhancement of alkaline phosphatase as proposed by FCS experiments

2020 ◽  
Vol 117 (35) ◽  
pp. 21328-21335
Author(s):  
Zhijie Chen ◽  
Alan Shaw ◽  
Hugh Wilson ◽  
Maxime Woringer ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Theoretical Models ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Single Molecule Level ◽  
Diffusion Phenomena ◽  
Diffusion Enhancement

Theoretical and experimental observations that catalysis enhances the diffusion of enzymes have generated exciting implications about nanoscale energy flow, molecular chemotaxis, and self-powered nanomachines. However, contradictory claims on the origin, magnitude, and consequence of this phenomenon continue to arise. To date, experimental observations of catalysis-enhanced enzyme diffusion have relied almost exclusively on fluorescence correlation spectroscopy (FCS), a technique that provides only indirect, ensemble-averaged measurements of diffusion behavior. Here, using an anti-Brownian electrokinetic (ABEL) trap and in-solution single-particle tracking, we show that catalysis does not increase the diffusion of alkaline phosphatase (ALP) at the single-molecule level, in sharp contrast to the ∼20% enhancement seen in parallel FCS experiments usingp-nitrophenyl phosphate (pNPP) as substrate. Combining comprehensive FCS controls, ABEL trap, surface-based single-molecule fluorescence, and Monte Carlo simulations, we establish thatpNPP-induced dye blinking at the ∼10-ms timescale is responsible for the apparent diffusion enhancement seen in FCS. Our observations urge a crucial revisit of various experimental findings and theoretical models––including those of our own––in the field, and indicate that in-solution single-particle tracking and ABEL trap are more reliable means to investigate diffusion phenomena at the nanoscale.

scholarly journals Studying the Interaction of Receptor Tyrosine Kinases and Adaptor Proteins at the Single-Molecule Level with Single-Particle Tracking

2020 ◽  
Vol 118 (3) ◽  
pp. 97a
Author(s):  
Tim Niklas Baldering ◽  
Johanna Rahm ◽  
Sebastian Malkusch ◽  
Marina S. Dietz ◽  
Mike Heilemann
Keyword(s):  
Single Molecule ◽  
Particle Tracking ◽  
Single Particle ◽  
Tyrosine Kinases ◽  
Receptor Tyrosine Kinases ◽  
Adaptor Proteins ◽  
Single Particle Tracking ◽  
Single Molecule Level

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.

Enhancing the generating and collecting efficiency of single particle upconverting luminescence at low power excitation

Nanophotonics ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1993-2000 ◽  
Author(s):  
Chenshuo Ma ◽  
Chunyan Shan ◽  
Kevin Park ◽  
Aaron T. Mok ◽  
Paul J. Antonick ◽  
...  
Keyword(s):  
Single Molecule ◽  
Single Particle ◽  
Excitation Power ◽  
Single Particle Tracking ◽  
Rare Earth Ions ◽  
High Excitation ◽  
Single Molecule Level ◽  
Collecting Efficiency ◽  
High Luminescence Intensity

AbstractUpconverting luminescent nanoparticles are photostable, nonblinking, and low chemically toxic fluorophores that are emerging as promising fluorescent probes at the single molecule level. High luminescence intensity upconversion nanoparticles (UCNPs) have previously been achieved by doping with high amounts of rare-earth ions using high excitation power (>2.5 MW/cm2). However, such particles are inadequate for in vitro live-cell imaging and single-particle tracking, as high excitation power can cause photodamage. Here, we compared UCNP luminescence intensities with different dopant concentrations and presented more efficient (about seven times) UCNPs at low excitation power by increasing the concentrations of Yb3+ and Tm3+ dopants (NaYF4: 60% Yb3+, 8% Tm3+) and adding a core-shell structure.

Interfacial Electron Transfer from CdSe/ZnS Quantum Dots to TiO2Nanoparticles: Linker Dependence at Single Molecule Level

2013 ◽  
Vol 25 (4) ◽  
pp. 1064-1073 ◽  
Author(s):  
Yu-Pin Chang ◽  
Po-Yu Tsai ◽  
Hsin-Lung Lee ◽  
King-Chuen Lin
Keyword(s):  
Quantum Dots ◽  
Electron Transfer ◽  
Single Molecule ◽  
Interfacial Electron Transfer ◽  
Single Molecule Level

scholarly journals Simple nanofluidic devices for high-throughput, non-equilibrium studies at the single-molecule level

2017 ◽  
Author(s):  
Carel Fijen ◽  
Mattia Fontana ◽  
Serge G. Lemay ◽  
Klaus Mathwig ◽  
Johannes Hohlbein
Keyword(s):  
High Throughput ◽  
Single Molecule ◽  
Single Particle Tracking ◽  
Biological Interactions ◽  
Dynamic Heterogeneity ◽  
High Throughput Analysis ◽  
Equilibrium Studies ◽  
Single Molecule Level ◽  
Nanofluidic Devices ◽  
And Performance

ABSTRACTSingle-molecule detection schemes offer powerful means to overcome static and dynamic heterogeneity inherent to complex samples. Probing chemical and biological interactions and reactions with high throughput and time resolution, however, remains challenging and often requires surface-immobilized entities. Here, utilizing camera-based fluorescence microscopy, we present glass-made nanofluidic devices in which fluorescently labelled molecules flow through nanochannels that confine their diffusional movement. The first design features an array of parallel nanochannels for high-throughput analysis of molecular species under equilibrium conditions allowing us to record 200.000 individual localization events in just 10 minutes. Using these localizations for single particle tracking, we were able to obtain accurate flow profiles including flow speeds and diffusion coefficients inside the channels.A second design featuring a T-shaped nanochannel enables precise mixing of two different species as well as the continuous observation of chemical reactions. We utilized the design to visualize enzymatically driven DNA synthesis in real time and at the single-molecule level. Based on our results, we are convinced that the versatility and performance of the nanofluidic devices will enable numerous applications in the life sciences.

scholarly journals Fluorescence Quenching of Quantum Dots by DNA Nucleotides and Amino Acids

2011 ◽  
Vol 64 (5) ◽  
pp. 512 ◽  
Author(s):  
Daniel Siegberg ◽  
Dirk-Peter Herten
Keyword(s):  
Amino Acids ◽  
Quantum Dots ◽  
Fluorescence Quenching ◽  
Single Molecule ◽  
Fluorescence Emission ◽  
Single Particle Tracking ◽  
Quenching Mechanism ◽  
Widespread Application ◽  
Biochemical Compounds ◽  
Single Molecule Fluorescence Spectroscopy

Quantum dots found widespread application in the biosciences as bright and highly photo-stable fluorescent probes, i.e. for single-particle tracking. In this work we used ensemble spectroscopy and single-molecule techniques to study the quenching of quantum dots by various biochemical compounds that are usually present in living cells and might thus influence the experiments. We found not only nucleotides such as cytosine, guanine, and thymine can significantly influence the fluorescence emission of CdSe and CdTe quantum dots, but also amino acids, like asparagine and tryptophan. Bulk studies on fluorescence quenching indicated a static quenching mechanism. Interestingly, we could also show by single-molecule fluorescence spectroscopy that quenching of the quantum dots can be irreversible, suggesting either a redox-reaction between quantum dot and quencher or strong binding of the quencher to the surface of the bio-conjugated quantum dots.

scholarly journals BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

2014 ◽  
Vol 207 (5) ◽  
pp. 599-613 ◽  
Author(s):  
Marcel Reuter ◽  
Alex Zelensky ◽  
Ihor Smal ◽  
Erik Meijering ◽  
Wiggert A. van Cappellen ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Molecule ◽  
Fluorescent Proteins ◽  
Single Particle Tracking ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Physical Interaction ◽  
Single Molecule Level ◽  
Before And After ◽  
Endogenous Loci

Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2.

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