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Results for “Single Molecule Imaging”

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At least 109 records · Page 6Linked to original sources

PubMed2006

Visualizing odorant receptor trafficking in living cells down to the single-molecule level.

Despite the importance of trafficking for regulating G protein-coupled receptor signaling, for many members of the seven transmembrane helix protein family, such as odorant receptors, little is known about this process in live cells. Here, the complete life cycle of the human odorant receptor OR17-40 was directly monitored in living cells by ensemble and single-molecule imaging, using a double-labeling strategy. While the overall, intracellular trafficking of the receptor was visualized continuously by using a GFP tag, selective imaging of cell surface receptors was achieved by pulse-labeling an acyl carrier protein tag. We found that OR17-40 efficiently translocated to the plasma membrane only at low expression, whereas at higher biosynthesis the receptor accumulated in intracellular compartments. Receptors in the plasma membrane showed high turnover resulting from constitutive internalization along the clathrin pathway, even in the absence of ligand. Single-molecule microscopy allowed monitoring of the early, dynamic processes in odorant receptor signaling. Although mobile receptors initially diffused either freely or within domains of various sizes, binding of an agonist or an antagonist increased partitioning of receptors into small domains of approximately 190 nm, which likely are precursors of clathrin-coated pits. The binding of a ligand, therefore, resulted in modulation of the continuous, constitutive internalization. After endocytosis, receptors were directed to early endosomes for recycling. This unique mechanism of continuous internalization and recycling of OR17-40 might be instrumental in allowing rapid recovery of odor perception.

Cell Membrane↗

PubMed2003

Atomic-resolution STM structure of DNA and localization of the retinoic acid binding site.

Single-molecule imaging by scanning tunnelling microscopy (STM) yields the atomic-resolution (0.6A) structure of individual B-type DNA molecules. The strong correlation between these STM structures and those predicted from the known base sequence indicates that sequencing of single DNA molecules using STM may be feasible. There is excellent agreement between the STM and X-ray structures, but subtle differences exist due to radial distortions. We show that the interactions of other molecules with DNA, their binding configurations, and the structure of these complexes can be studied at the single-molecule level. The anti-cancer drug retinoic acid (RA) binds selectively to the minor groove of DNA with up to 6 RA molecules per DNA turn and with the plane of the RA molecule approximately parallel to the DNA symmetry axis. Similar studies for other drug molecules will be valuable in the a priori evaluation of the effectiveness of anti-cancer drugs.

Antineoplastic Agents↗

PubMed2002

Imaging and manipulation of biological structures with the AFM.

Many biologists have dreamt of physically touching and manipulating the biomolecules they were investigating. With the invention of the atomic force microscope (AFM), this dream has come true. Here, recent applications of the AFM to image and to manipulate biological systems at the nanometer scale are reviewed. Macromolecular biological assemblies as well as individual biomolecules can be subjected to controlled nanomanipulation. Examples of AFM application in imaging and nanomanipulation include the extraction of chromosomal DNA for genetic analysis, the disruption of antibody--antigen bonds, the dissection of biological membranes, the nanodissection of protein complexes, and the controlled modulation of protein conformations. Also reviewed is the novel combination of single molecule imaging and force spectroscopy which allows biomolecules to be imaged, and inter- and intramolecular forces to be measured. Future application of these nanotechniques will reveal new information on the structure, function and assembly of biomolecules.

Antigen-Antibody Complex↗

PubMed2006

Three-dimensional mapping of a deformation field inside a nanocrystal.

Coherent X-ray diffraction imaging is a rapidly advancing form of microscopy: diffraction patterns, measured using the latest third-generation synchrotron radiation sources, can be inverted to obtain full three-dimensional images of the interior density within nanocrystals. Diffraction from an ideal crystal lattice results in an identical copy of this continuous diffraction pattern at every Bragg peak. This symmetry is broken by the presence of strain fields, which arise from the epitaxial contact forces that are inevitable whenever nanocrystals are prepared on a substrate. When strain is present, the diffraction copies at different Bragg peaks are no longer identical and contain additional information, appearing as broken local inversion symmetry about each Bragg point. Here we show that one such pattern can nevertheless be inverted to obtain a 'complex' crystal density, whose phase encodes a projection of the lattice deformation. A lead nanocrystal was crystallized in ultrahigh vacuum from a droplet on a silica substrate and equilibrated close to its melting point. A three-dimensional image of the density, obtained by inversion of the coherent X-ray diffraction, shows the expected facetted morphology, but in addition reveals a real-space phase that is consistent with the three-dimensional evolution of a deformation field arising from interfacial contact forces. Quantitative three-dimensional imaging of lattice strain on the nanometre scale will have profound consequences for our fundamental understanding of grain interactions and defects in crystalline materials. Our method of measuring and inverting diffraction patterns from nanocrystals represents a vital step towards the ultimate goal of atomic resolution single-molecule imaging that is a prominent justification for development of X-ray free-electron lasers.

Journal Article↗

PubMed2006

Formation of signal transduction complexes during immobile phase of NGFR movements.

Nerve growth factor (NGF) is a secreted neurotrophin involved in the differentiation, growth, and maintenance of neurons. Here, we have used single-molecule imaging to characterize the behavior of Cy3-tagged NGF after binding to receptor complexes on the surfaces of PC12 cells. We show that NGF-receptor complexes have two distinct diffusive states, characterized as mobile and immobile phase. The transition between the two diffusive states occurred reversibly with duration times determined by a single rate limiting process. The abrupt transition to the immobile phase often occurred simultaneously with the clustering of NGF-receptor complexes. Immobilization depended on the phosphorylation of the TrkA NGF-receptor. Using dual-color imaging, it was demonstrated that the membrane recruitment of the intercellular signaling protein occurs with NGF-receptor complexes in the immobile phase indicating signal transduction occurs during this phase. Thus, NGF signaling is performed through a repetitive random process to induce transient formation of signaling complexes.

Animals↗

PubMed2006

Single-molecule visualization of environment-sensitive fluorophores inserted into cell membranes by staphylococcal gamma-hemolysin.

Single-molecule imaging of the entrance of a protein into the hydrophobic environment of a cell membrane was investigated. The pre-stem of LukF, one of the two components of the pore-forming toxin staphylococcal gamma-hemolysin, was specifically labeled with 6-bromoacetyl-2-dimethylaminonaphthalene (Badan), an environment-sensitive fluorophore. Incubation of this derivative with erythrocyte ghost membranes resulted in a pronounced increase in fluorescence indicating insertion of Badan into the hydrophobic interior of the lipid bilayers. However, the increase in fluorescence was completely dependent on the interaction of Badan-labeled LukF with the gamma-hemolysin second component. Individual spots of Badan fluorescence on erythrocyte membranes were visualized that were associated with single pores. Analyses of the intensities of these fluorescent spots and their photobleaching independently showed that a single pore contained 3-4 LukF molecules. Thus, environment-sensitive fluorophore signals can be used to study the insertion of specific protein domains into cell membranes at the single-molecule lever, and the use of this approach in the present study revealed that a single gamma-hemolysin pore opening contains at least three LukF molecules.

2-Naphthylamine↗

PubMed2003

Optical bioimaging: from living tissue to a single molecule: single-molecule visualization of cell signaling processes of epidermal growth factor receptor.

Single-molecule imaging is an ideal technology to study molecular mechanisms of biological reactions in vitro. Recently, this technology has been extended to real-time observation of fluorescent dye-labeled molecules in living cells. Total internal reflection fluorescence microscopy is the major technique for this purpose. Using this technique, we have studied the process of early signal transduction of epidermal growth factor (EGF) in single molecules: binding of EGF to its receptor (EGFR) on the cell surface, dimerization of EGFR induced by binding of EGF, fluctuation of the structure of EGFR clusters, activation of EGFR through tyrosine phosphorylations on its cytoplasmic domain, and recognition of activated EGFR by a cytoplasmic adaptor protein, Grb2. EGF induces intracellular calcium response, sometimes caused by less than one hundred EGF molecules. Single-molecule studies suggested that this highly sensitive response to EGF was due to the amplification of the EGFR signal using dynamic clustering, reorganization of the dimers, and lateral mobility of EGFR on the cell surface. Through these studies, single-molecule analysis has proven to be a powerful technology to analyze intracellular protein systems.

Animals↗

PubMed2004

High-resolution imaging of single fluorescent molecules with the optical near-field of a metal tip.

We show that a concentration of light at a metal tip allows near-field optical imaging of single fluorescent dye molecules at very high resolution, despite strong quenching effects. Details as small as 10 nm were observed in the fluorescence patterns of single Cy-3 dyes bound to the termini of DNA. Data evaluation by model fitting determines the positions of the dyes to an accuracy even better than 1 nm and also yields their 3D orientation. The metal tip simultaneously provides high-resolution topographic imaging complementing the optical signal for a detailed surface examination.

Journal Article↗

PubMed2000

Observing single biomolecules at work with the atomic force microscope.

Progress in the application of the atomic force microscope (AFM) to imaging and manipulating biomolecules is the result of improved instrumentation, sample preparation methods and image acquisition conditions. Biological membranes can be imaged in their native state at a lateral resolution of 0.5-1 nm and a vertical resolution of 0. 1-0.2 nm. Conformational changes that are related to functions can be resolved to a similar resolution, complementing atomic structure data acquired by other methods. The unique capability of the AFM to directly observe single proteins in their native environments provides insights into the interactions of proteins that form functional assemblies. In addition, single molecule force spectroscopy combined with single molecule imaging provides unprecedented possibilities for analyzing intramolecular and intermolecular forces. This review discusses recent examples that illustrate the power of AFM.

Animals↗

PubMed2001

Single-molecule immunoassay and DNA diagnosis.

Many assays relevant to disease diagnosis are based on electrophoresis, where the migration velocity is used for distinguishing molecules of different size or charge. However, standard gel electrophoresis is not only slow but also insensitive. We describe a single-molecule imaging procedure to measure the electrophoretic mobilities of up to 100000 distinct molecules every second. The results correlate well with capillary electrophoresis (CE) experiments and afford confident discrimination between normal (16.5 kbp) and abnormal (6.1 kbp) mitochondrial DNA fragments, or beta-phycoerythrin-labeled digoxigenin (BP-D) and its immunocomplex (anti-D-BP-D). This demonstrates that virtually all electrophoresis diagnostic protocols from slab gels to CE should be adaptable to single-molecule detection. This opens up the prossibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction (PCR) or other biological amplification.

Antigen-Antibody Complex↗

PubMed2001

Simultaneous topographic and fluorescence imaging of single DNA molecules for DNA analysis with a scanning near-field optical/atomic force microscope.

High-resolution fluorescence imaging of lambda-phage DNA molecules, intercalated with the dye YOYO-1, has been performed by a SNOM/AFM based on a bent-type optical fiber probe. A modified design of the optical probe has been made, and successful near-field optical resolution has been obtained for the strongly stretched lambda-phage DNA molecules. The best optical resolution was estimated at 45 nm for the dye-intercalated single lambda-DNA molecules by a mean width evaluation. In our comparison between the far-field fluorescence and high-resolution near-field fluorescence images for the DNA, it has been found that the near-field images much better defined the intercalation state of the dye. Finally, the relation between the DNA shapes and the dye distribution states, and the discrimination between the double-stranded and single-stranded DNA molecules, are discussed by comparing the topography and fluorescence images of the SNOM/AFM.

Benzoxazoles↗

PubMed2001

Direct imaging of single-molecules: from dynamics of a single DNA chain to the study of complex DNA-protein interactions.

Recent years have seen significant advances in the characterization and manipulation of individual molecules. The combination of single-molecule fluorescence and micromanipulation enables one to study physical and biological systems at new length scales, to unravel qualitative mechanisms, and to measure kinetic parameters that cannot be addressed by traditional biochemistry. DNA is one of the most studied biomolecules. Imaging single DNA molecules eliminates important limitations of classical techniques and provides a new method for testing polymer dynamics and DNA-protein interactions. Here we review some applications of this new approach to physical and biological problems, focusing on videomicroscopy observations of individual DNA chains extended in a shear flow. We will first describe data obtained on the stretching, relaxation and dynamics of a single tethered polymer in a shear flow, to demonstrate that the deformation of sheared tethered chains is partially governed by the thermally driven fluctuations of the chain transverse to the flow direction. Next, we will show how single-molecule videomicroscopy can be used to study in real time DNA folding into chromatin, a complex association of DNA and proteins responsible for the packaging of DNA in the nucleus of an eukaryotic cell.

DNA↗

PubMed1989

Real-time imaging of single DNA molecules with fluorescence microscopy.

A fluorescence microscopy technique was used to image the dynamics of individual DNA molecules. Lambda, calf thymus, cosmid (circular), and T4 DNA were studied with the fluorescent dye acridine orange. Experiments with DNAase I were conducted, and the results indicate that these observations correspond to DNA molecules. The results of experiments with circular DNA provide strong evidence that these were single DNA molecules. Molecules were observed free in solution or attached to a glass or copper surface at one or several points. The Brownian motion of these molecules was observed, indicating that DNA in solution exists in a partially supercoiled state. Some molecules appeared stretched and were attached to the surface by their termini; the lengths of these molecules were measured. Such molecules also exhibited elastic behavior upon breaking. The power of this technique is demonstrated in images of cosmid DNA molecules, catenanes, and DNA extending from T4 phage particles. These results suggest immediate applications to molecular biology, such as examining the dynamics of protein-DNA interactions. Areas of ongoing research are discussed.

DNA↗

PubMed2004

Formins: processive cappers of growing actin filaments.

Taking the advantage of single-molecule imaging, our recent study has revealed surprisingly long processive movement of a Formin protein, mDia1, surfing along with the growing end of actin filaments in living cells. This finding provides direct evidence for the ability of Formins to function as processive cappers that has been postulated from several lines of evidence in biochemical studies. With nucleating filaments from the profilin-actin pool, Formins may effectively generate long actin filaments, and contribute to the generation of the specific actin-based structures, that is, the contractile ring in cytokinesis, actin stress fibers in animal cells, and yeast actin cables. Furthermore, Formins have the potential to function as actin polymerization-driven molecular motors. Although much remains to be tested about the role of this novel molecular mobilization mechanism, cells might utilize actin polymerization energy for cell shape change and/or trafficking via Formin motors.

Actin Cytoskeleton↗

PubMed2001

Single molecule nanomanipulation of biomolecules.

The development of nanomanipulation techniques has given investigators the ability to manipulate single biomolecules and to record mechanical events of biomolecules at the single molecule level. The techniques were developed to elucidate the mechanism of molecular motors. We can directly monitor the unitary process of the mechanical work and the energy conversion processes by combining these techniques with the single molecule imaging techniques. Our results strongly suggest that the sliding movement of the actomyosin motor is driven by Brownian movement. Other groups have reported data that are more consistent with the lever arm model. These methods and imaging techniques enable us to monitor the behavior of biomolecules at work and will be applied to other molecular machines.

Actin Cytoskeleton↗

PubMed2004

GroEL mediates protein folding with a two successive timer mechanism.

GroEL encapsulates nonnative substrate proteins in a central cavity capped by GroES, providing a safe folding cage. Conventional models assume that a single timer lasting approximately 8 s governs the ATP hydrolysis-driven GroEL chaperonin cycle. We examine single molecule imaging of GFP folding within the cavity, binding release dynamics of GroEL-GroES, ensemble measurements of GroEL/substrate FRET, and the initial kinetics of GroEL ATPase activity. We conclude that the cycle consists of two successive timers of approximately 3 s and approximately 5 s duration. During the first timer, GroEL is bound to ATP, substrate protein, and GroES. When the first timer ends, the substrate protein is released into the central cavity and folding begins. ATP hydrolysis and phosphate release immediately follow this transition. ADP, GroES, and substrate depart GroEL after the second timer is complete. This mechanism explains how GroES binding to a GroEL-substrate complex encapsulates the substrate rather than allowing it to escape into solution.

Adenosine Triphosphatases↗

PubMed2003

Picket and other fences in biological membranes.

Nakada et al. revisit the controversial question of whether membrane lipids are able to diffuse from the axon of a neuron into the soma. Using single molecule imaging of a fluorescent phospholipid, the authors show that a diffusion barrier in the axon initial segment blocks the diffusion of lipids.

Animals↗

PubMed2005

Linker histone H1 per se can induce three-dimensional folding of chromatin fiber.

Higher-order architectures of chromosomes play important roles in the regulation of genome functions. To understand the molecular mechanism of genome packing, an in vitro chromatin reconstitution method and a single-molecule imaging technique (atomic force microscopy) were combined. In 50 mM NaCl, well-stretched beads-on-a-string chromatin fiber was observed. However, in 100 mM NaCl, salt-induced interaction between nucleosomes caused partial aggregation. Addition of histone H1 promoted a further folding of the fiber into thicker fibers 20-30 nm in width. Micrococcal nuclease digestion of these thicker fibers produced an approximately 170 bp fragment of nucleosomal DNA, which was approximately 20 bp longer than in the absence of histone H1 ( approximately 150 bp), indicating that H1 is correctly placed at the linker region. The width of the fiber depended on the ionic strength. Widths of 20 nm in 50 mM NaCl became 30 nm as the ionic strength was changed to 100 mM. On the basis of these results, a flexible model of chromatin fiber formation was proposed, where the mode of the fiber compaction changes depending both on salt environment and linker histone H1. The biological significance of this property of the chromatin architecture will be apparent in the closed segments ( approximately 100 kb) between SAR/MAR regions.

Chromatin↗