CiteULike: melting's library [165 articles]

Advances in Single-Molecule Fluorescence Methods for Molecular Biology

Chirlmin Joo — 2008-07-21T10:32:59-00:00

Annual Review of Biochemistry, Vol. 77, No. 1. (2008), pp. 51-76.

Ever since their introduction two decades ago, single-molecule (SM) fluorescence methods have matured and branched out to address numerous biological questions, which were inaccessible via ensemble measurements. Among the current arsenal, SM fluorescence techniques have capabilities of probing the dynamic interactions of nucleic acids and proteins via Forster (fluorescence) resonance energy transfer (FRET), tracking single particles over microns of distances, and deciphering the rotational motion of multisubunit systems. In this exciting era of transitioning from in vitro to in vivo and in situ conditions, it is anticipated that SM fluorescence methodology will become a common tool of molecular biology.

The structural basis of myosin V processive movement as revealed by electron cryomicroscopy.

N Volkmann — 2008-08-01T23:11:30-00:00

Molecular cell, Vol. 19, No. 5. (2 September 2005), pp. 595-605.

The processive motor myosin V has a relatively high affinity for actin in the presence of ATP and, thus, offers the unique opportunity to visualize some of the weaker, hitherto inaccessible, actin bound states of the ATPase cycle. Here, electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional (3D) reconstructions provide the atomic models of myosin V in both weak and strong actin bound states. One structure shows that ATP binding opens the long cleft dividing the actin binding region of the motor domain, thus destroying the strong binding actomyosin interface while rearranging loop 2 as a tether. Nucleotide analogs showed a second new state in which the lever arm points upward, in a prepower-stroke configuration (lever arm up) bound to actin before phosphate release. Our findings reveal how the structural elements of myosin V work together to allow myosin V to step along actin for multiple ATPase cycles without dissociating.

Myosin V processivity: multiple kinetic pathways for head-to-head coordination.

JE Baker — 2008-07-31T00:53:23-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 101, No. 15. (13 April 2004), pp. 5542-5546.

Myosin V, a double-headed molecular motor, transports organelles within cells by walking processively along actin, a process that requires coordination between the heads. To understand the mechanism underlying this coordination, processive runs of single myosin V molecules were perturbed by varying nucleotide content. Contrary to current views, our results show that the two heads of a myosin V molecule communicate, not through any one mechanism but through an elaborate system of cooperative mechanisms involving multiple kinetic pathways. These mechanisms introduce redundancy and safeguards that ensure robust processivity under differing physiologic demands.

Load-dependent kinetics of myosin-V can explain its high processivity

Claudia Veigel — 2005-09-01T16:26:17-00:00

Nature Cell Biology, Vol. 7, No. 9. (14 August 2005), pp. 861-869.

Neck length and processivity of myosin V.

T Sakamoto — 2008-07-30T22:27:40-00:00

The Journal of biological chemistry, Vol. 278, No. 31. (1 August 2003), pp. 29201-29207.

Myosin V is an unconventional myosin that transports cargo such as vesicles, melanosomes, or mRNA on actin filaments. It is a two-headed myosin with an unusually long neck that has six IQ motifs complexed with calmodulin. In vitro studies have shown that myosin V moves processively on actin, taking multiple 36-nm steps that coincide with the helical repeat of actin. This allows the molecule to "walk" across the top of an actin filament, a feature necessary for moving large vesicles along an actin filament bound to the cytoskeleton. The extended neck length of the two heads is thought to be critical for taking 36-nm steps for processive movements. To test this hypothesis we have expressed myosin V heavy meromyosin-like fragments containing 6IQ motifs, as well as ones that shorten (2IQ, 4IQ) or lengthen (8IQ) the neck region or alter the spacing between 3rd and 4th IQ motifs. The step size was proportional to neck length for the 2IQ, 4IQ, 6IQ, and 8IQ molecules, but the molecule with the altered spacing took shorter than expected steps. Total internal reflection fluorescence microscopy was used to determine whether the heavy meromyosin IQ molecules were capable of processive movements on actin. At saturating ATP concentrations, all molecules except for the 2IQ mutant moved processively on actin. When the ATP concentration was lowered to 10 microm or less, the 2IQ mutant demonstrated some processive movements but with reduced run lengths compared with the other mutants. Its weak processivity was also confirmed by actin landing assays.

Processivity of chimeric class V myosins.

EB Krementsova — 2008-07-30T22:26:07-00:00

The Journal of biological chemistry, Vol. 281, No. 9. (3 March 2006), pp. 6079-6086.

Unconventional myosin V takes many 36-nm steps along an actin filament before it dissociates, thus ensuring its ability to move cargo intracellularly over long distances. In the present study we assessed the structural features that affect processive run length by analyzing the properties of chimeras of mouse myosin V and a non-processive class V myosin from yeast (Myo4p) (Reck-Peterson, S. L., Tyska, M. J., Novick, P. J., and Mooseker, M. S. (2001) J. Cell Biol. 153, 1121-1126). Surprisingly a chimera containing the yeast motor domain on the neck and rod of mouse myosin V (Y-MD) showed longer run lengths than mouse wild type at low salt. Run lengths of mouse myosin V showed little salt dependence, whereas those of Y-MD decreased steeply with ionic strength, similar to a chimera containing yeast loop 2 in the mouse myosin V backbone. Loop 2 binds to acidic patches on actin in the weak binding states of the cycle (Volkmann, N., Liu, H., Hazelwood, L., Krementsova, E. B., Lowey, S., Trybus, K. M., and Hanein, D. (2005) Mol. Cell 19, 595-605). Constructs containing yeast loop 2, which has no net charge compared with +6 for wild type, showed a higher K(m) for actin in steady-state ATPase assays. The results imply that a positively charged loop 2 and a high affinity for actin are important to maintain processivity near physiologic ionic strength.

A kinetic model describing the processivity of myosin-V.

KI Skau — 2008-07-30T22:24:53-00:00

Biophysical journal, Vol. 91, No. 7. (1 October 2006), pp. 2475-2489.

The precise details of how myosin-V coordinates the biochemical reactions and mechanical motions of its two head elements to engineer effective processive molecular motion along actin filaments remain unresolved. We compare a quantitative kinetic model of the myosin-V walk, consisting of five basic states augmented by two further states to allow for futile hydrolysis and detachments, with experimental results for run lengths, velocities, and dwell times and their dependence on bulk nucleotide concentrations and external loads in both directions. The model reveals how myosin-V can use the internal strain in the molecule to synchronize the motion of the head elements. Estimates for the rate constants in the reaction cycle and the internal strain energy are obtained by a computational comparison scheme involving an extensive exploration of the large parameter space. This scheme exploits the fact that we have obtained analytic results for our reaction network, e.g., for the velocity but also the run length, diffusion constant, and fraction of backward steps. The agreement with experiment is often reasonable but some open problems are highlighted, in particular the inability of such a general model to reproduce the reported dependence of run length on ADP concentration. The novel way that our approach explores parameter space means that any confirmed discrepancies should give new insights into the reaction network model.

Dynamic multiple-target tracing to probe spatiotemporal cartography of cell membranes.

Arnauld Sergé — 2008-07-28T08:57:39-00:00

Nature methods (6 July 2008)

Although the highly dynamic and mosaic organization of the plasma membrane is well-recognized, depicting a resolved, global view of this organization remains challenging. We present an analytical single-particle tracking (SPT) method and tool, multiple-target tracing (MTT), that takes advantage of the high spatial resolution provided by single-fluorophore sensitivity. MTT can be used to generate dynamic maps at high densities of tracked particles, thereby providing global representation of molecular dynamics in cell membranes. Deflation by subtracting detected peaks allows detection of lower-intensity peaks. We exhaustively detected particles using MTT, with performance reaching theoretical limits, and then reconnected trajectories integrating the statistical information from past trajectories. We demonstrate the potential of this method by applying it to the epidermal growth factor receptor (EGFR) labeled with quantum dots (Qdots), in the plasma membrane of live cells. We anticipate the use of MTT to explore molecular dynamics and interactions at the cell membrane.

Robust single-particle tracking in live-cell time-lapse sequences.

Khuloud Jaqaman — 2008-07-24T11:49:31-00:00

Nature methods (20 July 2008)

Single-particle tracking (SPT) is often the rate-limiting step in live-cell imaging studies of subcellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose solution identifies the overall most likely set of particle trajectories throughout a movie. Using this approach, we show that the GTPase dynamin differentially affects the kinetics of long- and short-lived endocytic structures and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.

Single-particle tracking: connecting the dots

Michael Saxton — 2008-07-30T17:45:22-00:00

Nat Meth, Vol. 5, No. 8. (2008), pp. 671-672.

Unconstrained Steps of Myosin VI Appear Longest among Known Molecular Motors

Yusuf Ali — 2008-07-30T17:34:48-00:00

Biophys. J., Vol. 86, No. 6. (1 June 2004), pp. 3804-3810.

Myosin VI is a two-headed molecular motor that moves along an actin filament in the direction opposite to most other myosins. Previously, a single myosin VI molecule has been shown to proceed with steps that are large compared to its neck size: either it walks by somehow extending its neck or one head slides along actin for a long distance before the other head lands. To inquire into these and other possible mechanism of motility, we suspended an actin filament between two plastic beads, and let a single myosin VI molecule carrying a bead duplex move along the actin. This configuration, unlike previous studies, allows unconstrained rotation of myosin VI around the right-handed double helix of actin. Myosin VI moved almost straight or as a right-handed spiral with a pitch of several micrometers, indicating that the molecule walks with strides slightly longer than the actin helical repeat of 36 nm. The large steps without much rotation suggest kinesin-type walking with extended and flexible necks, but how to move forward with flexible necks, even under a backward load, is not clear. As an answer, we propose that a conformational change in the lifted head would facilitate landing on a forward, rather than backward, site. This mechanism may underlie stepping of all two-headed molecular motors including kinesin and myosin V. 10.1529/biophysj.103.037416

Entropic benefit of a cross-link in protein association.

MH Zaman — 2008-07-30T00:58:41-00:00

Proteins, Vol. 48, No. 2. (1 August 2002), pp. 341-351.

We introduce a method to estimate the loss of configurational entropy upon insertion of a cross-link to a dimeric system. First, a clear distinction is established between the loss of entropy upon tethering and binding, two quantities that are often considered to be equivalent. By comparing the probability distribution of the center-to-center distances for untethered and cross-linked versions, we are able to calculate the loss of translational entropy upon cross-linking. The distribution function for the untethered helices is calculated from the probability that a given helix is closer to its partner than to all other helices, the "Nearest Neighbor" method. This method requires no assumptions about the nature of the solvent, and hence resolves difficulties normally associated with calculations for systems in liquids. Analysis of the restriction of angular freedom upon tethering indicates that the loss of rotational entropy is negligible. The method is applied in the context of the folding of a ten turn helical coiled coil with the tether modeled as a Gaussian chain or a flexible amino acid chain. After correcting for loop closure entropy in the docked state, we estimate the introduction of a six-residue tether in the coiled coil results in an effective concentration of the chain to be about 4 or 100 mM, depending upon whether the helices are denatured or pre-folded prior to their association. Thus, tethering results in significant stabilization for systems with millimolar or stronger dissociation constants.

Fluorescent Probes for Chemical Transformations on the Single-Molecule Level

Gregor Jung — 2008-07-25T21:37:51-00:00

Ann NY Acad Sci, Vol. 1130, No. 1. (1 May 2008), pp. 131-137.

We highlight our recent achievements in the design of fluorescent dyes for the investigation of chemical reactions on the single-molecule level. These fluorophores are tailored to undergo changes in their photophysical properties upon chemical transformations. Three examples are presented: electrophilic aromatic substitution, phosphoester cleavage, and oxidation of double-bonds. Thin-layer chromatography and fluorescence correlation spectroscopy are used to separate and characterize the different reaction products, respectively. We are planning to develop more fluorescent synthons which enable us to perform single-molecule chemistry of various reactions. 10.1196/annals.1430.006

Energetics of a Forced Thermal Ratchet

Hideki Kamegawa — 2008-07-29T07:55:11-00:00

Physical Review Letters, Vol. 80, No. 24. (15 June 1998), 5251.

A myosin motor that selects bundled actin for motility.

S Nagy — 2008-07-29T19:23:30-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 28. (15 July 2008), pp. 9616-9620.

Eukaryotic cells organize their contents through trafficking along cytoskeletal filaments. The leading edge of a typical metazoan cytoskeleton consists of a dense and complex arrangement of cortical actin. A dendritic mesh is found across the broad lamellopodium, with long parallel bundles at microspikes and filopodia. It is currently unclear whether and how myosin motors identify the few actin filaments that lead to the correct destination, when presented with many similar alternatives within the cortex. Here we show that myosin X, an actin-based motor that concentrates at the distal tips of filopodia, selects the fascin-actin bundle at the filopodial core for motility. Myosin X moves individual actin filaments poorly in vitro, often supercoiling actin into plectonemes. However, single myosin X motors move robustly and processively along fascin-actin bundles. This selection requires only parallel, closely spaced filaments, as myosin X is also processive on artificial actin bundles formed by molecular crowding. Myosin X filopodial localization is perturbed in fascin-depleted HeLa cells, demonstrating that fascin bundles also direct motility in vivo. Our results demonstrate that myosin X recognizes the local structural arrangement of filaments in long bundles, providing a mechanism for sorting cargo to distant target sites.

A beta alpha-barrel built by the combination of fragments from different folds.

TA Bharat — 2008-07-29T19:17:47-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 105, No. 29. (22 July 2008), pp. 9942-9947.

Combinatorial assembly of protein domains plays an important role in the evolution of proteins. There is also evidence that protein domains have come together from stable subdomains. This concept of modular assembly could be used to construct new well folded proteins from stable protein fragments. Here, we report the construction of a chimeric protein from parts of a (betaalpha)(8)-barrel enzyme from histidine biosynthesis pathway (HisF) and a protein of the (betaalpha)(5)-flavodoxin-like fold (CheY) from Thermotoga maritima that share a high structural similarity. We expected this construct to fold into a full (betaalpha)(8)-barrel. Our results show that the chimeric protein is a stable monomer that unfolds with high cooperativity. Its three-dimensional structure, which was solved to 3.1 A resolution by x-ray crystallography, confirms a barrel-like fold in which the overall structures of the parent proteins are highly conserved. The structure further reveals a ninth strand in the barrel, which is formed by residues from the HisF C terminus and an attached tag. This strand invades between beta-strand 1 and 2 of the CheY part closing a gap in the structure that might be due to a suboptimal fit between the fragments. Thus, by a combination of parts from two different folds and a small arbitrary fragment, we created a well folded and stable protein.

Subdiffraction Multicolor Imaging of the Nuclear Periphery with 3D Structured Illumination Microscopy

Lothar Schermelleh — 2008-06-06T16:49:53-00:00

Science, Vol. 320, No. 5881. (6 June 2008), pp. 1332-1336.

Fluorescence light microscopy allows multicolor visualization of cellular components with high specificity, but its utility has until recently been constrained by the intrinsic limit of spatial resolution. We applied three-dimensional structured illumination microscopy (3D-SIM) to circumvent this limit and to study the mammalian nucleus. By simultaneously imaging chromatin, nuclear lamina, and the nuclear pore complex (NPC), we observed several features that escape detection by conventional microscopy. We could resolve single NPCs that colocalized with channels in the lamin network and peripheral heterochromatin. We could differentially localize distinct NPC components and detect double-layered invaginations of the nuclear envelope in prophase as previously seen only by electron microscopy. Multicolor 3D-SIM opens new and facile possibilities to analyze subcellular structures beyond the diffraction limit of the emitted light. 10.1126/science.1156947

How vision begins: An odyssey — PNAS

Dong-Gen Luo — 2008-07-29T19:11:58-00:00

Proceedings of the National Academy of Science, Vol. 105, No. 29. (22 July 2008), pp. 9855-9862.

Retinal rods and cones, which are the front-end light detectors in the eye, achieve wonders together by being able to signal single-photon absorption and yet also able to adjust their function to brightness changes spanning 109-fold. How these cells detect light is now quite well understood. Not surprising for almost any biological process, the intial step of seeing reveals a rich complexity as the probing goes deeper. The odyssey continues, but the knowledge gained so far is already nothing short of remarkable in qualitative and quantitative detail. It has also indirectly opened up the mystery of odorant sensing. Basic science aside, clinical ophthalmology has benefited tremendously from this endeavor as well. This article begins by recapitulating the key developments in this understanding from the mid-1960s to the late 1980s, during which period the advances were particularly rapid and fit for an intricate detective story. It then highlights some details discovered more recently, followed by a comparison between rods and cones.

From the first protein structures to our current knowledge of protein folding: delights and scepticisms.

Alan R Fersht — 2008-06-27T07:04:03-00:00

Nature reviews. Molecular cell biology (25 June 2008)

Every breakthrough that opens new vistas also removes the ground from under the feet of other scientists. The scientific joy of those who have seen the new light is accompanied by the dismay of those whose way of life has been changed for ever. The publication of the first structures of proteins at atomic resolution 50 years ago astounded and inspired scientists in every field, but caused others to flee or scoff. That advance and every subsequent paradigm-shifting breakthrough in protein science have met with some resistance before universal acceptance. I relate these events and their impact on the field of protein folding.

Counterion-dependent microrheological properties of F-actin solutions across the isotropic-nematic phase transition

Jun He — 2008-07-29T18:55:43-00:00

Physical Review E (Statistical, Nonlinear, and Soft Matter Physics), Vol. 78, No. 1. (2008)

We studied microrheological properties of F-actin across the isotropic-nematic phase transition region by video particle tracking (VPT) and by laser deflection particle tracking (LDPT). Both methods track the motion of thermally driven micron-sized beads, and convert the temporal mean square displacement (MSD) to shear moduli. The two methods give consistent results for the elastic modulus G and less so for the loss modulus G. As the nematic order parameter increases with actin concentration, G||[prime]" align="middle"> (measured parallel to the nematic director) and G[perpendicular][prime]" align="middle"> (perpendicular to the director) grow apart, with G[perpendicular][prime]" align="middle"> larger than G||[prime]" align="middle">. The moduli scale with actin concentration as G||[prime]" align="middle">~c0.54±0.13 and G[perpendicular][prime]" align="middle">~c1.38±0.15. Furthermore, G and G dependence on [Mg2+] were measured and compared for 1 mg/ml isotropic and 4 mg/ml nematic F-actin solutions, respectively. In the isotropic phase, G increases with [Mg2+] up to 6 mM and then plateaus. In the nematic phase, G[perpendicular][prime]" align="middle"> is larger than G||[prime]" align="middle">, and both G[perpendicular][prime]" align="middle"> and G||[prime]" align="middle"> increase with [Mg2+] progressively up to 16 mM, above which F-actin form large bundles. In both isotropic and nematic phases, G only weakly depends on [Mg2+]. In conclusion, particle tracking microrheology reveals rich rheological features of F-actin affected by the isotropic-nematic phase transition and by tuning weak electrostatic interactions among the protein filaments.

Fluctuation theorem and large deviation function for a solvable model of a molecular motor

D Lacoste — 2008-07-29T18:54:22-00:00

Physical Review E (Statistical, Nonlinear, and Soft Matter Physics), Vol. 78, No. 1. (2008)

We study a discrete stochastic model of a molecular motor. This discrete model can be viewed as a minimal ratchet model. We extend our previous work on this model, by further investigating the constraints imposed by the fluctuation theorem on the operation of a molecular motor far from equilibrium. In this work, we show the connections between different formulations of the fluctuation theorem. One formulation concerns the generating function of the currents while another one concerns the corresponding large deviation function, which we have calculated exactly for this model. A third formulation concerns the ratio of the probability of observing a velocity v to the same probability of observing a velocity −v. Finally, we show that all the formulations of the fluctuation theorem can be understood from the notion of entropy production.

Model of DNA Bending by Cooperative Binding of Proteins

SM Rappaport — 2008-07-29T18:53:22-00:00

Physical Review Letters, Vol. 101, No. 3. (2008)

We present a model of nonspecific cooperative binding of proteins to DNA in which the binding of isolated proteins generates local bends but binding of proteins at neighboring sites on DNA straightens the polymer. We solve the statistical mechanical problem and calculate the effective persistence length, site occupancy, and cooperativity. Cooperativity leads to nonmonotonic variation of the persistence length with protein concentration, and to an unusual shape of the binding isotherm. The results are in qualitative agreement with recent single molecule experiments on nucleoid protein HU-DNA complexes.

Lifeact: a versatile marker to visualize F-actin.

Julia Riedl — 2008-06-20T12:26:56-00:00

Nature methods (8 June 2008)

Live imaging of the actin cytoskeleton is crucial for the study of many fundamental biological processes, but current approaches to visualize actin have several limitations. Here we describe Lifeact, a 17-amino-acid peptide, which stained filamentous actin (F-actin) structures in eukaryotic cells and tissues. Lifeact did not interfere with actin dynamics in vitro and in vivo and in its chemically modified peptide form allowed visualization of actin dynamics in nontransfectable cells.

Interaction between motor domains can explain the complex dynamics of heterodimeric kinesins

Rahul Das — 2008-07-01T16:56:27-00:00

Physical Review E (Statistical, Nonlinear, and Soft Matter Physics), Vol. 77, No. 6. (2008)

Motor proteins are active enzyme molecules that play a crucial role in many biological processes. They transform chemical energy into mechanical work and move unidirectionally along rigid cytoskeleton filaments. Single-molecule experiments indicate that motor proteins, consisting of two motor domains, move in a hand-over-hand mechanism where each subunit changes between trailing and leading positions in alternating steps, and it is assumed that these subunits do not interact with each other. However, recent experiments on heterodimeric kinesins suggest that the motion of motor domains is not independent, but rather strongly coupled and coordinated, although the mechanism of these interactions is not known. We propose a simple discrete stochastic model to describe the dynamics of homodimeric and heterodimeric two-headed motor proteins. It is argued that interactions between motor domains modify original free energy landscapes for each motor subunit, while motor proteins still move via the hand-over-hand mechanism but with different transition rates specified by the new free energy profiles. Our calculations of biophysical properties agree with experimental observations. Several ways to test the theoretical model are proposed.

A Photoactivatable Push-Pull Fluorophore for Single-Molecule Imaging in Live Cells

Samuel Lord — 2008-06-24T15:06:26-00:00

J. Am. Chem. Soc. (24 June 2008)

Abstract: We have reengineered a red-emitting dicyanomethylenedihydrofuran pushpull fluorophore so that it is dark until photoactivated with a short burst of low-intensity violet light. Photoactivation of the dark fluorogen leads to conversion of an azide to an amine, which shifts the absorption to long wavelengths. After photoactivation, the fluorophore is bright and photostable enough to be imaged on the single-molecule level in living cells. This proof-of-principle demonstration provides a new class of bright photoactivatable fluorophores, as are needed for super-resolution imaging schemes that require active control of single molecule emission.

Kinetic Mechanism and Regulation of Myosin VI

De La — 2008-06-26T21:42:20-00:00

J. Biol. Chem., Vol. 276, No. 34. (17 August 2001), pp. 32373-32381.

Myosin VI is the only pointed end-directed myosin identified and is likely regulated by heavy chain phosphorylation (HCP) at the actin-binding site in vivo. We undertook a detailed kinetic analysis of the actomyosin VI ATPase cycle to determine whether there are unique adaptations to support reverse directionality and to determine the molecular basis of regulation by HCP. ADP release is the rate-limiting step in the cycle. ATP binds slowly and with low affinity. At physiological nucleotide concentrations, myosin VI is strongly bound to actin and populates the nucleotide-free (rigor) and ADP-bound states. Therefore, myosin VI is a high duty ratio motor adapted for maintaining tension and has potential to be processive. A mutant mimicking HCP increases the rate of Pi release, which lowers the KATPase but does not affect ADP release. These measurements are the first to directly measure the steps regulated by HCP for any myosin. Measurements with double-headed myosin VI demonstrate that the heads are not independent, and the native dimer hydrolyzes multiple ATPs per diffusional encounter with an actin filament. We propose an alternating site model for the stepping and processivity of two-headed high duty ratio myosins. 10.1074/jbc.M104136200

HaloTag: A Novel Protein Labeling Technology for Cell Imaging and Protein Analysis

Georgyi Los — 2008-06-06T17:18:18-00:00

ACS Chem. Biol. (6 June 2008)

We have designed a modular protein tagging system that allows different functionalities to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells. The protein tag (HaloTag) is a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands). The synthetic ligands comprise a chloroalkane linker attached to a variety of useful molecules, such as fluorescent dyes, affinity handles, or solid surfaces. Covalent bond formation between the protein tag and the chloroalkane linker is highly specific, occurs rapidly under physiological conditions, and is essentially irreversible. We demonstrate the utility of this system for cellular imaging and protein immobilization by analyzing multiple molecular processes associated with NF-ºB-mediated cellular physiology, including imaging of subcellular protein translocation and capture of proteinprotein and proteinDNA complexes.

Long single [alpha]-helical tail domains bridge the gap between structure and function of myosin VI

Benjamin Spink — 2008-06-20T08:36:10-00:00

Nat Struct Mol Biol, Vol. 15, No. 6. (June 2008), pp. 591-597.

The importance of surfaces in single-molecule bioscience

Mari Visnapuu — 2008-05-02T21:24:43-00:00

Molecular BioSystems, Vol. 4, No. 5. (2008), pp. 394-403.

The last ten years have witnessed an explosion of new techniques that can be used to probe the dynamic behavior of individual biological molecules, leading to discoveries that would not have been possible with more traditional biochemical methods. A common feature among these single-molecule approaches is the need for the biological molecules to be anchored to a solid support surface. This must be done under conditions that minimize nonspecific adsorption without compromising the biological integrity of the sample. In this review we highlight why surface attachments are a critical aspect of many single-molecule studies and we discuss current methods for anchoring biomolecules. Finally, we provide a detailed description of a new method developed by our laboratory for anchoring and organizing hundreds of individual DNA molecules on a surface, allowing 'high-throughput' studies of protein-DNA interactions at the single-molecule level.

Spherical nanosized focal spot unravels the interior of cells.

Roman Schmidt — 2008-05-27T14:47:59-00:00

Nature methods (18 May 2008)

The resolution of any linear imaging system is given by its point spread function (PSF) that quantifies the blur of an object point in the image. The sharper the PSF, the better the resolution is. In standard fluorescence microscopy, however, diffraction dictates a PSF with a cigar-shaped main maximum, called the focal spot, which extends over at least half the wavelength of light (lambda = 400-700 nm) in the focal plane and >lambda along the optical axis (z). Although concepts have been developed to sharpen the focal spot both laterally and axially, none of them has reached their ultimate goal: a spherical spot that can be arbitrarily downscaled in size. Here we introduce a fluorescence microscope that creates nearly spherical focal spots of 40-45 nm (lambda/16) in diameter. Fully relying on focused light, this lens-based fluorescence nanoscope unravels the interior of cells noninvasively, uniquely dissecting their sub-lambda-sized organelles.

Multiple events on single molecules: Unbiased estimation in single-molecule biophysics

Daniel Koster — 2006-02-08T13:31:49-00:00

PNAS, Vol. 103, No. 6. (7 February 2006), pp. 1750-1755.

Most analyses of single-molecule experiments consist of binning experimental outcomes into a histogram and finding the parameters that optimize the fit of this histogram to a given data model. Here we show that such an approach can introduce biases in the estimation of the parameters, thus great care must be taken in the estimation of model parameters from the experimental data. The bias can be particularly large when the observations themselves are not statistically independent and are subjected to global constraints, as, for example, when the iterated steps of a motor protein acting on a single molecule must not exceed the total molecule length. We have developed a maximum-likelihood analysis, respecting the experimental constraints, which allows for a robust and unbiased estimation of the parameters, even when the bias well exceeds 100%. We demonstrate the potential of the method for a number of single-molecule experiments, focusing on the removal of DNA supercoils by topoisomerase IB, and validate the method by numerical simulation of the experiment.

Tracking Single Kinesin Molecules in the Cytoplasm of Mammalian Cells

Dawen Cai — 2008-05-14T17:06:47-00:00

Biophys. J., Vol. 92, No. 12. (15 June 2007), pp. 4137-4144.

Understanding dynamic cellular processes requires precise knowledge of the distribution, transport, and interactions of individual molecules in living cells. Despite recent progress in in vivo imaging, it has not been possible to express and directly track single molecules in the cytoplasm of live cells. Here, we overcome these limitations by combining fluorescent protein-labeling with high resolution total internal reflection fluorescence microcopy, using the molecular motor Kinesin-1 as model system. First, we engineered a three-tandem monomeric Citrine tag for genetic labeling of individual molecules and expressed this motor in COS cells. Detailed analysis of the quantized photobleaching behavior of individual fluorescent spots demonstrates that we are indeed detecting single proteins in the cytoplasm of live cells. Tracking the movement of individual cytoplasmic molecules reveals that individual Kinesin-1 motors in vivo move with an average speed of 0.78 +/- 0.11 microm/s and display an average run length of 1.17 +/- 0.38 microm, which agrees well with in vitro measurements. Thus, Kinesin-1's speed and processivity are not upregulated or hindered by macromolecular crowding. Second, we demonstrate that standard deviation maps of the fluorescence intensity computed from single molecule image sequences can be used to reveal important physiological information about infrequent cellular events in the noisy fluorescence background of live cells. Finally, we show that tandem fluorescent protein tags enable single-molecule, in vitro analyses of extracted, mammalian-expressed proteins. Thus, by combining direct genetic labeling and single molecule imaging in vivo, our work establishes an important new biophysical method for observing single molecules expressed and localized in the mammalian cytoplasm. 10.1529/biophysj.106.100206

Detection of single-molecule interactions using correlated thermal diffusion.

AD Mehta — 2008-05-05T22:00:54-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 94, No. 15. (22 July 1997), pp. 7927-7931.

Observation of discrete, single-molecule binding events allows one to bypass assumptions required to infer single-molecule properties from studies of ensembles of molecules. Optically trapped beads and glass microneedles have been applied to detect single-molecule binding events, but it remains difficult to identify signs of binding events given the large displacements induced by thermal forces. Here, we exploit thermal diffusion by using correlation between motion of optically trapped beads attached to both ends of a single actin filament to track binding events of individual myosin molecules. We use correlated diffusion to measure the stiffness of a single myosin molecule and estimate its thermal fluctuation in a poststroke state as comparable in amplitude to the measured stroke distance. The use of correlated diffusion to measure kinetics of single-molecule interactions and the stiffness of the interacting moieties should be applicable to any pair of interacting molecules, and not limited to biological motors.

Molecular motors interacting with their own tracks

Max Artyomov — 2008-05-02T22:08:07-00:00

Physical Review E (Statistical, Nonlinear, and Soft Matter Physics), Vol. 77, No. 4. (2008)

Dynamics of molecular motors that move along linear lattices and interact with them via reversible destruction of specific lattice bonds is investigated theoretically by analyzing exactly solvable discrete-state “burnt-bridge” models. Molecular motors are viewed as diffusing particles that can asymmetrically break or rebuild periodically distributed weak links when passing over them. Our explicit calculations of dynamic properties show that coupling the transport of the unbiased molecular motor with the bridge-burning mechanism leads to a directed motion that lowers fluctuations and produces a dynamic transition in the limit of low concentration of weak links. Interaction between the backward biased molecular motor and the bridge-burning mechanism yields a complex dynamic behavior. For the reversible dissociation the backward motion of the molecular motor is slowed down. There is a change in the direction of the molecular motor's motion for some range of parameters. The molecular motor also experiences nonmonotonic fluctuations due to the action of two opposing mechanisms: the reduced activity after the burned sites and locking of large fluctuations. Large spatial fluctuations are observed when two mechanisms are comparable. The properties of the molecular motor are different for the irreversible burning of bridges where the velocity and fluctuations are suppressed for some concentration range, and the dynamic transition is also observed. Dynamics of the system is discussed in terms of the effective driving forces and transitions between different diffusional regimes.

Optical lock-in detection of fluorescence resonance energy transfer using synthetic and genetically-encoded optical switches.

Shu Mao — 2008-05-02T17:06:06-00:00

Biophysical Journal (15 February 2008)

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. However, the effectiveness and sensitivity of determining FRET can be reduced by photobleaching, cross talk, autofluorescence and unlabeled, endogenous proteins. We present a FRET-imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS) that substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A Snap-tag approach is used to generate a GFP-alkylguaninetransferase (AGT) fusion protein that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this GFP-AGT(NitroBIPS) complex employing optical lock-in detection of FRET allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins.

Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics

Hari Shroff — 2008-04-29T17:52:50-00:00

Nat Meth, Vol. 5, No. 5. (May 2008), pp. 417-423.

Rapid Membrane Fusion of Individual Virus Particles with Supported Lipid Bilayers

Laura Wessels — 2008-05-02T01:42:06-00:00

Biophys. J., Vol. 93, No. 2. (15 July 2007), pp. 526-538.

Many enveloped viruses employ low-pH-triggered membrane fusion during cell penetration. Solution-based in vitro assays in which viruses fuse with liposomes have provided much of our current biochemical understanding of low-pH-triggered viral membrane fusion. Here, we extend this in vitro approach by introducing a fluorescence assay using single particle tracking to observe lipid mixing between individual virus particles (influenza or Sindbis) and supported lipid bilayers. Our single-particle experiments reproduce many of the observations of the solution assays. The single-particle approach naturally separates the processes of membrane binding and membrane fusion and therefore allows measurement of details that are not available in the bulk assays. We find that the dynamics of lipid mixing during individual Sindbis fusion events is faster than 30 ms. Although neither virus binds membranes at neutral pH, under acidic conditions, the delay between membrane binding and lipid mixing is less than half a second for nearly all virus-membrane combinations. The delay between binding and lipid mixing lengthened only for Sindbis virus at the lowest pH in a cholesterol-dependent manner, highlighting the complex interaction between lipids, virus proteins, and buffer conditions in membrane fusion. 10.1529/biophysj.106.097485

Three-dimensional Resolution Doubling in Widefield Fluorescence Microscopy by Structured Illumination.

Mats Gustafsson — 2008-04-23T23:47:49-00:00

Biophysical Journal (13 March 2008)

Structured illumination microscopy is a method that can increase the spatial resolution of wide-field fluorescence microscopy beyond its classical limit by using spatially structured illumination light. Here we describe how this method can be applied in three dimensions to double the axial as well as the lateral resolution, with true optical sectioning. A grating is used to generate three mutually coherent light beams, which interfere in the specimen to form an illumination pattern that varies both laterally and axially. The spatially structured excitation intensity causes normally unreachable high-resolution information to become encoded into the observed images through spatial frequency mixing. This new information is computationally extracted and used to generate a three-dimensional reconstruction with twice as high resolution, in all three dimensions, as is possible in a conventional widefield microscope. The method has been demonstrated on both test objects and biological specimens, and has produced the first light microscopy images of the synaptonemal complex in which the lateral elements are clearly resolved.

The molecular structure of green fluorescent protein.

F Yang — 2008-04-23T21:45:03-00:00

Nature biotechnology, Vol. 14, No. 10. (October 1996), pp. 1246-1251.

The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods. The protein is in the shape of a cylinder, comprising 11 strands of beta-sheet with an alpha-helix inside and short helical segments on the ends of the cylinder. This motif, with beta-structure on the outside and alpha-helix on the inside, represents a new protein fold, which we have named the beta-can. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr66 with reduction of its C alpha-C beta bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests specific side chains that could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.

Localization Accuracy in Single-Molecule Microscopy

Raimund Ober — 2008-04-23T20:52:50-00:00

Biophysical Journal, Vol. 86, No. 2. (1 February 2004), pp. 1185-1200.

One of the most basic questions in single-molecule microscopy concerns the accuracy with which the location of a single molecule can be determined. Using the Fisher information matrix it is shown that the limit of the localization accuracy for a single molecule is given by [IMG]f1.gif" BORDER="0">, wherelambda em, na,gamma , A, and t denote the emission wavelength of the single molecule, the numerical aperture of the objective, the efficiency of the optical system, the emission rate of the single molecule and the acquisition time, respectively. Using Monte Carlo simulations it is shown that estimation algorithms can come close to attaining the limit given in the expression. Explicit quantitative results are also provided to show how the limit of the localization accuracy is reduced by factors such as pixelation of the detector and noise sources in the detection system. The results demonstrate what is achievable by single-molecule microscopy and provide guidelines for experimental design.

Predicting Allosteric Communication in Myosin via a Pathway ofConserved Residues

Susan Tang — 2007-09-04T11:46:14-00:00

Journal of Molecular Biology, Vol. In Press, Accepted Manuscript

Macromolecular-scale resolution in biological fluorescence microscopy.

G Donnert — 2006-10-24T15:04:26-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, No. 31. (1 August 2006), pp. 11440-11445.

We demonstrate far-field fluorescence microscopy with a focal-plane resolution of 15-20 nm in biological samples. The 10- to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitation-depletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to approximately 140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffraction-unlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences.

Nanoscale Resolution in the Focal Plane of an Optical Microscope

Volker Westphal — 2008-04-19T20:19:15-00:00

Physical Review Letters, Vol. 94, No. 14. (2005)

Utilizing single fluorescent molecules as probes, we prove the ability of a far-field microscope to attain spatial resolution down to 16 nm in the focal plane, corresponding to about 1/50 of the employed wavelength. The optical bandwidth expansion by nearly an order of magnitude is realized by a saturated depletion through stimulated emission of the molecular fluorescent state. We demonstrate that en route to the molecular scale, the resolving power increases with the square root of the saturation level, which constitutes a new law regarding the resolution of an emerging class of far-field light microscopes that are not limited by diffraction.

Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission.

TA Klar — 2006-10-24T15:01:41-00:00

Proceedings of the National Academy of Sciences of the United States of America, Vol. 97, No. 15. (18 July 2000), pp. 8206-8210.

The diffraction barrier responsible for a finite focal spot size and limited resolution in far-field fluorescence microscopy has been fundamentally broken. This is accomplished by quenching excited organic molecules at the rim of the focal spot through stimulated emission. Along the optic axis, the spot size was reduced by up to 6 times beyond the diffraction barrier. The simultaneous 2-fold improvement in the radial direction rendered a nearly spherical fluorescence spot with a diameter of 90-110 nm. The spot volume of down to 0.67 attoliters is 18 times smaller than that of confocal microscopy, thus making our results also relevant to three-dimensional photochemistry and single molecule spectroscopy. Images of live cells reveal greater details.

Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories

Samuel Hess — 2007-10-31T13:05:26-00:00

Proceedings of the National Academy of Sciences, Vol. 104, No. 44. (30 October 2007), pp. 17370-17375.

Organization in biological membranes spans many orders of magnitude in length scale, but limited resolution in far-field light microscopy has impeded distinction between numerous biomembrane models. One canonical example of a heterogeneously distributed membrane protein is hemagglutinin (HA) from influenza virus, which is associated with controversial cholesterol-rich lipid rafts. Using fluorescence photoactivation localization microscopy, we are able to image distributions of tens of thousands of HA molecules with subdiffraction resolution (approx40 nm) in live and fixed fibroblasts. HA molecules form irregular clusters on length scales from approx40 nm up to many micrometers, consistent with results from electron microscopy. In live cells, the dynamics of HA molecules within clusters is observed and quantified to determine an effective diffusion coefficient. The results are interpreted in terms of several established models of biological membranes. 10.1073/pnas.0708066104

Nanoscale resolution in GFP-based microscopy

Katrin Willig — 2006-08-24T16:08:22-00:00

Nature Methods, Vol. 3, No. 9. (23 August 2006), pp. 721-723.

Far-Field Optical Nanoscopy

Stefan Hell — 2008-04-15T11:32:00-00:00

Science, Vol. 316, No. 5828. (25 May 2007), pp. 1153-1158.

In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization. 10.1126/science.1137395

Fundamental improvement of resolution with a 4Pi-confocal fluorescence microscope using two-photon excitation

Stefan Hell — 2008-04-17T22:10:13-00:00

Optics Communications, Vol. 93, No. 5-6. (15 October 1992), pp. 277-282.

A new type of confocal fluorescence microscope with an unprecedented resolution is proposed. Calculations demonstrate that an axial resolution on the order of 100 nm can be achieved in a confocal microscope with enlarged aperture when two-photon excitation is applied. This axial resolution is up to four times higher than that of a confocal fluorescence microscope. The proposed microscope yields the highest point resolution ever achieved in far-field light microscopy.

The complete genome of an individual by massively parallel DNA sequencing

David Wheeler — 2008-04-16T19:24:49-00:00

Nature, Vol. 452, No. 7189. (17 April 2008), pp. 872-876.

Single-molecule high-resolution imaging with photobleaching

Matthew Gordon — 2008-04-16T17:49:42-00:00

Proceedings of the National Academy of Sciences, Vol. 101, No. 17. (27 April 2004), pp. 6462-6465.

Conventional light microscopy is limited in its resolving power by the Rayleigh limit to length scales on the order of 200 nm. On the other hand, spectroscopic techniques such as fluorescence resonance energy transfer cannot be used to measure distances >10 nm, leaving a "gap" in the ability of optical techniques to measure distances on the 10- to 100-nm scale. We have previously demonstrated the ability to localize single dye molecules to a precision of 1.5 nm with subsecond time resolution. Here we locate the position of two dyes and determine their separation with 5-nm precision, using the quantal photobleaching behavior of single fluorescent dye molecules. By fitting images both before and after photobleaching of one of the dyes, we may localize both dyes simultaneously and compute their separation. Hence, we have circumvented the Rayleigh limit and achieved nanometer-scale resolution. Specifically, we demonstrate the technique by measuring the distance between single fluorophores separated by 10-20 nm via attachment to the ends of double-stranded DNA molecules immobilized on a surface. In addition to bridging the gap in optical resolution, this technique may be useful for biophysical or genomic applications, including the generation of super-high-density maps of single-nucleotide polymorphisms. 10.1073/pnas.0401638101