A fluorogenic array for temporally unlimited single-molecule tracking
NATURE CHEMICAL BIOLOGY
2019; 15 (4): 401-+
Satb1 integrates DNA binding site geometry and torsional stress to differentially target nucleosome-dense regions.
2019; 10 (1): 3221
Origins of chemoreceptor curvature sorting in Escherichia coli
The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.
View details for DOI 10.1038/s41467-019-11118-8
View details for PubMedID 31324780
ATAC-see reveals the accessible genome by transposase-mediated imaging and sequencing.
Bacterial chemoreceptors organize into large clusters at the cell poles. Despite a wealth of structural and biochemical information on the system's components, it is not clear how chemoreceptor clusters are reliably targeted to the cell pole. Here, we quantify the curvature-dependent localization of chemoreceptors in live cells by artificially deforming growing cells of Escherichia coli in curved agar microchambers, and find that chemoreceptor cluster localization is highly sensitive to membrane curvature. Through analysis of multiple mutants, we conclude that curvature sensitivity is intrinsic to chemoreceptor trimers-of-dimers, and results from conformational entropy within the trimer-of-dimers geometry. We use the principles of the conformational entropy model to engineer curvature sensitivity into a series of multi-component synthetic protein complexes. When expressed in E. coli, the synthetic complexes form large polar clusters, and a complex with inverted geometry avoids the cell poles. This demonstrates the successful rational design of both polar and anti-polar clustering, and provides a synthetic platform on which to build new systems.
View details for DOI 10.1038/ncomms14838
View details for Web of Science ID 000396857600001
View details for PubMedID 28322223
Molecular Architecture and Assembly Principles of Vibrio cholerae Biofilms
2012; 337 (6091): 236-239
Spatial organization of the genome plays a central role in gene expression, DNA replication, and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see), a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ, cell sorting, and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion, termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility, and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control.
View details for DOI 10.1038/nmeth.4031
View details for PubMedID 27749837
Strong triaxial coupling and anomalous Poisson effect in collagen networks
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2019; 116 (14): 6790–99
Strong triaxial coupling and anomalous Poisson effect in collagen networks.
Proceedings of the National Academy of Sciences of the United States of America
In their natural environment, microbes organize into communities held together by an extracellular matrix composed of polysaccharides and proteins. We developed an in vivo labeling strategy to allow the extracellular matrix of developing biofilms to be visualized with conventional and superresolution light microscopy. Vibrio cholerae biofilms displayed three distinct levels of spatial organization: cells, clusters of cells, and collections of clusters. Multiresolution imaging of living V. cholerae biofilms revealed the complementary architectural roles of the four essential matrix constituents: RbmA provided cell-cell adhesion; Bap1 allowed the developing biofilm to adhere to surfaces; and heterogeneous mixtures of Vibrio polysaccharide, RbmC, and Bap1 formed dynamic, flexible, and ordered envelopes that encased the cell clusters.
View details for DOI 10.1126/science.1222981
View details for Web of Science ID 000306323500059
View details for PubMedID 22798614
View details for PubMedCentralID PMC3513368
A fluorogenic array for temporally unlimited single-molecule tracking.
Nature chemical biology
While cells within tissues generate and sense 3D states of strain, the current understanding of the mechanics of fibrous extracellular matrices (ECMs) stems mainly from uniaxial, biaxial, and shear tests. Here, we demonstrate that the multiaxial deformations of fiber networks in 3D cannot be inferred solely based on these tests. The interdependence of the three principal strains gives rise to anomalous ratios of biaxial to uniaxial stiffness between 8 and 9 and apparent Poisson's ratios larger than 1. These observations are explained using a microstructural network model and a coarse-grained constitutive framework that predicts the network Poisson effect and stress-strain responses in uniaxial, biaxial, and triaxial modes of deformation as a function of the microstructural properties of the network, including fiber mechanics and pore size of the network. Using this theoretical approach, we found that accounting for the Poisson effect leads to a 100-fold increase in the perceived elastic stiffness of thin collagen samples in extension tests, reconciling the seemingly disparate measurements of the stiffness of collagen networks using different methods. We applied our framework to study the formation of fiber tracts induced by cellular forces. In vitro experiments with low-density networks showed that the anomalous Poisson effect facilitates higher densification of fibrous tracts, associated with the invasion of cancerous acinar cells. The approach developed here can be used to model the evolving mechanics of ECM during cancer invasion and fibrosis.
View details for PubMedID 30894480
Physical confinement induces malignant transformation in mammary epithelial cells.
2019; 217: 119307
We describe three optical tags, ArrayG, ArrayD and ArrayG/N, for intracellular tracking of single molecules over milliseconds to hours. ArrayG is a fluorogenic tag composed of a green fluorescent protein-nanobody array and monomeric wild-type green fluorescent protein binders that are initially dim but brighten ~26-fold on binding with the array. By balancing the rates of binder production, photobleaching and stochastic binder exchange, we achieve temporally unlimited tracking of single molecules. High-speed tracking of ArrayG-tagged kinesins and integrins for thousands of frames reveals novel dynamical features. Tracking of single histones at 0.5Hz for>1hour with the import competent ArrayG/N tag shows that chromosomal loci behave as Rouse polymers with visco-elastic memory and exhibit a non-Gaussian displacement distribution. ArrayD, based on a dihydrofolate reductase nanobody array and dihydrofolate reductase-fluorophore binder, enables dual-color imaging. The arrays combine brightness, fluorogenicity, fluorescence replenishment and extended fluorophore choice, opening new avenues for tracking single molecules in living cells.
View details for PubMedID 30858596
A Mutation in Histone H2B Represents a New Class of Oncogenic Driver.
The physical microenvironment of tumor cells plays an important role in cancer initiation and progression. Here, we present evidence that confinement - a new physical parameter that is apart from matrix stiffness - can also induce malignant transformation in mammary epithelial cells. We discovered that MCF10A cells, a benign mammary cell line that forms growth-arrested polarized acini in Matrigel, transforms into cancer-like cells within the same Matrigel material following confinement in alginate shell hydrogel microcapsules. The confined cells exhibited a range of tumor-like behaviors, including uncontrolled cellular proliferation and invasion. Additionally, 4-6 weeks after transplantation into the mammary fad pads of immunocompromised mice, the confined cells formed large palpable masses that exhibited histological features similar to that of carcinomas. Taken together, our findings suggest that physical confinement represents a previously unrecognized mechanism for malignancy induction in mammary epithelial cells and also provide a new, microcapsule-based, high throughput model system for testing new breast cancer therapeutics.
View details for DOI 10.1016/j.biomaterials.2019.119307
View details for PubMedID 31271857
Mechanisms of Plastic Deformation in Collagen Networks Induced by Cellular Forces
2018; 114 (2): 450–61
By examination of the cancer genomics database we identified a new set of mutations in core histones that frequently recur in cancer patient samples and are predicted to disrupt nucleosome stability. In support of this idea, we characterized a glutamate to lysine mutation of histone H2B at amino acid 76 (H2B-E76K) found particularly in bladder and head and neck cancer that disrupts the interaction between H2B and H4. Although H2B-E76K forms dimers with H2A, it does not form stable histone octamers with H3 and H4 in vitro and when reconstituted with DNA forms unstable nucleosomes with increased sensitivity to nuclease. Expression of the equivalent H2B mutant in yeast restricted growth at high temperature and led to defective nucleosome-mediated gene repression. Significantly, H2B-E76K expression in the normal mammary epithelial cell line MCF10A increased cellular proliferation, cooperated with PIK3CA to promote colony formation, caused a significant drift in gene expression and fundamental changes in chromatin accessibility particularly at gene regulatory elements. Taken together, these data demonstrate that mutations in the globular domains of core histones may give rise to an oncogenic program due to nucleosome dysfunction and deregulation of gene expression.
View details for DOI 10.1158/2159-8290.CD-19-0393
View details for PubMedID 31337617
Importin-beta modulates the permeability of the nuclear pore complex in a Ran-dependent manner
Contractile cells can reorganize fibrous extracellular matrices and form dense tracts of fibers between neighboring cells. These tracts guide the development of tubular tissue structures and provide paths for the invasion of cancer cells. Here, we studied the mechanisms of the mechanical plasticity of collagen tracts formed by contractile premalignant acinar cells and fibroblasts. Using fluorescence microscopy and second harmonic generation, we quantified the collagen densification, fiber alignment, and strains that remain within the tracts after cellular forces are abolished. We explained these observations using a theoretical fiber network model that accounts for the stretch-dependent formation of weak cross-links between nearby fibers. We tested the predictions of our model using shear rheology experiments. Both our model and rheological experiments demonstrated that increasing collagen concentration leads to substantial increases in plasticity. We also considered the effect of permanent elongation of fibers on network plasticity and derived a phase diagram that classifies the dominant mechanisms of plasticity based on the rate and magnitude of deformation and the mechanical properties of individual fibers. Plasticity is caused by the formation of new cross-links if moderate strains are applied at small rates or due to permanent fiber elongation if large strains are applied over short periods. Finally, we developed a coarse-grained model for plastic deformation of collagen networks that can be employed to simulate multicellular interactions in processes such as morphogenesis, cancer invasion, and fibrosis.
View details for PubMedID 29401442
View details for PubMedCentralID PMC5984980
Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration
2015; 7 (10): 1120-1134
Rapid disorganization of mechanically interacting systems of mammary acini
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2014; 111 (2): 658-663
Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impβ at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impβ, but not transportin-1 (TRN1), alters the pore's permeability in a Ran-dependent manner, suggesting that impβ is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impβ or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153. We propose that impβ and Nup153 interact at the NPC's nuclear face to form a Ran-regulated mesh that modulates NPC permeability.
View details for DOI 10.7554/eLife.04052
View details for Web of Science ID 000351864100002
View details for PubMedID 25748139
View details for PubMedCentralID PMC4375889
Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2013; 110 (46): 18519-18524
Cells and multicellular structures can mechanically align and concentrate fibers in their ECM environment and can sense and respond to mechanical cues by differentiating, branching, or disorganizing. Here we show that mammary acini with compromised structural integrity can interconnect by forming long collagen lines. These collagen lines then coordinate and accelerate transition to an invasive phenotype. Interacting acini begin to disorganize within 12.5 ± 4.7 h in a spatially coordinated manner, whereas acini that do not interact mechanically with other acini disorganize more slowly (in 21.8 ± 4.1 h) and to a lesser extent (P < 0.0001). When the directed mechanical connections between acini were cut with a laser, the acini reverted to a slowly disorganizing phenotype. When acini were fully mechanically isolated from other acini and also from the bulk gel by box-cuts with a side length <900 μm, transition to an invasive phenotype was blocked in 20 of 20 experiments, regardless of waiting time. Thus, pairs or groups of mammary acini can interact mechanically over long distances through the collagen matrix, and these directed mechanical interactions facilitate transition to an invasive phenotype.
View details for DOI 10.1073/pnas.1311312110
View details for Web of Science ID 000329614500032
View details for PubMedID 24379367
View details for PubMedCentralID PMC3896145
A single-molecule analysis reveals morphological targets for cellulase synergy
NATURE CHEMICAL BIOLOGY
2013; 9 (6): 356-?
The RAF serine/threonine kinases regulate cell growth through the MAPK pathway, and are targeted by small-molecule RAF inhibitors (RAFis) in human cancer. It is now apparent that protein multimers play an important role in RAF activation and tumor response to RAFis. However, the exact stoichiometry and cellular location of these multimers remain unclear because of the lack of technologies to visualize them. In the present work, we demonstrate that photoactivated localization microscopy (PALM), in combination with quantitative spatial analysis, provides sufficient resolution to directly visualize protein multimers in cells. Quantitative PALM imaging showed that CRAF exists predominantly as cytoplasmic monomers under resting conditions but forms dimers as well as trimers and tetramers at the cell membrane in the presence of active RAS. In contrast, N-terminal truncated CRAF (CatC) lacking autoinhibitory domains forms constitutive dimers and occasional tetramers in the cytoplasm, whereas a CatC mutant with a disrupted CRAF-CRAF dimer interface does not. Finally, artificially forcing CRAF to the membrane by fusion to a RAS CAAX motif induces multimer formation but activates RAF/MAPK only if the dimer interface is intact. Together, these quantitative results directly confirm the existence of RAF dimers and potentially higher-order multimers and their involvement in cell signaling, and showed that RAF multimer formation can result from multiple mechanisms and is a critical but not sufficient step for RAF activation.
View details for DOI 10.1073/pnas.1318188110
View details for Web of Science ID 000326830900054
View details for PubMedID 24158481
View details for PubMedCentralID PMC3831949
A physical sciences network characterization of non-tumorigenic and metastatic cells
The mechanisms of enzyme activity on solid substrates are not well understood. Unlike enzyme catalysis in aqueous solutions, enzyme activity on surfaces is complicated by adsorption steps and structural heterogeneities that make enzyme-substrate interactions difficult to characterize. Cellulase enzymes, which catalyze the depolymerization of cellulose, show binding specificities for different cellulose surface morphologies, but the influence of these specificities on the activity of multienzyme mixtures has remained unclear. We developed a metric to quantify binding-target arrangements determined by photoactivated localization microscopy, and we used that metric to show that combinations of cellulases designed to bind within similar but nonidentical morphologies can have synergistic activity. This phenomenon cannot be explained with the binary crystalline or amorphous classifications commonly used to characterize cellulase-binding targets. Our results reveal a strategy for improving the activity of cellulolytic mixtures and demonstrate a versatile method for investigating protein organization on heterogeneous surfaces.
View details for DOI 10.1038/nchembio.1227
View details for Web of Science ID 000319328200006
View details for PubMedID 23563526
Scaffold nucleoporins Nup188 and Nup192 share structural and functional properties with nuclear transport receptors.
To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.
View details for DOI 10.1038/srep01449
View details for Web of Science ID 000318061300001
View details for PubMedID 23618955
View details for PubMedCentralID PMC3636513
mMaple: A Photoconvertible Fluorescent Protein for Use in Multiple Imaging Modalities
2012; 7 (12)
Nucleocytoplasmic transport is mediated by nuclear pore complexes (NPCs) embedded in the nuclear envelope. About 30 different proteins (nucleoporins, nups) arrange around a central eightfold rotational axis to build the modular NPC. Nup188 and Nup192 are related and evolutionary conserved, large nucleoporins that are part of the NPC scaffold. Here we determine the structure of Nup188. The protein folds into an extended stack of helices where an N-terminal 130 kDa segment forms an intricate closed ring, while the C-terminal region is a more regular, superhelical structure. Overall, the structure has distant similarity with flexible S-shaped nuclear transport receptors (NTRs). Intriguingly, like NTRs, both Nup188 and Nup192 specifically bind FG-repeats and are able to translocate through NPCs by facilitated diffusion. This blurs the existing dogma of a clear distinction between stationary nups and soluble NTRs and suggests an evolutionary relationship between the NPC and the soluble nuclear transport machinery. DOI:http://dx.doi.org/10.7554/eLife.00745.001.
View details for DOI 10.7554/eLife.00745
View details for PubMedID 23795296
Scanning angle interference microscopy reveals cell dynamics at the nanoscale
2012; 9 (8): 825-?
Recent advances in fluorescence microscopy have extended the spatial resolution to the nanometer scale. Here, we report an engineered photoconvertible fluorescent protein (pcFP) variant, designated as mMaple, that is suited for use in multiple conventional and super-resolution imaging modalities, specifically, widefield and confocal microscopy, structured illumination microscopy (SIM), and single-molecule localization microscopy. We demonstrate the versatility of mMaple by obtaining super-resolution images of protein organization in Escherichia coli and conventional fluorescence images of mammalian cells. Beneficial features of mMaple include high photostability of the green state when expressed in mammalian cells and high steady state intracellular protein concentration of functional protein when expressed in E. coli. mMaple thus enables both fast live-cell ensemble imaging and high precision single molecule localization for a single pcFP-containing construct.
View details for DOI 10.1371/journal.pone.0051314
View details for Web of Science ID 000312290800051
View details for PubMedID 23240015
What does physics have to do with cancer?
NATURE REVIEWS CANCER
2011; 11 (9): 657-670
Emerging questions in cell biology necessitate nanoscale imaging in live cells. Here we present scanning angle interference microscopy, which is capable of localizing fluorescent objects with nanoscale precision along the optical axis in motile cellular structures. We use this approach to resolve nanotopographical features of the cell membrane and cytoskeleton as well as the temporal evolution, three-dimensional architecture and nanoscale dynamics of focal adhesion complexes.
View details for DOI 10.1038/NMETH.2077
View details for Web of Science ID 000307015700026
View details for PubMedID 22751201
Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking
2010; 467 (7315): 600-U126
Large-scale cancer genomics, proteomics and RNA-sequencing efforts are currently mapping in fine detail the genetic and biochemical alterations that occur in cancer. However, it is becoming clear that it is difficult to integrate and interpret these data and to translate them into treatments. This difficulty is compounded by the recognition that cancer cells evolve, and that initiation, progression and metastasis are influenced by a wide variety of factors. To help tackle this challenge, the US National Cancer Institute Physical Sciences-Oncology Centers initiative is bringing together physicists, cancer biologists, chemists, mathematicians and engineers. How are we beginning to address cancer from the perspective of the physical sciences?
View details for DOI 10.1038/nrc3092
View details for Web of Science ID 000294203400013
View details for PubMedID 21850037
Potential of light-harvesting proton pumps for bioenergy applications
CURRENT OPINION IN BIOTECHNOLOGY
2010; 21 (3): 265-270
The nuclear pore complex (NPC) mediates all exchange between the cytoplasm and the nucleus. Small molecules can passively diffuse through the NPC, whereas larger cargos require transport receptors to translocate. How the NPC facilitates the translocation of transport receptor/cargo complexes remains unclear. To investigate this process, we tracked single protein-functionalized quantum dot cargos as they moved through human NPCs. Here we show that import proceeds by successive substeps comprising cargo capture, filtering and translocation, and release into the nucleus. Most quantum dots are rejected at one of these steps and return to the cytoplasm, including very large cargos that abort at a size-selective barrier. Cargo movement in the central channel is subdiffusive and cargos that can bind more transport receptors diffuse more freely. Without Ran GTPase, a critical regulator of transport directionality, cargos still explore the entire NPC, but have a markedly reduced probability of exit into the nucleus, suggesting that NPC entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos. The overall selectivity of the NPC seems to arise from the cumulative action of multiple reversible substeps and a final irreversible exit step.
View details for DOI 10.1038/nature09285
View details for Web of Science ID 000282273100041
View details for PubMedID 20811366
PLASMON RULERS AS DYNAMIC MOLECULAR RULERS IN ENZYMOLOGY
METHODS IN ENZYMOLOGY, VOL 475: SINGLE MOLECULE TOOLS, PT B
2010; 475: 175-198
Concerns about the security and longevity of traditional energy sources have increased interest in alternative methods of energy production, particularly those which utilize abundantly available solar energy. Solar energy can be harvested either indirectly through the conversion of plant or algal byproducts into biofuels or directly using engineered microorganisms. Here we summarize the main features of light-harvesting proton pumps, which may provide a relatively simple way to boost the efficiency of energy-limited biological processes in fuel production. This family of proton pumps, which includes bacteriorhodopsin and proteorhodopsin, directly uses light energy to create a proton motive force (pmf) which can be used by other enzymes to facilitate active transport, regulate transmembrane proteins, or to generate ATP and NADH.
View details for DOI 10.1016/j.copbio.2010.03.007
View details for Web of Science ID 000278953600006
View details for PubMedID 20371172
Self-Organization of the Escherichia coli Chemotaxis Network Imaged with Super-Resolution Light Microscopy
2009; 7 (6)
This chapter provides an introduction to the concept of "plasmon rulers," pairs of biopolymer-linked tethered nanoparticles which act as nonblinking, nonbleaching rulers for dynamic molecular distance measurements. Plasmon rulers utilize the distance dependence of the plasmon coupling between individual noble metal particles to measure distances. Although the plasmon ruler approach is still an emerging technology, proof-of-principle experiments have demonstrated that plasmon rulers can already be used to investigate structural fluctuations in nucleoprotein complexes, monitor nuclease catalyzed DNA or RNA cleavage reactions, and detect DNA bending. The physical concepts underlying plasmon rulers are summarized, and effective assembly approaches as well as recent applications are discussed. Plasmon rulers are a useful addition to the single molecule biophysics toolbox, since they allow single biomolecules to be continuously monitored for days at high temporal resolutions.
View details for DOI 10.1016/S0076-6879(10)75008-4
View details for Web of Science ID 000280733800008
View details for PubMedID 20627158
Fabrication of 10 nm diameter hydrocarbon nanopores
APPLIED PHYSICS LETTERS
2008; 93 (18)
Optical measurement of mechanical forces inside short DNA loops
2008; 94 (6): 2179-2186
The Escherichia coli chemotaxis network is a model system for biological signal processing. In E. coli, transmembrane receptors responsible for signal transduction assemble into large clusters containing several thousand proteins. These sensory clusters have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here, we use photoactivated localization microscopy (PALM) to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW) with a precision of 15 nm. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One-third of Tar receptors are part of smaller lateral clusters and not of the large polar clusters. Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support the notion that stochastic self-assembly can create and maintain approximately periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.
View details for DOI 10.1371/journal.pbio.1000137
View details for Web of Science ID 000268398600014
View details for PubMedID 19547746
Controlling DNA capture and propagation through artificial nanopores
2007; 7 (9): 2824-2830
Knowledge of the mechanical properties of double-stranded DNA (dsDNA) is essential to understand the role of dsDNA looping in gene regulation and the mechanochemistry of molecular machines that operate on dsDNA. Here, we use a newly developed tool, force sensors with optical readout, to measure the forces inside short, strained loops composed of both dsDNA and single-stranded DNA. By varying the length of the loops and their proportion of dsDNA, it was possible to vary their internal forces from 1 pN to >20 pN. Surprisingly, internal loop forces changed erratically as the amount of dsDNA was increased for a given loop length, with the effect most notable in the smallest loop (57 nucleotides). Monte Carlo simulations based on the helical wormlike chain model accurately predict internal forces when more than half of the loop is dsDNA but fail otherwise. Mismatches engineered into the double-stranded regions increased flexibility, suggesting that Watson-Crick basepaired dsDNA can withstand high compressive forces without recourse to multibase melts. Fluorescence correlation spectroscopy further excluded transient melting (microsecond to millisecond duration) as a mechanism for relief of compressive forces in the tested dsDNAs. DNA loops with integrated force sensors may allow the comprehensive mapping of the elasticity of short dsDNAs as a function of both sequence and salt.
View details for DOI 10.1529/biophysj.107.114413
View details for Web of Science ID 000253676200022
View details for PubMedID 18065484
View details for PubMedCentralID PMC2257878
Tunable nanowire nonlinear optical probe
2007; 447 (7148): 1098-U8
Electrophorescing biopolymers across nanopores modulates the ionic current through the pore, revealing the polymer's diameter, length, and conformation. The rapidity of polymer translocation ( approximately 30,000 bp/ms) in this geometry greatly limits the information that can be obtained for each base. Here we show that the translocation speed of lambda-DNA through artificial nanopores can be reduced using optical tweezers. DNAs coupled to optically trapped beads were presented to nanopores. DNAs initially placed up to several micrometers from the pore could be captured. Subsequently, the optical tweezers reduced translocation speeds to 150 bp/ms, about 200-fold slower than free DNA. Moreover, the optical tweezers allowed us to "floss" single polymers back and forth through the pore. The combination of controlled sample presentation, greatly slowed translocation speeds, and repeated electrophoresis of single DNAs removes several barriers to using artificial nanopores for sequencing, haplotyping, and characterization of protein-DNA interactions.
View details for DOI 10.1021/nl0714334
View details for Web of Science ID 000249501900051
View details for PubMedID 17705552
Use of plasmon coupling to reveal the dynamics of DNA bending and cleavage by single EcoRV restriction enzymes
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (8): 2667-2672
One crucial challenge for subwavelength optics has been the development of a tunable source of coherent laser radiation for use in the physical, information and biological sciences that is stable at room temperature and physiological conditions. Current advanced near-field imaging techniques using fibre-optic scattering probes have already achieved spatial resolution down to the 20-nm range. Recently reported far-field approaches for optical microscopy, including stimulated emission depletion, structured illumination, and photoactivated localization microscopy, have enabled impressive, theoretically unlimited spatial resolution of fluorescent biomolecular complexes. Previous work with laser tweezers has suggested that optical traps could be used to create novel spatial probes and sensors. Inorganic nanowires have diameters substantially below the wavelength of visible light and have electronic and optical properties that make them ideal for subwavelength laser and imaging technology. Here we report the development of an electrode-free, continuously tunable coherent visible light source compatible with physiological environments, from individual potassium niobate (KNbO3) nanowires. These wires exhibit efficient second harmonic generation, and act as frequency converters, allowing the local synthesis of a wide range of colours via sum and difference frequency generation. We use this tunable nanometric light source to implement a novel form of subwavelength microscopy, in which an infrared laser is used to optically trap and scan a nanowire over a sample, suggesting a wide range of potential applications in physics, chemistry, materials science and biology.
View details for DOI 10.1038/nature05921
View details for Web of Science ID 000247564600034
View details for PubMedID 17597756
Light-powering Escherichia coli with proteorhodopsin
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2007; 104 (7): 2408-2412
Pairs of Au nanoparticles have recently been proposed as "plasmon rulers" based on the dependence of their light scattering on the interparticle distance. Preliminary work has suggested that plasmon rulers can be used to measure and monitor dynamic distance changes over the 1- to 100-nm length scale in biology. Here, we substantiate that plasmon rulers can be used to measure dynamical biophysical processes by applying the ruler to a system that has been investigated extensively by using ensemble kinetic measurements: the cleavage of DNA by the restriction enzyme EcoRV. Temporal resolutions of up to 240 Hz were obtained, and the end-to-end extension of up to 1,000 individual dsDNA enzyme substrates could be simultaneously monitored for hours. The kinetic parameters extracted from our single-molecule cleavage trajectories agree well with values obtained in bulk through other methods and confirm well known features of the cleavage process, such as DNA bending before cleavage. Previously unreported dynamical information is revealed as well, for instance, the degree of softening of the DNA just before cleavage. The unlimited lifetime, high temporal resolution, and high signal/noise ratio make the plasmon ruler a unique tool for studying macromolecular assemblies and conformational changes at the single-molecule level.
View details for DOI 10.1073/pnas.0607826104
View details for Web of Science ID 000244511200021
View details for PubMedID 17307879
ZnO-Al2O3 and ZnO-TiO2 core-shell nanowire dye-sensitized solar cells
JOURNAL OF PHYSICAL CHEMISTRY B
2006; 110 (45): 22652-22663
Proteorhodopsin (PR) is a light-powered proton pump identified by community sequencing of ocean samples. Previous studies have established the ecological distribution and enzymatic activity of PR, but its role in powering cells and participation in ocean energy fluxes remains unclear. Here, we show that when cellular respiration is inhibited by depleting oxygen or by the respiratory poison azide, Escherichia coli cells expressing PR become light-powered. Illumination of these cells with light coinciding with PR's absorption spectrum creates a proton motive force (pmf) that turns the flagellar motor, yielding cells that swim when illuminated with green light. By measuring the pmf of individual illuminated cells, we quantify the coupling between light-driven and respiratory proton currents, estimate the Michaelis-Menten constant (Km) of PR (10(3) photons per second/nm2), and show that light-driven pumping by PR can fully replace respiration as a cellular energy source in some environmental conditions. Moreover, sunlight-illuminated PR+ cells are less sensitive to azide than PR- cells, consistent with PR+ cells possessing an alternative means of maintaining cellular pmf and, thus, viability. Proteorhodopsin allows Escherichia coli cells to withstand environmental respiration challenges by harvesting light energy.
View details for DOI 10.1073/pnas.0611035104
View details for Web of Science ID 000244438500066
View details for PubMedID 17277079
Optical trapping and integration of semiconductor nanowire assemblies in water
2006; 5 (2): 97-101
We describe the construction and performance of dye-sensitized solar cells (DSCs) based on arrays of ZnO nanowires coated with thin shells of amorphous Al(2)O(3) or anatase TiO(2) by atomic layer deposition. We find that alumina shells of all thicknesses act as insulating barriers that improve cell open-circuit voltage (V(OC)) only at the expense of a larger decrease in short-circuit current density (J(SC)). However, titania shells 10-25 nm in thickness cause a dramatic increase in V(OC) and fill factor with little current falloff, resulting in a substantial improvement in overall conversion efficiency, up to 2.25% under 100 mW cm(-2) AM 1.5 simulated sunlight. The superior performance of the ZnO-TiO(2) core-shell nanowire cells is a result of a radial surface field within each nanowire that decreases the rate of recombination in these devices. In a related set of experiments, we have found that TiO(2) blocking layers deposited underneath the nanowire films yield cells with reduced efficiency, in contrast to the beneficial use of blocking layers in some TiO(2) nanoparticle cells. Raising the efficiency of our nanowire DSCs above 2.5% depends on achieving higher dye loadings through an increase in nanowire array surface area.
View details for DOI 10.1021/jp0648644
View details for Web of Science ID 000241905700051
View details for PubMedID 17092013
Calibration of dynamic molecular rule based on plasmon coupling between gold nanoparticles
2005; 5 (11): 2246-2252
Semiconductor nanowires have received much attention owing to their potential use as building blocks of miniaturized electrical, nanofluidic and optical devices. Although chemical nanowire synthesis procedures have matured and now yield nanowires with specific compositions and growth directions, the use of these materials in scientific, biomedical and microelectronic applications is greatly restricted owing to a lack of methods to assemble nanowires into complex heterostructures with high spatial and angular precision. Here we show that an infrared single-beam optical trap can be used to individually trap, transfer and assemble high-aspect-ratio semiconductor nanowires into arbitrary structures in a fluid environment. Nanowires with diameters as small as 20 nm and aspect ratios of more than 100 can be trapped and transported in three dimensions, enabling the construction of nanowire architectures that may function as active photonic devices. Moreover, nanowire structures can now be assembled in physiological environments, offering new forms of chemical, mechanical and optical stimulation of living cells.
View details for DOI 10.1038/nmat1563
View details for Web of Science ID 000235060000015
View details for PubMedID 16429143
Biocompatible force sensor with optical readout and dimensions of 6 nm(3)
2005; 5 (7): 1509-1514
Pairs of noble metal nanoparticles can be used to measure distances via the distance dependence of their plasmon coupling. These "plasmon rulers" offer exceptional photostability and brightness; however, the advantages and limitations of this approach remain to be explored. Here we report detailed plasmon peak versus separation calibration curves for 42- and 87-nm-diameter particle pairs, determine their measurement errors, and describe experimental procedures to improve their performance in biology, nanotechnology, and materials sciences.
View details for DOI 10.1021/nl051592s
View details for Web of Science ID 000233481700026
View details for PubMedID 16277462
The nonequilibrium thermodynamics of small systems
2005; 58 (7): 43-48
A molecular ruler based on plasmon coupling of single gold and silver nanoparticles
2005; 23 (6): 741-745
We have developed a nanoscopic force sensor with optical readout. The sensor consists of a single-stranded DNA oligomer flanked by two dyes. The DNA acts as a nonlinear spring: when the spring is stretched, the distance between the two dyes increases, resulting in reduced Förster resonance energy transfer. The sensor was calibrated between 0 and 20 pN using a combined magnetic tweezers/single-molecule fluorescence microscope. We show that it is possible to tune the sensor's force response by varying the interdye spacing and that the FRET efficiency of the sensors decreases with increasing force. We demonstrate the usefulness of these sensors by using them to measure the forces internal to a single polymer molecule, a small DNA loop. Partial conversion of the single-stranded DNA loop to a double-stranded form results in the accumulation of strain: a force of approximately 6 pN was measured in the loop upon hybridization. The sensors should allow measurement of forces internal to various materials, including programmable DNA self-assemblies, polymer meshes, and DNA-based machines.
View details for DOI 10.1021/nl050875h
View details for Web of Science ID 000230571300058
View details for PubMedID 16178266
Experimental test of Hatano and Sasa's nonequilibrium steady-state equality
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2004; 101 (42): 15038-15041
Forster Resonance Energy Transfer has served as a molecular ruler that reports conformational changes and intramolecular distances of single biomolecules. However, such rulers suffer from low and fluctuating signal intensities, limited observation time due to photobleaching, and an upper distance limit of approximately 10 nm. Noble metal nanoparticles have plasmon resonances in the visible range and do not blink or bleach. They have been employed as alternative probes to overcome the limitations of organic fluorophores, and the coupling of plasmons in nearby particles has been exploited to detect particle aggregation by a distinct color change in bulk experiments. Here we demonstrate that plasmon coupling can be used to monitor distances between single pairs of gold and silver nanoparticles. We followed the directed assembly of gold and silver nanoparticle dimers in real time and studied the kinetics of single DNA hybridization events. These "plasmon rulers" allowed us to continuously monitor separations of up to 70 nm for >3,000 s.
View details for DOI 10.1038/nbt1100
View details for Web of Science ID 000229628700036
View details for PubMedID 15908940
Identifying kinetic barriers to mechanical unfolding of the T-thermophila ribozyme
2003; 299 (5614): 1892-1895
Most natural processes occur far from equilibrium and cannot be treated within the framework of classical thermodynamics. In 1998, Oono and Paniconi [Oono, Y. & Paniconi, M. (1998) Prog. Theor. Phys. Suppl. 130, 29-44] proposed a general phenomenological framework, steady-state thermodynamics, encompassing nonequilibrium steady states and transitions between such states. In 2001, Hatano and Sasa [Hatano, T. & Sasa, S. (2001) Phys. Rev. Lett. 86, 3463-3466] derived a testable prediction of this theory. Specifically, they were able to show that the exponential average of Y, a quantity similar to a dissipated work, should be equal to zero for arbitrary transitions between nonequilibrium steady states, -ln = 0. We have tested this strong prediction by measuring the dissipation and fluctuations of microspheres optically driven through water. We have found that -ln approximately 0 for three different nonequilibrium systems, supporting Hatano and Sasa's proposed extension of thermodynamics to arbitrary steady states and irreversible transitions.
View details for DOI 10.1073/pnas.0406405101
View details for Web of Science ID 000224688700013
View details for PubMedID 15469914
Equilibrium information from nonequilibrium measurements in an experimental test of Jarzynski's equality
2002; 296 (5574): 1832-1835
Mechanical unfolding trajectories for single molecules of the Tetrahymena thermophila ribozyme display eight intermediates corresponding to discrete kinetic barriers that oppose mechanical unfolding with lifetimes of seconds and rupture forces between 10 and 30 piconewtons. Barriers are magnesium dependent and correspond to known intra- and interdomain interactions. Several barrier structures are "brittle," breakage requiring high forces but small (1 to 3 nanometers) deformations. Barrier crossing is stochastic, leading to variable unfolding paths. The response of complex RNA structures to locally applied mechanical forces may be analogous to the responses of RNA during translation, messenger RNA export from the nucleus, and viral replication.
View details for Web of Science ID 000181669700046
View details for PubMedID 12649482
Reversible unfolding of single RNA molecules by mechanical force
2001; 292 (5517): 733-737
Recent advances in statistical mechanical theory can be used to solve a fundamental problem in experimental thermodynamics. In 1997, Jarzynski proved an equality relating the irreversible work to the equilibrium free energy difference, DeltaG. This remarkable theoretical result states that it is possible to obtain equilibrium thermodynamic parameters from processes carried out arbitrarily far from equilibrium. We test Jarzynski's equality by mechanically stretching a single molecule of RNA reversibly and irreversibly between two conformations. Application of this equality to the irreversible work trajectories recovers the DeltaG profile of the stretching process to within k(B)T/2 (half the thermal energy) of its best independent estimate, the mean work of reversible stretching. The implementation and test of Jarzynski's equality provides the first example of its use as a bridge between the statistical mechanics of equilibrium and nonequilibrium systems. This work also extends the thermodynamic analysis of single molecule manipulation data beyond the context of equilibrium experiments.
View details for Web of Science ID 000176054300037
View details for PubMedID 12052949
Single-molecule studies of DNA mechanics
CURRENT OPINION IN STRUCTURAL BIOLOGY
2000; 10 (3): 279-285
Here we use mechanical force to induce the unfolding and refolding of single RNA molecules: a simple RNA hairpin, a molecule containing a three-helix junction, and the P5abc domain of the Tetrahymena thermophila ribozyme. All three molecules (P5abc only in the absence of Mg2+) can be mechanically unfolded at equilibrium, and when kept at constant force within a critical force range, are bi-stable and hop between folded and unfolded states. We determine the force-dependent equilibrium constants for folding/unfolding these single RNA molecules and the positions of their transition states along the reaction coordinate.
View details for Web of Science ID 000168478300053
View details for PubMedID 11326101
The role of RNA pseudoknot stem 1 length in the promotion of efficient-1 ribosomal frameshifting
JOURNAL OF MOLECULAR BIOLOGY
1999; 288 (3): 305-320
During the past decade, physical techniques such as optical tweezers and atomic force microscopy were used to study the mechanical properties of DNA at the single-molecule level. Knowledge of DNA's stretching and twisting properties now permits these single-molecule techniques to be used in the study of biological processes such as DNA replication and transcription.
View details for Web of Science ID 000087667000002
View details for PubMedID 10851197
Evidence for an RNA pseudoknot loop-helix interaction essential for efficient-1 ribosomal frameshifting
JOURNAL OF MOLECULAR BIOLOGY
1999; 288 (3): 321-335
The ribosomal frameshifting signal present in the genomic RNA of the coronavirus infectious bronchitis virus (IBV) contains a classic hairpin-type RNA pseudoknot that is believed to possess coaxially stacked stems of 11 bp (stem 1) and 6 bp (stem 2). We investigated the influence of stem 1 length on the frameshift process by measuring the frameshift efficiency in vitro of a series of IBV-based pseudoknots whose stem 1 length was varied from 4 to 13 bp in single base-pair increments. Efficient frameshifting depended upon the presence of a minimum of 11 bp; pseudoknots with a shorter stem 1 were either non-functional or had reduced frameshift efficiency, despite the fact that a number of them had a stem 1 with a predicted stability equal to or greater than that of the wild-type IBV pseudoknot. An upper limit for stem 1 length was not determined, but pseudoknots containing a 12 or 13 bp stem 1 were fully functional. Structure probing analysis was carried out on RNAs containing either a ten or 11 bp stem 1; these experiments confirmed that both RNAs formed pseudoknots and appeared to be indistinguishable in conformation. Thus the difference in frameshifting efficiency seen with the two structures was not simply due to an inability of the 10 bp stem 1 construct to fold into a pseudoknot. In an attempt to identify other parameters which could account for the poor functionality of the shorter stem 1-containing pseudoknots, we investigated, in the context of the 10 bp stem 1 construct, the influence on frameshifting of altering the slippery sequence-pseudoknot spacing distance, loop 2 length, and the number of G residues at the bottom of the 5'-arm of stem 1. For each parameter, it was possible to find a condition where a modest stimulation of frameshifting was observable (about twofold, from seven to a maximal 17 %), but we were unable to find a situation where frameshifting approached the levels seen with 11 bp stem 1 constructs (48-57 %). Furthermore, in the next smaller construct (9 bp stem 1), changing the bottom four base-pairs to G.C (the optimal base composition) only stimulated frameshifting from 3 to 6 %, an efficiency about tenfold lower than seen with the 11 bp construct. Thus stem 1 length is a major factor in determining the functionality of this class of pseudoknot and this has implications for models of the frameshift process.
View details for Web of Science ID 000080204600002
View details for PubMedID 10329144
RNA pseudoknots are structural elements that participate in a variety of biological processes. At -1 ribosomal frameshifting sites, several types of pseudoknot have been identified which differ in their organisation and functionality. The pseudoknot found in infectious bronchitis virus (IBV) is typical of those that possess a long stem 1 of 11-12 bp and a long loop 2 (30-164 nt). A second group of pseudoknots are distinguishable that contain stems of only 5 to 7 bp and shorter loops. The NMR structure of one such pseudoknot, that of mouse mammary tumor virus (MMTV), has revealed that it is kinked at the stem 1-stem 2 junction, and that this kinked conformation is essential for efficient frameshifting. We recently investigated the effect on frameshifting of modulating stem 1 length and stability in IBV-based pseudoknots, and found that a stem 1 with at least 11 bp was needed for efficient frameshifting. Here, we describe the sequence manipulations that are necessary to bypass the requirement for an 11 bp stem 1 and to convert a short non-functional IBV-derived pseudoknot into a highly efficient, kinked frameshifter pseudoknot. Simple insertion of an adenine residue at the stem 1-stem 2 junction (an essential feature of a kinked pseudoknot) was not sufficient to create a functional pseudoknot. An additional change was needed: efficient frameshifting was recovered only when the last nucleotide of loop 2 was changed from a G to an A. The requirement for an A at the end of loop 2 is consistent with a loop-helix contact similar to those described in other RNA tertiary structures. A mutational analysis of both partners of the proposed interaction, the loop 2 terminal adenine residue and two G.C pairs near the top of stem 1, revealed that the interaction was essential for efficient frameshifting. The specific requirement for a 3'-terminal A residue was lost when loop 2 was increased from 8 to 14 nt, suggesting that the loop-helix contact may be required only in those pseudoknots with a short loop 2.
View details for Web of Science ID 000080204600003
View details for PubMedID 10329145