Live-cell Single-molecule Imaging Research Articles

Understanding the Pathogenicity of Vibrio Cholerae via Two-Color Live-Cell Super-Resolution Microscopy

Cholera afflicts more than 5 million people annually. Here, we investigate the virulence pathway of this epidemic disease with live-cell single-molecule fluorescence imaging in Vibrio cholerae cells, the bacterium responsible for producing the cholera toxin. In the Gram-negative pathogen V. cholerae, virulence gene expression is under control of an unusual set of membrane proteins. Here, a membrane complex including two activators, ToxR and TcpP, binds the toxT promoter, recruits RNA polymerase, and activates toxT gene expression leading to activation of ToxT-controlled virulence genes. To circumvent the diffraction limit of light, which bounds the resolution of optical microscopy to ∼250 nm, we use single-molecule tracking and super-resolution techniques like Photoactivated Localization Microscopy (PALM) to achieve resolutions more than an order of magnitude better than the diffraction limit. We have created fusions of the membrane-bound transcription activators TcpP and ToxR with orthogonal photo-(re)activatable fluorescent proteins, and in this study, we examine the dynamics and co-localization patterns of single PAmCherry-TcpP and mCitrine-ToxR molecules in the virulence pathway. We also image V. cholerae cells that have knockouts of ToxR and/or TcpP to determine if the protein dynamics change in the absence of the binding partner. This work aims to identify characteristics of TcpP and ToxR motion to understand their regulatory behavior in the transcriptional activation of the gene toxT and subsequent activation of downstream virulence genes, and to establish a model for the formation of the ToxR/TcpP/toxT protein-DNA complex important in early pathogenesis. In addition to elucidating the regulatory pathway of V. cholerae, the impact of this work will be to further provide a general model for outer-membrane-bound transcription control in bacteria and nuclear-membrane-bound transcription in eukaryotic cells.

A Highly Specific Gold Nanoprobe for Live-Cell Single-Molecule Imaging in Confined Environments: Intracellular Tracking and Long-Term Single Integrin Tracking in Adhesion Sites

Single molecule tracking in live cells is the ultimate tool to study subcellular protein dynamics, but it is often limited by the probe size and photostability. Due to these issues, long-term tracking of proteins in confined and crowded environments, such as adhesion sites, synaptic clefts or intracellular spaces, remains challenging. We present a novel optical probe consisting of 5-nm gold nanoparticles functionalized with a small fragment of camelid antibodies that recognize widely used GFPs with a very high affinity (1). These small gold nanoparticles can be detected and tracked using photothermal imaging for arbitrarily long periods of time (1-2). Surface and intracellular GFP-proteins can effectively be labeled even in very crowded environments such as adhesion sites and cytoskeletal structures both in vitro and in live cell cultures. Comparison with performances obtained by superresolution methods such as PALM and STED are presented for single integrin tracking in and out adhesion sites (3). These nanobody-coated gold nanoparticles are single molecule probes with unparalleled capabilities; small size, perfect photostability, high specificity, and versatility afforded by combination with the vast existing library of GFP-tagged proteins.

The N-Terminal Membrane-Spanning Domain of the Escherichia coli DNA Translocase FtsK Hexamerizes at Midcell

ABSTRACTBacterial FtsK plays a key role in coordinating cell division with the late stages of chromosome segregation. The N-terminal membrane-spanning domain of FtsK is required for cell division, whereas the C-terminal domain is a fast double-stranded DNA (dsDNA) translocase that brings the replication termination region of the chromosome to midcell, where it facilitates chromosome unlinking by activating XerCD-dif site-specific recombination. Therefore, FtsK coordinates the late stages of chromosome segregation with cell division. Although the translocase is known to act as a hexamer on DNA, it is unknown when and how hexamers form, as is the number of FtsK molecules in the cell and within the divisome. Using single-molecule live-cell imaging, we show that newborn Escherichia coli cells growing in minimal medium contain ~40 membrane-bound FtsK molecules that are largely monomeric; the numbers increase proportionately with cell growth. After recruitment to the midcell, FtsK is present only as hexamers. Hexamers are observed in all cells and form before any visible sign of cell constriction. An average of 7 FtsK hexamers per cell are present at midcell, with the N-terminal domain being able to hexamerize independently of the translocase. Detergent-solubilized and purified FtsK N-terminal domains readily form hexamers, as determined by in vitro biochemistry, thereby supporting the in vivo data. The hexameric state of the FtsK N-terminal domain at the division site may facilitate assembly of a functional C-terminal DNA translocase on chromosomal DNA.

Molecular viewing of actin polymerizing actions and beyond: Combination analysis of single‐molecule speckle microscopy with modeling, FRAP and s‐FDAP (sequential fluorescence decay after photoactivation)

Live-cell single-molecule imaging is a powerful tool to elucidate the in vivo biochemistry of cytoskeletal proteins. However, it is often somewhat difficult to interpret how a bulk population of the observed molecule might behave as a whole. We review our recent studies in which the combination of image analysis with modeling and bulk kinetics measurements such as FRAP (fluorescence recovery after photobleaching) clarified basic problems in the regulation of actin remodeling pathways.

Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass.

Two- and Three-Dimensional Single-Molecule Super-Resolution Imaging in Live Bacteria Cells

Single-molecule imaging has extended the resolution of fluorescence microscopy down to the nanometer scale. This super-resolution technique is non-invasive, tolerates simple sample preparation, and takes advantage of high-specificity labeling schemes. We will discuss recent studies focused on imaging protein structure and dynamics in live bacterial cells, with attention to the particular challenges that this system presents: the organisms are small, have short cell cycles, live in specific environments, and their organization is relatively poorly understood. We have developed methods to extend the capabilities of single-molecule fluorescence microscopy to study motion and localization in live Caulobacter crescentus, Vibrio cholerae, and Bacteroides thetaiotaomicron.FtsZ is an essential protein that polymerizes at the mid-cell, recruits the division machinery, and may generate constrictive forces necessary for cytokinesis. Based on astigmatism and on the natural dynamics of the protein, we resolve in two and three dimensions the midplane Z-ring formed by FtsZ, in C. crescentus. We further apply live-cell single-molecule imaging to two prokaryotes of biomedical interest, V. cholerae, and the gut symbiont B.thetaiotaomicron. The V. cholerae protein TcpP is a rare example of a membrane-bound transcription factor, and we have investigated the dynamics and localization of TcpP, its binding partner ToxR, and the toxT gene they regulate, in order to elucidate the mechanism of membrane-bound transcription activation. The starch utilization system (Sus) allows B. thetaiotaomicron to catabolize complex carbohydrates, and we have investigated the response of Sus proteins to stimuli with live anaerobic cell single-molecule imaging.

Single-Molecule Imaging of NGF Axonal Transport in Microfluidic Devices

Nerve growth factor (NGF) plays a critical role in neuron survival and functioning. NGF binds and activates membrane receptors TrkA, endocytosed NGF-TrkA complex then recruits various motor proteins to transport along cytoskeletal tracks towards cell body, where downstream signaling processes are regulated. Our recent study revealed that the majority of endosomes contain a single NGF molecule, which makes single-molecule imaging an essential tool for NGF studies. However single-molecule imaging in live cells is often limited by the intrinsic background fluorescence. Here, we efficiently reduced the background by employing a microfluidic culture platform, which can guide the growth of neurons and allow separately controlled microenvironment for cell bodies or axon termini. Single-molecule imaging of Qdot-labeled NGF in live axons shows exclusive retrograde transport of cargoes toward cell body with a stop-and-go pattern. Measurements at various temperatures show that the rate of NGF retrograde transport decreased exponentially over the range of 36-14 degree Celsius. A 10-degree decrease in temperature resulted in a threefold decrease in the rate of NGF retrograde transport. Our successful measurements of NGF transport suggest that the microfluidic device can serve as a unique platform for single-molecule imaging of molecular processes in live neurons.

Cytoskeletal Control of Receptor Diffusion in Membrane Promotes CD36 Function and Signaling

Receptor clustering and organization into membrane microdomains is an essential feature of transmembrane signal transduction. The processes that govern the coalescence of receptors into functional aggregates, however, are poorly understood.CD36 is a clustering-responsive class B scavenger receptor in macrophages, where it binds to multivalent ligands such as oxidized low-density lipoprotein (oxLDL), apoptotic cells and malaria-infected erythrocytes. It is implicated in a wide range of processes, from lipid metabolism to innate immunity to tissue. Biochemical studies suggest that CD36 clustering at the cell surface upon engagement of multivalent ligands triggers signal transduction and receptor-ligand complex internalization. However, it is not known whether CD36 receptors at rest exist as monomers or as oligomers that facilitate the cellular response to ligand exposure, and what factors contribute to CD36 clustering.To address these questions, we combined quantitative live-cell single-molecule imaging and biochemical approaches to study the dynamics, oligomerization and signaling of CD36 in primary human macrophages. We found that unliganded CD36 receptors exist in the membrane as metastable oligomers that prime the cells to respond to ligand exposure. Temporal multi-scale analysis of single receptor trajectories combined with pharmacological perturbation of the cytoskeleton showed that the movement of CD36 in the membrane was controlled by the submembranous actomyosin meshwork and by microtubules. Specifically, a subset of receptors diffused within cytoskeleton-dependent linear channels which promoted receptor oligomerization by allowing free diffusion in one direction while imposing confinement along the perpendicular direction. Perturbation of this organization markedly decreased CD36-mediated signal transduction. These data demonstrate a critical role for the cytoskeleton in controlling CD36 signaling by organizing the diffusion of receptors within regions of the membrane that increase receptor collision and oligomerization frequency.

Single-molecule imaging of NGF axonal transport in microfluidic devices

Nerve growth factor (NGF) signaling begins at the nerve terminal, where it binds and activates membrane receptors and subsequently carries the cell-survival signal to the cell body through the axon. A recent study revealed that the majority of endosomes contain a single NGF molecule, which makes single-molecule imaging an essential tool for NGF studies. Despite being an increasingly popular technique, single-molecule imaging in live cells is often limited by background fluorescence. Here, we employed a microfluidic culture platform to achieve background reduction for single-molecule imaging in live neurons. Microfluidic devices guide the growth of neurons and allow separately controlled microenvironment for cell bodies or axon termini. Designs of microfluidic devices were optimized and a three-compartment device successfully achieved direct observation of axonal transport of single NGF when quantum dot labeled NGF (Qdot-NGF) was applied only to the distal-axon compartment while imaging was carried out exclusively in the cell-body compartment. Qdot-NGF was shown to move exclusively toward the cell body with a characteristic stop-and-go pattern of movements. Measurements at various temperatures show that the rate of NGF retrograde transport decreased exponentially over the range of 36-14 degrees C. A 10 degrees C decrease in temperature resulted in a threefold decrease in the rate of NGF retrograde transport. Our successful measurements of NGF transport suggest that the microfluidic device can serve as a unique platform for single-molecule imaging of molecular processes in neurons.

Intracellular Myosin Motor Protein Motion Using Laser Scanning Confocal Microscopy

We focus on live cell single molecule imaging and tracking with particular focus on measuring single myosin V steps along actin filaments within live cells. The goal of the work is to acquire information that will offer new insights into the mechanism of these motors' processivity. Conventionally, single molecule Myosin studies have been accomplished through total internal reflection fluorescence microscopy (TIRF).1,2 However, TIRF methods have very limited applicability to live cell in vivo studies. As such, we apply confocal laser scanning microcopy (CLSM) to imaging and tracking of Myosin V. CLSM also offers the capability to provide full 3-dimensional tracking of biomolecular motors. Our experiments have confirmed step size results from previous TIRF experiments. In terms of instrumentation, we overcome the nominally slow scanning speed of CLSM by the use of a fiber scanning technique that allows us to scan with image acquisition times of less than 100 ms. We make use of home-made streptavidin-functionalized quantum dots in conjunction with this tracking technique to increase the quantum efficiency and stability of the fluorophores. The QD fabrication and sample preparation techniques will also be presented.1. A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization,” Science 300 (5628), 2061-2065 (2003).2. T. Sakamoto, A. Yildez, P. R. Selvin, and J. R. Sellers, “Step-size is determined by neck length in myosin V,” Biochemistry 44 (49), 16203-16210 (2005).

Next-generation quantum dots

Quantum dots that are small and non-blinking offer new opportunities for dynamic single-molecule imaging in live cells.

Determinants of aquaporin-4 assembly in orthogonal arrays revealed by live-cell single-molecule fluorescence imaging

We investigated the molecular determinants of aquaporin-4 (AQP4) assembly in orthogonal arrays of particles (OAPs) by visualizing fluorescently labeled AQP4 mutants in cell membranes using quantum-dot single-particle tracking and total internal reflection fluorescence microscopy. The full-length ;long' (M1) form of AQP4 diffused freely in membranes and did not form OAPs, whereas the ;short' (M23) form of AQP4 formed OAPs and was nearly immobile. Analysis of AQP4 deletion mutants revealed progressive disruption of OAPs by the addition of three to seven residues at the AQP4-M23 N-terminus, with polyalanines as effective as native AQP4 fragments. OAPs disappeared upon downstream deletions of AQP4-M23, which, from analysis of point mutants, involves N-terminus interactions of residues Val24, Ala25 and Phe26. OAP formation was also prevented by introducing proline residues at sites just downstream from the hydrophobic N-terminus of AQP4-M23. AQP1, an AQP4 homolog that does not form OAPs, was induced to form OAPs upon replacement of its N-terminal domain with that of AQP4-M23. Our results indicate that OAP formation by AQP4-M23 is stabilized by hydrophobic intermolecular interactions involving N-terminus residues, and that absence of OAPs in AQP4-M1 results from non-selective blocking of this interaction by seven residues just upstream from Met23.

G-actin regulates rapid induction of actin nucleation by mDia1 to restore cellular actin polymers

mDia1 belongs to the formin family of proteins that share FH1 and FH2 domains. Although formins play a critical role in the formation of many actin-based cellular structures, the physiological regulation of formin-mediated actin assembly within the cell is still unknown. Here we show that cells possess an acute actin polymer restoration mechanism involving mDia1. By using single-molecule live-cell imaging, we found that several treatments including low-dose G-actin-sequestering drugs and unpolymerizable actin mutants activate mDia1 to initiate fast directional movement. The FH2 region, the core domain for actin nucleation, is sufficient to respond to latrunculin B (LatB) to increase its actin nucleation frequency. Simulation analysis revealed an unexpected paradoxical effect of LatB that leads to a several fold increase in free G-actin along with an increase in total G-actin. These results indicate that in cells, the actin nucleation frequency of mDia1 is enhanced not only by Rho, but also strongly through increased catalytic efficiency of the FH2 domain. Consistently, frequent actin nucleation by mDia1 was found around sites of vigorous actin disassembly. Another major actin nucleator, the Arp2/3 complex, was not affected by the G-actin increase induced by LatB. Taken together, we propose that transient accumulation of G-actin works as a cue to promote mDia1-catalyzed actin nucleation to execute rapid reassembly of actin filaments.

Single-molecule live-cell imaging of clathrin-based endocytosis.

Clathrin-coated vesicles carry traffic from the plasma membrane to endosomes. We report here the first real-time visualization of cargo sorting and endocytosis by clathrin-coated pits in living cells. We have visualized the formation of coats by monitoring the incorporation of fluorescently tagged clathrin or its adaptor AP-2 (adaptor protein 2), and have followed clathrin-mediated uptake of transferrin, single LDL (low-density lipoprotein) and single reovirus particles. The intensity of a cargo-loaded clathrin cluster grows steadily during its lifetime, and the time required to complete assembly is proportional to the size of the cargo particle. These results are consistent with a nucleation-growth mechanism and an approximately constant growth rate. There are no preferred nucleation sites. A proportion of the nucleation events appear to be abortive. Cargo incorporation occurs primarily or exclusively in a newly formed coated pit, and loading appears to commit that pit to finish assembly. Our data led to a model in which coated pits initiate randomly, but collapse with high likelihood unless stabilized, presumably by cargo capture.