Development of ultrafast camera-based single fluorescent-molecule imaging for cell biology.

The spatial resolution of fluorescence microscopy has recently been greatly enhanced. However, improvements in temporal resolution have been limited, despite their importance for examining living cells. Here, we developed an ultrafast camera system that enables the highest time resolutions in single fluorescent-molecule imaging to date, which were photon-limited by fluorophore photophysics: 33 and 100 µs with single-molecule localization precisions of 34 and 20 nm, respectively, for Cy3, the optimal fluorophore we identified. Using theoretical frameworks developed for the analysis of single-molecule trajectories in the plasma membrane (PM), this camera successfully detected fast hop diffusion of membrane molecules in the PM, previously detectable only in the apical PM using less preferable 40-nm gold probes, thus helping to elucidate the principles governing the PM organization and molecular dynamics. Furthermore, as described in the companion paper, this camera allows simultaneous data acquisitions for PALM/dSTORM at as fast as 1 kHz, with 29/19 nm localization precisions in the 640 × 640 pixel view-field.

Ultrafast single-molecule imaging reveals focal adhesion nano-architecture and molecular dynamics.

Using our newly developed ultrafast camera described in the companion paper, we reduced the data acquisition periods required for photoactivation/photoconversion localization microscopy (PALM, using mEos3.2) and direct stochastic reconstruction microscopy (dSTORM, using HMSiR) by a factor of ≈30 compared with standard methods, for much greater view-fields, with localization precisions of 29 and 19 nm, respectively, thus opening up previously inaccessible spatiotemporal scales to cell biology research. Simultaneous two-color PALM-dSTORM and PALM-ultrafast (10 kHz) single fluorescent-molecule imaging-tracking has been realized. They revealed the dynamic nanoorganization of the focal adhesion (FA), leading to the compartmentalized archipelago FA model, consisting of FA-protein islands with broad diversities in size (13-100 nm; mean island diameter ≈30 nm), protein copy numbers, compositions, and stoichiometries, which dot the partitioned fluid membrane (74-nm compartments in the FA vs. 109-nm compartments outside the FA). Integrins are recruited to these islands by hop diffusion. The FA-protein islands form loose ≈320 nm clusters and function as units for recruiting FA proteins.

Asymmetric polar localization dynamics of the serine chemoreceptor protein Tsr in Escherichia coli.

The spatial location of proteins in living cells can be critical for their function. For example, the E. coli chemotaxis machinery is localized to the cell poles. Here we describe the polar localization of the serine chemoreceptor Tsr using a strain synthesizing a fluorescent Tsr-Venus fusion at a low level from a single-copy chromosomal construct. Using photobleaching and imaging during recovery by new synthesis, we observed distinct asymmetry between a bright (old) pole and a dim (new) pole. The old pole was shown to be a more stable cluster and to recover after photobleaching faster, which is consistent with the hypothesis that newly synthesized Tsr proteins are inserted directly at or near the old pole. The new pole was shown to be a less stable cluster and to exchange proteins freely with highly mobile Tsr-Venus proteins diffusing in the membrane. We propose that the new pole arises from molecules escaping from the old pole and diffusing to the new pole where a more stable cluster forms over time. Our localization imaging data support a model in which a nascent new pole forms prior to stable cluster formation.

Confined Mobility of TonB and FepA in Escherichia coli Membranes.

The important process of nutrient uptake in Escherichia coli, in many cases, involves transit of the nutrient through a class of beta-barrel proteins in the outer membrane known as TonB-dependent transporters (TBDTs) and requires interaction with the inner membrane protein TonB. Here we have imaged the mobility of the ferric enterobactin transporter FepA and TonB by tracking them in the membranes of live E. coli with single-molecule resolution at time-scales ranging from milliseconds to seconds. We employed simple simulations to model/analyze the lateral diffusion in the membranes of E.coli, to take into account both the highly curved geometry of the cell and artifactual effects expected due to finite exposure time imaging. We find that both molecules perform confined lateral diffusion in their respective membranes in the absence of ligand with FepA confined to a region [Formula: see text] µm in radius in the outer membrane and TonB confined to a region [Formula: see text] µm in radius in the inner membrane. The diffusion coefficient of these molecules on millisecond time-scales was estimated to be [Formula: see text] µm2/s and [Formula: see text] µm2/s for FepA and TonB, respectively, implying that each molecule is free to diffuse within its domain. Disruption of the inner membrane potential, deletion of ExbB/D from the inner membrane, presence of ligand or antibody to FepA and disruption of the MreB cytoskeleton was all found to further restrict the mobility of both molecules. Results are analyzed in terms of changes in confinement size and interactions between the two proteins.

Single Molecule Studies of the Diffusion of Band 3 in Sickle Cell Erythrocytes.

Sickle cell disease (SCD) is caused by an inherited mutation in hemoglobin that leads to sickle hemoglobin (HbS) polymerization and premature HbS denaturation. Previous publications have shown that HbS denaturation is followed by binding of denatured HbS (a.k.a. hemichromes) to band 3, the consequent clustering of band 3 in the plane of the erythrocyte membrane that in turn promotes binding of autologous antibodies to the clustered band 3, and removal of the antibody-coated erythrocytes from circulation. Although each step of the above process has been individually demonstrated, the fraction of band 3 that is altered by association with denatured HbS has never been determined. For this purpose, we evaluated the lateral diffusion of band 3 in normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and hemoglobin SC erythrocytes (HbSC) in order to estimate the fraction of band 3 that was diffusing more slowly due to hemichrome-induced clustering. We labeled fewer than ten band 3 molecules per intact erythrocyte with a quantum dot to avoid perturbing membrane structure and we then monitored band 3 lateral diffusion by single particle tracking. We report here that the size of the slowly diffusing population of band 3 increases in the sequence: normal cells

Diffusion of glycophorin A in human erythrocytes.

Several lines of evidence suggest that glycophorin A (GPA) interacts with band 3 in human erythrocyte membranes including: i) the existence of an epitope shared between band 3 and GPA in the Wright b blood group antigen, ii) the fact that antibodies to GPA inhibit the diffusion of band 3, iii) the observation that expression of GPA facilitates trafficking of band 3 from the endoplasmic reticulum to the plasma membrane, and iv) the observation that GPA is diminished in band 3 null erythrocytes. Surprisingly, there is also evidence that GPA does not interact with band 3, including data showing that: i) band 3 diffusion increases upon erythrocyte deoxygenation whereas GPA diffusion does not, ii) band 3 diffusion is greatly restricted in erythrocytes containing the Southeast Asian Ovalocytosis mutation whereas GPA diffusion is not, and iii) most anti-GPA or anti-band 3 antibodies do not co-immunoprecipitate both proteins. To try to resolve these apparently conflicting observations, we have selectively labeled band 3 and GPA with fluorescent quantum dots in intact erythrocytes and followed their diffusion by single particle tracking. We report here that band 3 and GPA display somewhat similar macroscopic and microscopic diffusion coefficients in unmodified cells, however perturbations of band 3 diffusion do not cause perturbations of GPA diffusion. Taken together the collective data to date suggest that while weak interactions between GPA and band 3 undoubtedly exist, GPA and band 3 must have separate interactions in the membrane that control their lateral mobility.

Dynamics of the serine chemoreceptor in the Escherichia coli inner membrane: a high-speed single-molecule tracking study.

We investigated the mobility of the polar localized serine chemoreceptor, Tsr, labeled by the fluorescent protein Venus in the inner membrane of live Escherichia coli cells at observation rates up to 1000 Hz. A fraction (7%) of all Tsr molecules shows free diffusion over the entire cell surface with an average diffusion coefficient of 0.40 ± 0.01 µm(2) s(-1). The remaining molecules were found to be ultimately confined in compartments of size 290 ± 15 nm and showed restricted diffusion at an inner barrier found at 170 ± 10 nm. At the shortest length-scales (<170 nm), all Tsr molecules diffuse equally. Disruption of the cytoskeleton and rounding of the cells resulted in an increase in the mobile fraction of Tsr molecules and a fragmenting of the previously polar cluster of Tsr consistent with a curvature-based mechanism of Tsr cluster maintenance.

Biocompatible fluorescent silicon nanocrystals for single-molecule tracking and fluorescence imaging.

Fluorescence microscopy is used extensively in cell-biological and biomedical research, but it is often plagued by three major problems with the presently available fluorescent probes: photobleaching, blinking, and large size. We have addressed these problems, with special attention to single-molecule imaging, by developing biocompatible, red-emitting silicon nanocrystals (SiNCs) with a 4.1-nm hydrodynamic diameter. Methods for producing SiNCs by simple chemical etching, for hydrophilically coating them, and for conjugating them to biomolecules precisely at a 1:1 ratio have been developed. Single SiNCs neither blinked nor photobleached during a 300-min overall period observed at video rate. Single receptor molecules in the plasma membrane of living cells (using transferrin receptor) were imaged for ≥10 times longer than with other probes, making it possible for the first time to observe the internalization process of receptor molecules at the single-molecule level. Spatial variations of molecular diffusivity in the scale of 1-2 µm, i.e., a higher level of domain mosaicism in the plasma membrane, were revealed.

Energy-dependent motion of TonB in the Gram-negative bacterial inner membrane.

Gram-negative bacteria acquire iron with TonB-dependent uptake systems. The TonB-ExbBD inner membrane complex is hypothesized to transfer energy to outer membrane (OM) iron transporters. Fluorescence microscopic characterization of green fluorescent protein (GFP)-TonB hybrid proteins revealed an unexpected, restricted localization of TonB in the cell envelope. Fluorescence polarization measurements demonstrated motion of TonB in living cells, which likely was rotation. By determining the anisotropy of GFP-TonB in the absence and presence of inhibitors, we saw the dependence of its motion on electrochemical force and on the actions of ExbBD. We observed higher anisotropy for GFP-TonB in energy-depleted cells and lower values in bacteria lacking ExbBD. However, the metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings demonstrate that TonB undergoes energized motion in the bacterial cell envelope and that ExbBD couples this activity to the electrochemical gradient. The results portray TonB as an energized entity in a regular array underlying the OM bilayer, which promotes metal uptake through OM transporters by a rotational mechanism.

Single-molecule imaging in live bacteria cells.

Bacteria, such as Escherichia coli and Caulobacter crescentus, are the most studied and perhaps best-understood organisms in biology. The advances in understanding of living systems gained from these organisms are immense. Application of single-molecule techniques in bacteria have presented unique difficulties owing to their small size and highly curved form. The aim of this review is to show advances made in single-molecule imaging in bacteria over the past 10 years, and to look to the future where the combination of implementing such high-precision techniques in well-characterized and controllable model systems such as E. coli could lead to a greater understanding of fundamental biological questions inaccessible through classic ensemble methods.

Single-molecule study of molecular mobility in the cytoplasm of Escherichia coli.

The cytoplasm of bacterial cells is filled with individual molecules and molecular complexes that rely on diffusion to bring them together for interaction. The mobility of molecules in the cytoplasm has been characterized by several techniques mainly using fluorescent probes and ensemble methods. In order to probe the microenvrionment inside the cytoplasm as viewed by an individual molecule, we have studied single green fluorescent proteins (GFPs) diffusing in the cytoplasm of Escherichia coli cells at observation at rates ranging from 60 to 1000 Hz. Over long times the diffusion shows confinement due to the geometry of the cells themselves. A simulation in model cells using the actual distribution of cell sizes found in the experiments describes accurately the experimental results as well as reveals a short time diffusion coefficient that agrees well with that determined by ensemble methods. Higher short time diffusion coefficients can be obtained by filling the simulated cell with small spheres modeling cytoplasmic molecules and, depending on the density of particles included in the modeled cytoplasm, can approach the diffusion coefficient of GFPs found in water. Thus, single-molecule tracking combined with analysis using simple simulation of Brownian motion is able to reveal the main contributors to the GFP mobility in the cytoplasm of E. coli.

Analysis of the mobilities of band 3 populations associated with ankyrin protein and junctional complexes in intact murine erythrocytes.

Current models of the erythrocyte membrane depict three populations of band 3: (i) a population tethered to spectrin via ankyrin, (ii) a fraction attached to the spectrin-actin junctional complex via adducin, and (iii) a freely diffusing population. Because many studies of band 3 diffusion also distinguish three populations of the polypeptide, it has been speculated that the three populations envisioned in membrane models correspond to the three fractions observed in diffusion analyses. To test this hypothesis, we characterized band 3 diffusion by single-particle tracking in wild-type and ankyrin- and adducin-deficient erythrocytes. We report that ∼40% of total band 3 in wild-type murine erythrocytes is attached to ankyrin, whereas ∼33% is immobilized by adducin, and ∼27% is not attached to any cytoskeletal anchor. More detailed analyses reveal that mobilities of individual ankyrin- and adducin-tethered band 3 molecules are heterogeneous, varying by nearly 2 orders of magnitude and that there is considerable overlap in diffusion coefficients for adducin and ankyrin-tethered populations. Taken together, the data suggest that although the ankyrin- and adducin-immobilized band 3 can be monitored separately, significant heterogeneity still exists within each population, suggesting that structural and compositional properties likely vary considerably within each band 3 complex.

Hierarchical mesoscale domain organization of the plasma membrane.

Based on recent single-molecule imaging results in the living cell plasma membrane, we propose a hierarchical architecture of three-tiered mesoscale (2-300nm) domains to represent the fundamental functional organization of the plasma membrane: (i) membrane compartments of 40-300nm in diameter due to the partitioning of the entire plasma membrane by the actin-based membrane skeleton 'fence' and transmembrane protein 'pickets' anchored to the fence; (ii) raft domains (2-20nm); and (iii) dimers/oligomers and greater complexes of membrane-associated proteins (3-10nm). The basic molecular interactions required for the signal transduction function of the plasma membrane can be fundamentally understood and conveniently summarized as the cooperative actions of these mesoscale domains, where thermal fluctuations/movements of molecules and weak cooperativity play crucial roles.

Regulation of membrane-cytoskeletal interactions by tyrosine phosphorylation of erythrocyte band 3.

The cytoplasmic domain of band 3 serves as a center of erythrocyte membrane organization and constitutes the major substrate of erythrocyte tyrosine kinases. Tyrosine phosphorylation of band 3 is induced by several physiologic stimuli, including malaria parasite invasion, cell shrinkage, normal cell aging, and oxidant stress (thalassemias, sickle cell disease, glucose-6-phosphate dehydrogenase deficiency, etc). In an effort to characterize the biologic sequelae of band 3 tyrosine phosphorylation, we looked for changes in the polypeptide's function that accompany its phosphorylation. We report that tyrosine phosphorylation promotes dissociation of band 3 from the spectrin-actin skeleton as evidenced by: (1) a decrease in ankyrin affinity in direct binding studies, (2) an increase in detergent extractability of band 3 from ghosts, (3) a rise in band 3 cross-linkability by bis-sulfosuccinimidyl-suberate, (4) significant changes in erythrocyte morphology, and (5) elevation of the rate of band 3 diffusion in intact cells. Because release of band 3 from its ankyrin and adducin linkages to the cytoskeleton can facilitate changes in multiple membrane properties, tyrosine phosphorylation of band 3 is argued to enable adaptive changes in erythrocyte biology that permit the cell to respond to the above stresses.

Mobility of BtuB and OmpF in the Escherichia coli outer membrane: implications for dynamic formation of a translocon complex.

Diffusion of two Escherichia coli outer membrane proteins-the cobalamin (vitamin B12) receptor (BtuB) and the OmpF porin, which are implicated in the cellular import pathways of colicins and phages-was measured in vivo. The lateral mobility of these proteins is relevant to the mechanism of formation of the translocon for cellular import of colicins such as the rRNase colicin E3. The diffusion coefficient (D) of BtuB, the primary colicin receptor, complexed to fluorescent antibody or colicin, is 0.05±0.01 µm2/s and 0.10±0.02 µm2/s, respectively, over a timescale of 25-150 ms. Mutagenesis of the BtuB TonB box, which eliminates or significantly weakens the interaction between BtuB and the TonB energy-transducing protein that is anchored in the cytoplasmic membrane, resulted in a fivefold larger value of D, 0.27±0.06 µm2/s for antibody-labeled BtuB, indicating a cytoskeletal-like interaction of TonB with BtuB. OmpF has a diffusion coefficient of 0.006±0.002 µm2/s, ∼10-fold smaller than that of BtuB, and is restricted within a domain of diameter 100 nm, showing it to be relatively immobile compared to BtuB. Thus, formation of the outer membrane translocon for cellular import of the nuclease colicins is a demonstrably dynamic process, because it depends on lateral diffusion of BtuB and collisional interaction with relatively immobile OmpF.

Analysis of the kinetics of band 3 diffusion in human erythroblasts during assembly of the erythrocyte membrane skeleton.

During definitive erythropoiesis, erythroid precursors undergo differentiation through multiple nucleated states to an enucleated reticulocyte, which loses its residual RNA/organelles to become a mature erythrocyte. Over the course of these transformations, continuous changes in membrane proteins occur, including shifts in protein abundance, rates of expression, isoform prominence, states of phosphorylation, and stability. In an effort to understand when assembly of membrane proteins into an architecture characteristic of the mature erythrocyte occurs, we quantitated the lateral diffusion of the most abundant membrane protein, band 3 (AE1), during each stage of erythropoiesis using single particle tracking. Analysis of the lateral trajectories of individual band 3 molecules revealed a gradual reduction in mobility of the anion transporter as erythroblasts differentiated. Evidence for this progressive immobilization included a gradual decline in diffusion coefficients as determined at a video acquisition rate of 120 frames/s and a decrease in the percentage of compartment sizes >100 nm. Because complete acquisition of the properties of band 3 seen in mature erythrocytes is not observed until circulating erythrocytes are formed, we suggest that membrane maturation involves a gradual and cooperative assembly process that is not triggered by the synthesis of any single protein.

Neuronal elasticity as measured by atomic force microscopy.

A cell's form and function is determined to a great extent by its cellular membrane and the underlying cytoskeleton. Understanding changes in the cellular membrane and cytoskeleton can provide insight into aging and disease of the cell. The atomic force microscope (AFM) allows unparalled resolution for the imaging of these cellular components and the ability to probe their mechanical properties. This report describes our progress toward the use of AFM as a tool in neuroscience applications. Elasticity measurements are reported on living chick embryo dorsal root ganglion and sympathetic neurons in vitro. The neuronal cellular body and growth cones regions are examined for variations in cellular maturity. In addition, cellular changes due to exposure to various environmental conditions and neurotoxins are investigated. This report includes data obtained on different AFM systems, using various AFM techniques and thus also provides knowledge of AFM instruments and methodology.

Imaging of the diffusion of single band 3 molecules on normal and mutant erythrocytes.

Membrane-spanning proteins may interact with a variety of other integral and peripheral membrane proteins via a diversity of protein-protein interactions. Not surprisingly, defects or mutations in any one of these interacting components can impact the physical and biological properties on the entire complex. Here we use quantum dots to image the diffusion of individual band 3 molecules in the plasma membranes of intact human erythrocytes from healthy volunteers and patients with defects in one of their membrane components, leading to well-known red cell pathologies (hereditary spherocytosis, hereditary elliptocytosis, hereditary hydrocytosis, Southeast Asian ovalocytosis, and hereditary pyropoikilocytosis). After characterizing the motile properties of the major subpopulations of band 3 in intact normal erythrocytes, we demonstrate that the properties of these subpopulations of band 3 change significantly in diseased cells, as evidenced by changes in the microscopic and macroscopic diffusion coefficients of band 3 and in the compartment sizes in which the different band 3 populations can diffuse. Because the above membrane abnormalities largely arise from defects in other membrane components (eg, spectrin, ankyrin), these data suggest that single particle tracking of band 3 might constitute a useful tool for characterizing the general structural integrity of the human erythrocyte membrane.

Single molecule studies of molecular diffusion in cellular membranes: determining membrane structure.

Since the advent of single particle/molecule microscopies, researchers have applied these techniques to understanding the fluid membranes of cells. By observing diffusion of membrane proteins and lipids in live cell membranes of eukaryotic cells, it has been found that membranes contain a mosaic of fluid compartments. Such structure may be instrumental in understanding key characteristics of the membrane. Recent single molecule observations on prokaryotic cell membranes will also be discussed.

Dynamics of neutrophil membrane compliance and microstructure probed with a micropipet-based piconewton force transducer.

A novel biointerface probe was implemented to study the deformability of the neutrophil membrane and cortical cytoskeleton. Piconewton scale forces are applied to the cell using an ultrasensitive and tunable force transducer comprised of an avidin-coated microsphere attached to a biotinylated and swollen red blood cell. Deformations of freshly isolated human neutrophils were observed on the stage of an inverted phase contrast microscope. Force versus probe indentation curves over a cycle of contact, indentation, and retraction revealed three distinct material responses. Small probe deformations (approximately 500 nm) tested over a range of rates (e.g. 100-500 nm/s) revealed predominantly an elastic response. An initial low-slope region in the force-indentation curves (approximately 0.005 pN/nm), typically extending 0.5-1.0 microm from the cell surface was interpreted as probe contact with microvilli extensions. Further deformation yielded a slope of 0.054+/-0.006 pN/nm, indicative of a stiffer cortical membrane. Disrupting cytoskeletal actin organization by pretreatment with cytochalasin D, reduced the slope by 40% to 0.033+/-0.007 pN/nm and introduced hysteresis in the recovery phase. Modeling the neutrophil as a liquid drop with constant surface tension yielded values of cortical tension of 0.035 pN/nm for resting and 0.02 pN/nm for cytochalasin-treated neutrophils. These data demonstrate the utility of the biointerface probe for measuring local surface compliance and microstructure of living cells.