Investigating intracellular dynamics of FtsZ cytoskeleton with photoactivation single-molecule tracking

Using photoactivatable fluorescent protein as an intracellular protein label for single-molecule tracking offers several advantages over the traditional methods. Here we demonstrate the technique of photoactivation single-molecule tracking by investigating the mobility dynamics of intracellular FtsZ protein molecules in live Escherichia coli cells. FtsZ is a prokaryotic cytoskeleton protein (a homolog of tubulin) and plays important roles in cytokinesis. We demonstrate two heterogeneous subpopulations of FtsZ molecules with distinct diffusional dynamics. The FtsZ molecules forming the Z-rings near the center of the cell were mostly stationary, consistent with the assumption that they are within polymeric filamentous structures. The rest of the FtsZ molecules, on the other hand, undergo Brownian motion spanning the whole cell length. Surprisingly, the diffusion of FtsZ is spatially restricted to helical-shaped regions, implying an energy barrier for free diffusion. Consistently, the measured mean-square displacements of FtsZ showed anomalous diffusion characteristics. These results demonstrated the feasibility and advantages of photoactivation single-molecule tracking, and suggested new levels of complexity in the prokaryotic membrane organization.     

Measurement of Mesoscale Conformational Dynamics of Freely Diffusing Molecules with Tracking FCS

Few techniques are suited to probe the structure and dynamics of molecular complexes at the mesoscale level ($～$100–1000 nm). We have developed a single-molecule technique that uses tracking fluorescence correlation spectroscopy (tFCS) to probe the conformation and dynamics of mesoscale molecular assemblies. tFCS measures the distance fluctuations between two fluorescently labeled sites within an untethered, freely diffusing biomolecule. To achieve subdiffraction spatial resolution, we developed a feedback scheme that allows us to maintain the molecule at an optimal position within the laser intensity gradient for fluorescence correlation spectroscopy. We characterized tFCS spatial sensitivity by measuring the Brownian end-to-end dynamics of DNA molecules as short as 1000 bp. We demonstrate that tFCS detects changes in the compaction of reconstituted nucleosome arrays and can assay transient protein-mediated interactions between distant sites in an individual DNA molecule. Our measurements highlight the applicability of tFCS to a wide variety of biochemical processes involving mesoscale conformational dynamics.     

Identification of Binding Sites for Efflux Pump Inhibitors of the AcrAB-TolC Component AcrA

The overexpression of multidrug efflux pumps is an important mechanism of clinical resistance in Gram-negative bacteria. Recently, four small molecules were discovered that inhibit efflux in Escherichia coli and interact with the AcrAB-TolC efflux pump component AcrA. However, the binding site(s) for these molecules was not determined. Here, we combine ensemble docking and molecular dynamics simulations with tryptophan fluorescence spectroscopy, site-directed mutagenesis, and antibiotic susceptibility assays to probe binding sites and effects of binding of these molecules. We conclude that clorobiocin and SLU-258 likely bind at a site located between the lipoyl and β-barrel domains of AcrA.     

Four-Dimensional Spatial Nanometry of Single Particles in Living Cells Using Polarized Quantum Rods

Single particle tracking is widely used to study protein movement with high spatiotemporal resolution both in vitro and in cells. Quantum dots, which are semiconductor nanoparticles, have recently been employed in single particle tracking because of their intense and stable fluorescence. Although single particles inside cells have been tracked in three spatial dimensions (X, Y, Z), measurement of the angular orientation of a molecule being tracked would significantly enhance our understanding of the molecule’s function. In this study, we synthesized highly polarized, rod-shaped quantum dots (Qrods) and developed a coating method that optimizes the Qrods for biological imaging. We describe a Qrod-based single particle tracking technique that blends optical nanometry with nanomaterial science to simultaneously measure the three-dimensional and angular movements of molecules. Using Qrods, we spatially tracked a membrane receptor in living cells in four dimensions with precision close to the single-digit range in nanometers and degrees.     

Four-Dimensional Spatial Nanometry of Single Particles in Living Cells Using Polarized Quantum Rods

Single particle tracking is widely used to study protein movement with high spatiotemporal resolution both in vitro and in cells. Quantum dots, which are semiconductor nanoparticles, have recently been employed in single particle tracking because of their intense and stable fluorescence. Although single particles inside cells have been tracked in three spatial dimensions (X, Y, Z), measurement of the angular orientation of a molecule being tracked would significantly enhance our understanding of the molecule’s function. In this study, we synthesized highly polarized, rod-shaped quantum dots (Qrods) and developed a coating method that optimizes the Qrods for biological imaging. We describe a Qrod-based single particle tracking technique that blends optical nanometry with nanomaterial science to simultaneously measure the three-dimensional and angular movements of molecules. Using Qrods, we spatially tracked a membrane receptor in living cells in four dimensions with precision close to the single-digit range in nanometers and degrees.     

Visualizing the Disassembly of S. cerevisiae Rad51 Nucleoprotein Filaments

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into elongated nucleoprotein filaments on DNA. We have used total internal reflection fluorescence microscopy and a DNA curtain assay to investigate the dynamics of individual Saccharomyces cerevisiae Rad51 nucleoprotein filaments. For these experiments the DNA molecules were end-labeled with single fluorescent semiconducting nanocrystals. The assembly and disassembly of the Rad51 nucleoprotein filaments were visualized by tracking the location of the labeled DNA end in real time. Using this approach, we have analyzed yeast Rad51 under a variety of different reaction conditions to assess parameters that impact the stability of the nucleoprotein filament. We show that Rad51 readily dissociates from DNA in the presence of ADP or in the absence of nucleotide cofactor, but that free ATP in solution confers a fivefold increase in the stability of the nucleoprotein filaments. We also probe how protein dissociation is coupled to ATP binding and hydrolysis by examining the effects of ATP concentration, and by the use of the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido) triphosphate and ATPase active-site mutants. Finally, we demonstrate that the Rad51 gain-of-function mutant I345T dissociates from DNA with kinetics nearly identical to that of wild-type Rad51, but assembles 30% more rapidly. Together, these results provide a framework for studying the biochemical behaviors of S. cerevisiae Rad51 nucleoprotein filaments at the single-molecule level.     

Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking

Visualization of specific molecules and their interactions in real time and space is essential to delineate how cellular dynamics and the signaling circuit are orchestrated. Spatial regulation of conformational dynamics and structural plasticity of protein interactions is required to rewire signaling circuitry in response to extracellular cues. We introduce a method for optically imaging intracellular protein interactions at nanometer spatial resolution in live cells, using photoactivatable complementary fluorescent (PACF) proteins. Subsets of complementary fluorescent protein molecules were activated, localized, and then bleached; this was followed by the assembly of superresolution images from aggregate position of sum interactive molecules. Using PACF, we obtained precise localization of dynamic microtubule plus-end hub protein EB1 dimers and their distinct distributions at the leading edges and in the cell bodies of migrating cells. We further delineated the structure–function relationship of EB1 by generating EB1-PACF dimers (EB1^wt:EB1^wt, EB1^wt:EB1^mt, and EB1^mt:EB1^mt) and imaging their precise localizations in culture cells. Surprisingly, our analyses revealed critical role of a previously uncharacterized EB1 linker region in tracking microtubule plus ends in live cells. Thus PACF provides a unique approach to delineating spatial dynamics of homo- or heterodimerized proteins at the nanometer scale and establishes a platform to report the precise regulation of protein interactions in space and time in live cells.     

Femtosecond Hydration Map of Intrinsically Disordered α-Synuclein

Protein hydration water plays a fundamentally important role in protein folding, binding, assembly, and function. Little is known about the hydration water in intrinsically disordered proteins that challenge the conventional sequence-structure-function paradigm. Here, by combining experiments and simulations, we show the existence of dynamical heterogeneity of hydration water in an intrinsically disordered presynaptic protein, namely α-synuclein, implicated in Parkinson’s disease. We took advantage of nonoccurrence of cysteine in the sequence and incorporated a number of cysteine residues at the N-terminal segment, the central amyloidogenic nonamyloid-β component (NAC) domain, and the C-terminal end of α-synuclein. We then labeled these cysteine variants using environment-sensitive thiol-active fluorophore and monitored the solvation dynamics using femtosecond time-resolved fluorescence. The site-specific femtosecond time-resolved experiments allowed us to construct the hydration map of α-synuclein. Our results show the presence of three dynamically distinct types of water: bulk, hydration, and confined water. The amyloidogenic NAC domain contains dynamically restrained water molecules that are strikingly different from the water molecules present in the other two domains. Atomistic molecular dynamics simulations revealed longer residence times for water molecules near the NAC domain and supported our experimental observations. Additionally, our simulations allowed us to decipher the molecular origin of the dynamical heterogeneity of water in α-synuclein. These simulations captured the quasi-bound water molecules within the NAC domain originating from a complex interplay between the local chain compaction and the sequence composition. Our findings from this synergistic experimental simulation approach suggest longer trapping of interfacial water molecules near the amyloidogenic hotspot that triggers the pathological conversion into amyloids via chain sequestration, chain desolvation, and entropic liberation of ordered water molecules.     

Light-up properties of complexes between thiazole orange-small molecule conjugates and aptamers

The full understanding of dynamics of cellular processes hinges on the development of efficient and non-invasive labels for intracellular RNA species. Light-up aptamers binding fluorogenic ligands show promise as specific labels for RNA species containing those aptamers. Herein, we took advantage of existing, non-light-up aptamers against small molecules and demonstrated a new class of light-up probes in vitro. We synthesized two conjugates of thiazole orange dye to small molecules (GMP and AMP) and characterized in vitro their interactions with corresponding RNA aptamers. The conjugates preserved specific binding to aptamers while showing several 100-fold increase in fluorescence of the dye (the ‘light-up’ property). In the presence of free small molecules, conjugates can be displaced from aptamers serving also as fluorescent sensors. Our in vitro results provide the proof-of-concept that the small-molecule conjugates with light-up properties can serve as a general approach to label RNA sequences containing aptamers.     

Plasmid segregation: spatial awareness at the molecular level

In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel insight into the workings of the par system from Escherichia coli plasmid R1. Despite its relative simplicity, the plasmid partition spindle shares characteristics with the mitotic machinery of eukaryotic cells.     

Ab Initio Derivation of the FRET Equations Resolves Old Puzzles and Suggests Measurement Strategies

Quantitative imaging methods based on Förster resonance energy transfer (FRET) rely on the determination of an apparent FRET efficiency (E_app), as well as donor and acceptor concentrations, to uncover the identity and relative abundance of the supramolecular (or quaternary) structures of associating macromolecules. Theoretical work has provided “structure-based” relationships between E_app distributions and the quaternary structure models that underlie them. By contrast, the body of work that predicates the “signal-based” dependence of E_app on directly measurable quantities (i.e., fluorescence emission of donors and acceptors) relies largely on plausibility arguments, one of which is the seemingly obvious assumption that the fraction of fluorescent molecules in the ground state pretty nearly equals the total concentration of molecules. In this work, we use the kinetic models of fluorescence in the presence and absence of FRET to rigorously derive useful relationships between E_app and measurable fluorescence signals. Analysis of these relationships reveals a few anticipated results and some unexpected explanations for known experimental FRET puzzles, and it provides theoretical foundations for optimizing measurement strategies.     

Balbiani ring mRNPs diffuse through and bind to clusters of large intranuclear molecular structures

A detailed conception of intranuclear messenger ribonucleoprotein particle (mRNP) dynamics is required for the understanding of mRNP processing and gene expression outcome. We used complementary state-of-the-art fluorescence techniques to quantify native mRNP mobility at the single particle level in living salivary gland cell nuclei. Molecular beacons and fluorescent oligonucleotides were used to specifically label BR2.1 mRNPs by an in vivo fluorescence in situ hybridization approach. We characterized two major mobility components of the BR2.1 mRNPs. These components with diffusion coefficients of 0.3 ± 0.02 μm²/s and 0.73 ± 0.03 μm²/s were observed independently of the staining method and measurement technique used. The mobility analysis of inert tracer molecules revealed that the gland cell nuclei contain large molecular nonchromatin structures, which hinder the mobility of large molecules and particles. The mRNPs are not only hindered by these mobility barriers, but in addition also interact presumably with these structures, what further reduces their mobility and effectively leads to the occurrence of the two diffusion coefficients. In addition, we provide evidence that the remarkably high mobility of the large, 50 nm-sized BR2.1 mRNPs was due to the absence of retarding chromatin.     

Tracking of time-varying genomic regulatory networks with a LASSO-Kalman smoother

It is widely accepted that cellular requirements and environmental conditions dictate the architecture of genetic regulatory networks. Nonetheless, the status quo in regulatory network modeling and analysis assumes an invariant network topology over time. In this paper, we refocus on a dynamic perspective of genetic networks, one that can uncover substantial topological changes in network structure during biological processes such as developmental growth. We propose a novel outlook on the inference of time-varying genetic networks, from a limited number of noisy observations, by formulating the network estimation as a target tracking problem. We overcome the limited number of observations (small n large p problem) by performing tracking in a compressed domain. Assuming linear dynamics, we derive the LASSO-Kalman smoother, which recursively computes the minimum mean-square sparse estimate of the network connectivity at each time point. The LASSO operator, motivated by the sparsity of the genetic regulatory networks, allows simultaneous signal recovery and compression, thereby reducing the amount of required observations. The smoothing improves the estimation by incorporating all observations. We track the time-varying networks during the life cycle of the Drosophila melanogaster. The recovered networks show that few genes are permanent, whereas most are transient, acting only during specific developmental phases of the organism.     

Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment

The work presented here describes a new and simple method based on site-directed fluorescence labeling using the BADAN label that permits the examination of protein-lipid interactions in great detail. We applied this technique to a membrane-embedded, mainly α-helical reference protein, the M13 major coat protein. Using a high-throughput approach, 40 site-specific cysteine mutants were prepared of the 50-residues long protein. The steady-state fluorescence spectra were analyzed using a three-component spectral model that enabled the separation of Stokes shift contributions from water and internal label dynamics, and protein topology. We found that most of the fluorescence originated from BADAN labels that were hydrogen-bonded to water molecules even within the hydrophobic core of the membrane. Our spectral decomposition method revealed the embedment and topology of the labeled protein in the membrane bilayer under various conditions of headgroup charge and lipid chain length, as well as key characteristics of the membrane such as hydration level and local polarity, provided by the local dielectric constant.     

Photodynamics of Red Fluorescent Proteins Studied by Fluorescence Correlation Spectroscopy

Red fluorescent proteins are important tools in fluorescence-based life science research. Recently, we have introduced eqFP611, a red fluorescent protein with advantageous properties from the sea anemone Entacmaea quadricolor. Here, we have studied the submillisecond light-driven intramolecular dynamics between bright and dark states of eqFP611 and, for comparison, drFP583 (DsRed) by using fluorescence correlation spectroscopy on protein solutions. A three-state model with one dark and two fluorescent states describes the power-dependence of the flickering dynamics of both proteins at different excitation wavelengths. It involves two light-driven conformational transitions. We have also studied the photodynamics of individual (monomeric) eqFP611 molecules immobilized on surfaces. The flickering rates and dark state fractions of eqFP611 bound to polyethylene glycol-covered glass surfaces were identical to those measured in solution, showing that the bound FPs behaved identically. A second, much slower flickering process was observed on the 10-ms timescale. Deposition of eqFP611 molecules on bare glass surfaces yielded bright fluorescence without any detectable flickering and a >10-fold decreased photobleaching yield. These observations underscore the intimate connection between protein motions and photophysical processes in fluorescent proteins.     

Theory of Picosecond-Laser-Induced Fluorescence from Highly Excited Complexes with Small Numbers of Chromophores

The problem of singlet excitation kinetics and dynamics, especially at high excitation intensities, among a small number of chromophores of a given system has been addressed. A specific scheme for the kinetics is suggested and applied to CPII, a small chlorophyll (Chl)a/b antenna complex the fluorescence lifetime of which has been reported to be independent of excitation intensity over a wide intensity range of picosecond pulses. We have modeled the kinetics from the point of view that Chla molecules in CPII are Förster coupled so that a second excitation received by the group of Chla's either creates a state with two localized excitons or raises the first one to a doubly excited state. The data on CPII can be understood on the basis of a kinetic model that does not exclude exciton annihilation during the excitation pulse. The implied annihilation rate is consistent with our theoretical estimates of that rate obtained by applying excitation transfer theory to pairs of molecules both initially excited.     

Wide-field photon counting fluorescence lifetime imaging microscopy: application to photosynthesizing systems

Fluorescence lifetime imaging microscopy (FLIM) is a technique that visualizes the excited state kinetics of fluorescence molecules with the spatial resolution of a fluorescence microscope. We present a scanningless implementation of FLIM based on a time- and space-correlated single photon counting (TSCSPC) method employing a position-sensitive quadrant anode detector and wide-field illumination. The standard time-correlated photon counting approach leads to picosecond temporal resolution, making it possible to resolve complex fluorescence decays. This allows parallel acquisition of time-resolved images of biological samples under minimally invasive low-excitation conditions (<10mW/cm²). In this way unwanted photochemical reactions induced by high excitation intensities and distorting the decay kinetics are avoided. Comparably low excitation intensities are practically impossible to achieve with a conventional laser scanning microscope, where focusing of the excitation beam into a tight spot is required. Therefore, wide-field FLIM permits to study Photosystem II (PS II) in a way so far not possible with a laser scanning microscope. The potential of the wide-field TSCSPC method is demonstrated by presenting FLIM measurements of the fluorescence dynamics of photosynthetic systems in living cells of the chlorophyll d-containing cyanobacterium Acaryochloris marina.