Photobleaching FRET-FLIM-ICS for quaternary structure quantification on cells. Theory and simulations.

The oligomerization of proteins is an important biological control mechanism and has several functions in activity and stability of enzymes, structural proteins, ion channels and transcription factors. The determination of the relevant oligomeric states in terms of geometry (spatial extent), oligomer size (monomer or dimer or oligomer) and affinity (amounts of monomer, dimer and oligomer) is a challenging biophysical problem. Förster resonance energy transfer and fluorescence fluctuation spectroscopy are powerful tools that are sensitive to proximity and oligomerization respectively. Here it is proposed to combine image-based lifetime-detected Forster resonance energy transfer with image correlation spectroscopy and photobleaching to determine distances, oligomer sizes and oligomer distributions. Simulations for simple oligomeric forms illustrate the potential to improve the discrimination between different quaternary states in the cellular milieu.

Spectral Relaxation Imaging Microscopy II: Complex Dynamics.

The dynamics of condensed matter can be measured by the time-dependent Stokes shift of a suitable fluorescent probe. The time-dependent spectral correlation function is typically described by one or more spectral relaxation correlation times, which, in liquid solvents, characterize the timescales of the dipolar relaxation processes around the excited-state probe. The phasor plot provides a powerful approach to represent and analyze time and frequency-domain data acquired as images, thus providing a spatial map of spectral dynamics in a complex structure such as a living cell. Measurements of the phase and modulation at two emission wavelength channels were shown to be sufficient to extract a single excited-state lifetime and a single spectral relaxation correlation time, supplying estimates of the mean rate of excited-state depopulation and the mean rate of spectral shift. In the present contribution, two more issues were addressed. First, the provision of analytic formulae allowing extraction of the initial generalized polarization and the relaxed generalized polarization, which characterize the fluorescence spectrum of the unrelaxed state and the fully relaxed state. Second, improved methods of model discrimination and model parameter extraction for more complex spectral relaxation phenomena. The analysis workflow was illustrated with examples from the literature.

Transfer function approach to understanding periodic forcing of signal transduction networks.

Signal transduction networks are responsible for transferring biochemical signals from the extracellular to the intracellular environment. Understanding the dynamics of these networks helps understand their biological processes. Signals are often delivered in pulses and oscillations. Therefore, understanding the dynamics of these networks under pulsatile and periodic stimuli is useful. One tool to do this is the transfer function. This tutorial outlines the basic theory behind the transfer function approach and walks through some examples of simple signal transduction networks.

Label-free third harmonic generation imaging and quantification of lipid droplets in live filamentous fungi.

We report the utilization of Third-Harmonic Generation microscopy for label-free live cell imaging of lipid droplets in the hypha of filamentous fungus Phycomyces blakesleeanus. THG microscopy images showed bright spherical features dispersed throughout the hypha cytoplasm in control conditions and a transient increase in the number of bright features after complete nitrogen starvation. Colocalization analysis of THG and lipid-counterstained images disclosed that the cytoplasmic particles were lipid droplets. Particle Size Analysis and Image Correlation Spectroscopy were used to quantify the number density and size of lipid droplets. The two analysis methods both revealed an increase from 16 × 10-3 to 23 × 10-3 lipid droplets/µm2 after nitrogen starvation and a decrease in the average size of the droplets (range: 0.5-0.8 µm diameter). In conclusion, THG imaging, followed by PSA and ICS, can be reliably used for filamentous fungi for the in vivo quantification of lipid droplets without the need for labeling and/or fixation. In addition, it has been demonstrated that ICS is suitable for THG microscopy.

Intrinsically Fluorescent Anti-Cancer Drugs.

At present, about one-third of the total protein targets in the pharmaceutical research sector are kinase-based. While kinases have been attractive targets to combat many diseases, including cancer, selective kinase inhibition has been challenging, because of the high degree of structural homology in the active site where many kinase inhibitors bind. Despite efficacy as cancer drugs, kinase inhibitors can exhibit limited target specificity and rationalizing their target profiles in the context of precise molecular mechanisms or rearrangements is a major challenge for the field. Spectroscopic approaches such as infrared, Raman, NMR and fluorescence have the potential to provide significant insights into drug-target and drug-non-target interactions because of sensitivity to molecular environment. This review places a spotlight on the significance of fluorescence for extracting information related to structural properties, discovery of hidden conformers in solution and in target-bound state, binding properties (e.g., location of binding sites, hydrogen-bonding, hydrophobicity), kinetics as well as dynamics of kinase inhibitors. It is concluded that the information gleaned from an understanding of the intrinsic fluorescence from these classes of drugs may aid in the development of future drugs with improved side-effects and less disease resistance.

Dynamic Cellular Cartography: Mapping the Local Determinants of Oligodendrocyte Transcription Factor 2 (OLIG2) Function in Live Cells Using Massively Parallel Fluorescence Correlation Spectroscopy Integrated with Fluorescence Lifetime Imaging Microscopy (mpFCS/FLIM).

Compartmentalization and integration of molecular processes through diffusion are basic mechanisms through which cells perform biological functions. To characterize these mechanisms in live cells, quantitative and ultrasensitive analytical methods with high spatial and temporal resolution are needed. Here, we present quantitative scanning-free confocal microscopy with single-molecule sensitivity, high temporal resolution (∼10 µs/frame), and fluorescence lifetime imaging capacity, developed by integrating massively parallel fluorescence correlation spectroscopy with fluorescence lifetime imaging microscopy (mpFCS/FLIM); we validate the method, use it to map in live cell location-specific variations in the concentration, diffusion, homodimerization, DNA binding, and local environment of the oligodendrocyte transcription factor 2 fused with the enhanced Green Fluorescent Protein (OLIG2-eGFP), and characterize the effects of an allosteric inhibitor of OLIG2 dimerization on these determinants of OLIG2 function. In particular, we show that cytoplasmic OLIG2-eGFP is largely monomeric and freely diffusing, with the fraction of freely diffusing OLIG2-eGFP molecules being fD,freecyt = (0.75 ± 0.10) and the diffusion time τD,freecyt = (0.5 ± 0.3) ms. In contrast, OLIG2-eGFP homodimers are abundant in the cell nucleus, constituting ∼25% of the nuclear pool, some fD,boundnuc = (0.65 ± 0.10) of nuclear OLIG2-eGFP is bound to chromatin DNA, whereas freely moving OLIG2-eGFP molecules diffuse at the same rate as those in the cytoplasm, as evident from the lateral diffusion times τD,freenuc = τD,freecyt = (0.5 ± 0.3) ms. OLIG2-eGFP interactions with chromatin DNA, revealed through their influence on the apparent diffusion behavior of OLIG2-eGFP, τD,boundnuc (850 ± 500) ms, are characterized by an apparent dissociation constant Kd,appOLIG2-DNA = (45 ± 30) nM. The apparent dissociation constant of OLIG2-eGFP homodimers was estimated to be Kd,app(OLIG2-eGFP)2 ≈ 560 nM. The allosteric inhibitor of OLIG2 dimerization, compound NSC 50467, neither affects OLIG2-eGFP properties in the cytoplasm nor does it alter the overall cytoplasmic environment. In contrast, it significantly impedes OLIG2-eGFP homodimerization in the cell nucleus, increasing five-fold the apparent dissociation constant, Kd,app,NSC50467(OLIG2-eGFP)2 ≈ 3 µM, thus reducing homodimer levels to below 7% and effectively abolishing OLIG2-eGFP specific binding to chromatin DNA. The mpFCS/FLIM methodology has a myriad of applications in biomedical research and pharmaceutical industry. For example, it is indispensable for understanding how biological functions emerge through the dynamic integration of location-specific molecular processes and invaluable for drug development, as it allows us to quantitatively characterize the interactions of drugs with drug targets in live cells.

Red-Edge Excitation Shift Spectroscopy (REES): Application to Hidden Bound States of Ligands in Protein-Ligand Complexes.

Ligand-protein binding is responsible for the vast majority of bio-molecular functions. Most experimental techniques examine the most populated ligand-bound state. The determination of less populated, intermediate, and transient bound states is experimentally challenging. However, hidden bound states are also important because these can strongly influence ligand binding and unbinding processes. Here, we explored the use of a classical optical spectroscopic technique, red-edge excitation shift spectroscopy (REES) to determine the number, population, and energetics associated with ligand-bound states in protein-ligand complexes. We describe a statistical mechanical model of a two-level fluorescent ligand located amongst a finite number of discrete protein microstates. We relate the progressive emission red shift with red-edge excitation to thermodynamic parameters underlying the protein-ligand free energy landscape and to photo-physical parameters relating to the fluorescent ligand. We applied the theoretical model to published red-edge excitation shift data from small molecule inhibitor-kinase complexes. The derived thermodynamic parameters allowed dissection of the energetic contribution of intermediate bound states to inhibitor-kinase interactions.

Uptake quantification of gold nanoparticles inside of cancer cells using high order image correlation spectroscopy.

The application of gold nanoparticles (AuNPs) in cancer therapeutics and diagnostics has recently reached a clinical level. Functional use of the AuNP in theranostics first requires effective uptake into the cells, but accurate quantification of AuNPs cellular uptake in real-time is still a challenge due to the destructive nature of existing characterization methods. The optical imaging-based quantification method is highly desirable. Here, we propose the use of high-order image correlation spectroscopy (HICS) as an optical imaging-based nanoparticle quantification technique. Coupled with dark field microscopy (DFM), a non-destructive and easy quantification method could be achieved. We demonstrate HICS analysis on 80 nm AuNPs coated with cetyltrimethylammonium bromide (CTAB) uptake in HeLa cells to calculate the percentage of aggregate species (dimer) in the total uptake and their relative scattering quantum yield inside the cells, the details of which are not available with other quantification techniques. The total particle uptake kinetics measured were in a reasonable agreement with the literature.

Effects of Rationally Designed Physico-Chemical Variants of the Peptide PuroA on Biocidal Activity towards Bacterial and Mammalian Cells.

Antimicrobial peptides (AMPs) often exhibit wide-spectrum activities and are considered ideal candidates for effectively controlling persistent and multidrug-resistant wound infections. PuroA, a synthetic peptide based on the tryptophan (Trp)-rich domain of the wheat protein puroindoline A, displays strong antimicrobial activities. In this work, a number of peptides were designed based on PuroA, varying in physico-chemical parameters of length, number of Trp residues, net charge, hydrophobicity or amphipathicity, D-versus L-isomers of amino acids, cyclization or dimerization, and were tested for antimicrobial potency and salt and protease tolerance. Selected peptides were assessed for effects on biofilms of methicillin-resistant Staphylococcus aureus (MRSA) and selected mammalian cells. Peptide P1, with the highest amphipathicity, six Trp and a net charge of +7, showed strong antimicrobial activity and salt stability. Peptides W7, W8 and WW (seven to eight residues) were generally more active than PuroA and all diastereomers were protease-resistant. PuroA and certain variants significantly inhibited initial biomass attachment and eradicated preformed biofilms of MRSA. Further, P1 and dimeric PuroA were cytotoxic to HeLa cells. The work has led to peptides with biocidal effects on common human pathogens and/or anticancer potential, also offering great insights into the relationship between physico-chemical parameters and bioactivities, accelerating progress towards rational design of AMPs for therapeutics.

Deducing the Conformational Properties of a Tyrosine Kinase Inhibitor in Solution by Optical Spectroscopy and Computational Chemistry.

Dacomitinib (PF-00299804) was recently approved by the Food and Drug Administration (FDA) as a tyrosine kinase inhibitor (TKI). Unfortunately, side effects and disease resistance eventually result from its use. Off-target effects in some kinase inhibitors have arisen from drug conformational plasticity; however, the conformational states of Dacomitinib in solution are presently unknown. To fill this gap, we have used computational chemistry to explore optimized molecular geometry, properties, and ultraviolet-visible (UV-Vis) absorption spectra of Dacomitinib in dimethyl sulfoxide (DMSO) solution. Potential energy scans led to the discovery of two planar and two twisted conformers of Dacomitinib. The simulated UV-Vis spectral signatures of the planar conformers reproduced the two experimental spectral bands at 275 and 343 nm in solution. It was further discovered that Dacomitinib forms conformers through its three flexible linkers of two C-NH-C bridges, which control the orientations of the 3-chloro-4-fluoroaniline ring (Ring C) and the quinazoline ring (Rings A and B) and the 4-piperidin-1-yl-buten-2-nal side chain, and one C-O-C local bridge which controls the methoxy group locally. When in isolation, these flexible linkers form close hexagon and pentagon loops through strong intramolecular hydrogen bonding so that the "planar" conformers Daco-P1 and Daco-P2 are more stable in isolation. Such flexibility of the ligand and its ability to dock and bind with protein also depend on their interaction with the environment, in addition to their energy and spectra in isolation. However, an accurate quantum mechanical study on drug/ligand conformers in isolation provides necessary reference information for the ability to form a complex with proteins.

Does frequency-dependent cell proliferation exhibit a Fano-type resonance?

We examined PC12 cell proliferation in environments with temporally varying epidermal growth factor concentrations by means of a microfluidic system. Our measurements revealed frequency-dependent cell behaviour over an observation period of three days. The cell population either increased, decreased or remained constant depending on the frequency of epidermal growth factor applied. A plot of the apparent proliferation rate as a function of growth-factor frequency was mathematically described by the Fano line-shape formula. In the context of linear response theory, these results imply that the PC12 cells compute zero, first and second-order time derivatives of the ligand concentration and utilise this information to decide to proliferate or die. We discuss a physical model based on periodic forcing of coupled oscillators that accounts for these observations. Our results and analysis suggest the possibility to influence cell fate by controlling the dynamics of the extracellular environment.

In-cell structural dynamics of an EGF receptor during ligand-induced dimer-oligomer transition.

The epidermal growth factor receptor (EGFR) is a membrane protein that regulates cell proliferation, differentiation and survival, and is a drug target for cancer therapy. Ligand-induced activation of the EGFR kinase is generally regarded to require ligand-bound-dimers, while phosphorylation and down-stream signalling is modulated by oligomers. Recent work has unveiled changes in EGFR dynamics from ligand-induced dimerization in membranes extracted from cells, however, less is known about the changes in EGFR dynamics that accompany the ligand-induced oligomerization in a live cell environment. Here, we determine the dynamics of a c-terminal GFP tag attached to EGFR in the unliganded dimer and in the liganded oligomers. By means of the single-frequency polarized phasor ellipse approach we extracted two correlation times on the sub-nanosecond and super-nanosecond timescales, respectively. EGF binding to the EGFR-GFP dimer lengthened the sub-nanosecond correlation time (from 0.1 to 1.3 ns) and shortened the super-nanosecond correlation time (from 210 to 56 ns) of the c-terminal GFP probe. The sub-nanosecond depolarization processes were assigned to electronic energy migration between proximal GFPs in the EGFR dimer or oligomer, while the super-nanosecond correlation times were assigned to nanosecond fluctuations of the GFP probe in the EGFR complex. Accordingly, these results show that ligand binding increased the average separation between the c-terminal tags and increased their rotational mobility. We propose that the dynamics are linked to an inhibitory function of the c-terminal tail in the un-liganded dimer and to the requirement of facile stochastic switching between kinase activation and cytoplasmic adaptor/effector binding in the active oligomers.

Spectroscopic and Microscopic Approaches for Investigating the Dynamic Interactions of Anti-microbial Peptides With Membranes and Cells.

The emergence of microbes resistant to conventional antibiotics is a burgeoning threat to humanity with significant impacts on the health of people and on the health system itself. Antimicrobial peptides (AMPs) hold promise as potential future alternatives to conventional drugs because they form an integral part of the defense systems of other species in the animal, plant, and fungal kingdoms. To aid the design of the next generation of AMPs optimized for human use, we must first understand the mechanism of action of existing AMPs with their targets, ideally in the context of the complex landscape of the living (microbial) cell. Advances in lasers, optics, detectors, fluid dynamics and various probes has enabled the experimentalist to measure the kinetics of molecule-membrane, molecule-molecule, and molecule-cell interactions with increasing spatial and temporal resolution. The purpose of this review is to highlight studies into these dynamic interactions with a view to improving our understanding of AMP mechanisms.

Using fluorescence lifetime dequenching to estimate the average quinary stoichiometry of proteins in living cells.

Biological proteins are understood in terms of five structural levels-primary, secondary, tertiary, quaternary and quinary. The quinary structure is defined as the set of macromolecular interactions that are transient in vivo. This includes non-covalent protein-protein interactions occurring within the crowded intracellular environment. For much of twentieth century science, the canonical approach to studying biological proteins involved test tube environments. These uncrowded in vitro studies inadvertently failed to replicate and observe the quinary structures present within the original cells. Consequently, contemporary literature surrounding the fifth level of protein organisation is lacking. In particular, there is a lack of literature on the size of transient clusters within living cells. In an attempt to reconcile this gap in knowledge, we propose a quantitative method for estimating the average quinary stoichiometry in living cells. The method is based on lifetime self-quenching of fluorescently-labelled proteins in living cells. Close approach of two or more proteins in a quinary complex will result in self-quenching of the fluorescence lifetime from the fluorescent labels. Our method utilises the random mixing of proteins during cell division to mix fluorescently labelled with unlabelled proteins. Such mixing reduces the probability of adjacency between labelled proteins and, hence, decreases the probability of fluorescence lifetime quenching from labels. By monitoring fluorescence lifetime dequenching during multiple cell divisions, we can determine the average quinary structure in living proliferating cells. We demonstrate this method with a case study on cultured HeLa cells. The average quinary stoichiometry was found to be between five and six. That is, at any given point in time, there are five or six weakly interacting partners in the immediate neighbourhood of any given protein.

The architecture of EGFR's basal complexes reveals autoinhibition mechanisms in dimers and oligomers.

Our current understanding of epidermal growth factor receptor (EGFR) autoinhibition is based on X-ray structural data of monomer and dimer receptor fragments and does not explain how mutations achieve ligand-independent phosphorylation. Using a repertoire of imaging technologies and simulations we reveal an extracellular head-to-head interaction through which ligand-free receptor polymer chains of various lengths assemble. The architecture of the head-to-head interaction prevents kinase-mediated dimerisation. The latter, afforded by mutation or intracellular treatments, splits the autoinhibited head-to-head polymers to form stalk-to-stalk flexible non-extended dimers structurally coupled across the plasma membrane to active asymmetric tyrosine kinase dimers, and extended dimers coupled to inactive symmetric kinase dimers. Contrary to the previously proposed main autoinhibitory function of the inactive symmetric kinase dimer, our data suggest that only dysregulated species bear populations of symmetric and asymmetric kinase dimers that coexist in equilibrium at the plasma membrane under the modulation of the C-terminal domain.

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy.

Confocal microscopy provides an accessible methodology to capture sub-cellular interactions critical for the characterization and further development of pre-clinical agents labeled with fluorescent probes. With recent advancements in antibody based cytotoxic drug delivery systems, understanding the alterations induced by these agents within the realm of receptor aggregation and internalization is of critical importance. This protocol leverages the well-established methodology of fluorescent immunocytochemistry and the open source FIJI distribution of ImageJ, with its inbuilt autocorrelation and image mathematical functions, to perform spatial image correlation spectroscopy (ICS). This protocol quantitates the fluorescent intensity of labeled receptors as a function of the beam area of the confocal microscope. This provides a quantitative measure of the state of target molecule aggregation on the cell surface. This methodology is focused on the characterization of static cells with potential to expand into temporal investigations of receptor aggregation. This protocol presents an accessible methodology to provide quantification of clustering events occurring at the cell surface, utilizing well established techniques and non-specialized imaging apparatus.

Fluorescence-based approaches for monitoring membrane receptor oligomerization.

Membrane protein structures are highly under-represented relative to water-soluble protein structures in the protein databank. This is especially the case because membrane proteins represent more than 30% of proteins encoded in the human genome yet contribute to less than 10% of currently known structures (Torres et al. in Trends Biol Sci 28:137-144, 2003). Obtaining high-resolution structures of membrane proteins by traditional methods such as NMR and x-ray crystallography is challenging, because membrane proteins are difficult to solubilise, purify and crystallize. Consequently, development of methods to examine protein structure in situ is highly desirable. Fluorescence is highly sensitive to protein structure and dynamics (Lakowicz in Principles of fluorescence spectroscopy, Springer, New York, 2007). This is mainly because of the time a fluorescence probe molecule spends in the excited state. Judicious choice and placement of fluorescent molecule(s) within a protein(s) enables the experimentalist to obtain information at a specific site(s) in the protein (complex) of interest. Moreover, the inherent multi-dimensional nature of fluorescence signals across wavelength, orientation, space and time enables the design of experiments that give direct information on protein structure and dynamics in a biological setting. The purpose of this review is to introduce the reader to approaches to determine oligomeric state or quaternary structure at the cell membrane surface with the ultimate goal of linking the oligomeric state to the biological function. In the first section, we present a brief overview of available methods for determining oligomeric state and compare their advantages and disadvantages. In the second section, we highlight some of the methods developed in our laboratory to address contemporary questions in membrane protein oligomerization. In the third section, we outline our approach to determine the link between protein oligomerization and biological activity.

Conformational Plasticity in Tyrosine Kinase Inhibitor-Kinase Interactions Revealed with Fluorescence Spectroscopy and Theoretical Calculations.

To understand drug-protein dynamics, it is necessary to account for drug molecular flexibility and binding site plasticity. Herein, we exploit fluorescence from a tyrosine kinase inhibitor, AG1478, as a reporter of its conformation and binding site environment when complexed with its cognate kinase. Water-soluble kinases, aminoglycoside phosphotransferase APH(3')-Ia and mitogen-activated protein kinase 14 (MAPK14), were chosen for this study. On the basis of our prior work, the AG1478 conformation (planar or twisted) was inferred from the fluorescence excitation spectrum and the polarity of the AG1478-binding site was deduced from the fluorescence emission spectrum, while red-edge excitation shift (REES) probed the heterogeneity of the binding site (protein conformation and hydration) distributions in the protein conformational ensemble. In the AG1478-APH(3')-Ia complex, both twisted (or partially twisted) and planar AG1478 conformations were evidenced from emission wavelength-dependent excitation spectra. The binding site environment provided by APH(3')-Ia was moderately polar (λmax = 480 nm) with evidence for considerable heterogeneity (REES = 34 nm). In contrast, in the AG1478-MAPK14 complex, AG1478 was in a predominantly planar conformation with a lower degree of conformational heterogeneity. The binding site environment provided by the MAPK14 protein was of relatively low polarity (λmax = 430 nm) with a smaller degree of heterogeneity (REES = 11 nm). The results are compared with the available X-ray data and discussed in the context of our current understanding of tyrosine kinase inhibitor conformation and protein conformational ensembles.

Antimicrobial peptides: biochemical determinants of activity and biophysical techniques of elucidating their functionality.

Antimicrobial peptides (AMPs) have been established over millennia as powerful components of the innate immune system of many organisms. Due to their broad spectrum of activity and the development of host resistance against them being unlikely, AMPs are strong candidates for controlling drug-resistant pathogenic microbial pathogens. AMPs cause cell death through several independent or cooperative mechanisms involving membrane lysis, non-lytic activity, and/or intracellular mechanisms. Biochemical determinants such as peptide length, primary sequence, charge, secondary structure, hydrophobicity, amphipathicity and host cell membrane composition together influence the biological activities of peptides. A number of biophysical techniques have been used in recent years to study the mechanisms of action of AMPs. This work appraises the molecular parameters that determine the biocidal activity of AMPs and overviews their mechanisms of actions and the diverse biochemical, biophysical and microscopy techniques utilised to elucidate these.

One-step growth of thin film SnS with large grains using MOCVD.

Thin film tin sulphide (SnS) films were produced with grain sizes greater than 1 µm using a one-step metal organic chemical vapour deposition process. Tin-doped indium oxide (ITO) was used as the substrate, having a similar work function to molybdenum typically used as the back contact, but with potential use of its transparency for bifacial illumination. Tetraethyltin and ditertiarybutylsulphide were used as precursors with process temperatures 430-470 °C to promote film growth with large grains. The film stoichiometry was controlled by varying the precursor partial pressure ratios and characterised with energy dispersive X-ray spectroscopy to optimise the SnS composition. X-ray diffraction and Raman spectroscopy were used to determine the phases that were present in the film and revealed that small amounts of ottemannite Sn2S3 was present when SnS was deposited on to the ITO using optimised growth parameters. Interaction at the SnS/ITO interface to form Sn2S3 was deduced to have resulted for all growth conditions.