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Browsing by Author "Tsekouras, Konstantinos"
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Item Counting Photobleach Steps and the Dynamics of Bacterial Predators(Office of the Vice Chancellor for Research, 2016-04-08) Jashnsaz, Hossein; Tsekouras, Konstantinos; Al Juboori, Mohammed; Weistuch, Corey; Miller, Nick; Nguyen, Tyler; McCoy, Bryan; Perkins, Stephanie; Anderson, Gregory; Presse, StevePhotobleach (PB) counting is used to enumerate proteins by monitoring how the light intensity in some regions decreases by quanta as individual fluorophores photobleach. While it is straightforward in theory, PB counting is often difficult because fluorescence traces are noisy. In this work, we quantify the sources of noise that arise during photobleach counting to construct a principled likelihood function of observing the data given a model. Noise in the signal could arise from background fluorescence, variable fluorophore emission, and fluorophore blinking. In addition, in a completely different direction, we explore the role of hydrodynamic interactions on the dynamics of bacterial predators. Our study shows that Bdellovibrio (BV) - a model predatory bacterium - is susceptible to self-generated hydrodynamic forces. Near surfaces and defects, these hydrodynamic interactions co-localize BV with its prey, and this may enhance BV’s hunting efficiency.Item The heat released during catalytic turnover enhances the diffusion of an enzyme(Nature Publishing Group, 2015-01-08) Riedel, Clement; Gabizon, Ronen; Wilson, Christian A. M.; Hamadani, Kambiz; Tsekouras, Konstantinos; Marqusee, Susan; Pressé, Steve; Bustamante, Carlos; Department of Cellular & Integrative Physiology, IU School of MedicineRecent studies have shown that the diffusivity of enzymes increases in a substrate-dependent manner during catalysis,. Although this observation has been reported and characterized for several different systems–, the precise origin of this phenomenon is unknown. Calorimetric methods are often used to determine enthalpies from enzyme-catalysed reactions and can therefore provide important insight into their reaction mechanisms,. The ensemble averages involved in traditional bulk calorimetry cannot probe the transient effects that the energy exchanged in a reaction may have on the catalyst. Here we obtain single-molecule fluorescence correlation spectroscopy data and analyse them within the framework of a stochastic theory to demonstrate a mechanistic link between the enhanced diffusion of a single enzyme molecule and the heat released in the reaction. We propose that the heat released during catalysis generates an asymmetric pressure wave that results in a differential stress at the protein–solvent interface that transiently displaces the centre-of-mass of the enzyme (chemoacoustic effect). This novel perspective on how enzymes respond to the energy released during catalysis suggests a possible effect of the heat of reaction on the structural integrity and internal degrees of freedom of the enzyme.Item Hydrodynamic Hunters(Cell Press, 2017-03-28) Jashnsaz, Hossein; Al Juboori, Mohammed; Weistuch, Corey; Miller, Nicholas; Nguyen, Tyler; Meyerhoff, Viktoria; McCoy, Bryan; Perkins, Stephanie; Wallgren, Ross; Ray, Bruce D.; Tsekouras, Konstantinos; Anderson, Gregory G.; Pressé, Steve; Physics, School of ScienceThe Gram-negative Bdellovibrio bacteriovorus (BV) is a model bacterial predator that hunts other bacteria and may serve as a living antibiotic. Despite over 50 years since its discovery, it is suggested that BV probably collides into its prey at random. It remains unclear to what degree, if any, BV uses chemical cues to target its prey. The targeted search problem by the predator for its prey in three dimensions is a difficult problem: it requires the predator to sensitively detect prey and forecast its mobile prey’s future position on the basis of previously detected signal. Here instead we find that rather than chemically detecting prey, hydrodynamics forces BV into regions high in prey density, thereby improving its odds of a chance collision with prey and ultimately reducing BV’s search space for prey. We do so by showing that BV’s dynamics are strongly influenced by self-generated hydrodynamic flow fields forcing BV onto surfaces and, for large enough defects on surfaces, forcing BV in orbital motion around these defects. Key experimental controls and calculations recapitulate the hydrodynamic origin of these behaviors. While BV’s prey (Escherichia coli) are too small to trap BV in hydrodynamic orbit, the prey are also susceptible to their own hydrodynamic fields, substantially confining them to surfaces and defects where mobile predator and prey density is now dramatically enhanced. Colocalization, driven by hydrodynamics, ultimately reduces BV’s search space for prey from three to two dimensions (on surfaces) even down to a single dimension (around defects). We conclude that BV’s search for individual prey remains random, as suggested in the literature, but confined, however—by generic hydrodynamic forces—to reduced dimensionality.Item Inferring diffusion dynamics from FCS in heterogeneous nuclear environments(Elsevier, 2015-07-07) Tsekouras, Konstantinos; Siegel, Amanda P.; Day, Richard N.; Pressé, Steve; Department of Physics, School of ScienceFluorescence correlation spectroscopy (FCS) is a noninvasive technique that probes the diffusion dynamics of proteins down to single-molecule sensitivity in living cells. Critical mechanistic insight is often drawn from FCS experiments by fitting the resulting time-intensity correlation function, G(t), to known diffusion models. When simple models fail, the complex diffusion dynamics of proteins within heterogeneous cellular environments can be fit to anomalous diffusion models with adjustable anomalous exponents. Here, we take a different approach. We use the maximum entropy method to show-first using synthetic data-that a model for proteins diffusing while stochastically binding/unbinding to various affinity sites in living cells gives rise to a G(t) that could otherwise be equally well fit using anomalous diffusion models. We explain the mechanistic insight derived from our method. In particular, using real FCS data, we describe how the effects of cell crowding and binding to affinity sites manifest themselves in the behavior of G(t). Our focus is on the diffusive behavior of an engineered protein in 1) the heterochromatin region of the cell's nucleus as well as 2) in the cell's cytoplasm and 3) in solution. The protein consists of the basic region-leucine zipper (BZip) domain of the CCAAT/enhancer-binding protein (C/EBP) fused to fluorescent proteins.Item A novel method to accurately locate and count large numbers of steps by photobleaching(The American Society for Cell Biology, 2016-11-07) Tsekouras, Konstantinos; Custer, Thomas C.; Jashnsaz, Hossein; Walter, Nils G.; Pressé, Steve; Department of Physics, School of SciencePhotobleaching event counting is a single-molecule fluorescence technique that is increasingly being used to determine the stoichiometry of protein and RNA complexes composed of many subunits in vivo as well as in vitro. By tagging protein or RNA subunits with fluorophores, activating them, and subsequently observing as the fluorophores photobleach, one obtains information on the number of subunits in a complex. The noise properties in a photobleaching time trace depend on the number of active fluorescent subunits. Thus, as fluorophores stochastically photobleach, noise properties of the time trace change stochastically, and these varying noise properties have created a challenge in identifying photobleaching steps in a time trace. Although photobleaching steps are often detected by eye, this method only works for high individual fluorophore emission signal-to-noise ratios and small numbers of fluorophores. With filtering methods or currently available algorithms, it is possible to reliably identify photobleaching steps for up to 20-30 fluorophores and signal-to-noise ratios down to ∼1. Here we present a new Bayesian method of counting steps in photobleaching time traces that takes into account stochastic noise variation in addition to complications such as overlapping photobleaching events that may arise from fluorophore interactions, as well as on-off blinking. Our method is capable of detecting ≥50 photobleaching steps even for signal-to-noise ratios as low as 0.1, can find up to ≥500 steps for more favorable noise profiles, and is computationally inexpensive.Item Quantitative Kinetic Models from Intravital Microscopy: A Case Study Using Hepatic Transport(ACS, 2019-08-29) Tsekouras, Konstantinos; Day, Richard; Dunn, Kenneth W.; Pressé, Steve; Physics, School of ScienceThe liver performs critical physiological functions, including metabolizing and removing substances, such as toxins and drugs, from the bloodstream. Hepatotoxicity itself is intimately linked to abnormal hepatic transport, and hepatotoxicity remains the primary reason drugs in development fail and approved drugs are withdrawn from the market. For this reason, we propose to analyze, across liver compartments, the transport kinetics of fluorescein-a fluorescent marker used as a proxy for drug molecules-using intravital microscopy data. To resolve the transport kinetics quantitatively from fluorescence data, we account for the effect that different liver compartments (with different chemical properties) have on fluorescein's emission rate. To do so, we develop ordinary differential equation transport models from the data where the kinetics is related to the observable fluorescence levels by "measurement parameters" that vary across different liver compartments. On account of the steep non-linearities in the kinetics and stochasticity inherent to the model, we infer kinetic and measurement parameters by generalizing the method of parameter cascades. For this application, the method of parameter cascades ensures fast and precise parameter estimates from noisy time traces.Item Unraveling the Thousand Word Picture: An Introduction to Super-Resolution Data Analysis(ACS Publications, 2017-06-14) Lee, Antony; Tsekouras, Konstantinos; Calderon, Christopher; Bustamante, Carlos; Pressé, Steve; Chemistry and Chemical Biology, School of ScienceSuper-resolution microscopy provides direct insight into fundamental biological processes occurring at length scales smaller than light’s diffraction limit. The analysis of data at such scales has brought statistical and machine learning methods into the mainstream. Here we provide a survey of data analysis methods starting from an overview of basic statistical techniques underlying the analysis of super-resolution and, more broadly, imaging data. We subsequently break down the analysis of super-resolution data into four problems: the localization problem, the counting problem, the linking problem, and what we’ve termed the interpretation problem.