Physics
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Item CONSEQUENCES OF NUCLEAR CONFINEMENT IN CANCER METASTASIS(2021) Baird, Michelle; Waterman, Clare M; Stroka, Kimberly M; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Malignant melanoma is characterized by its mutational heterogeneity and aggressive metastatic spread. During metastasis, melanoma cells migrate through diverse microenvironments, including regions of dense tissue confinement to reach the vasculature. Microenvironmental confinement of tumor cells causes nuclear deformation, which can lead to loss of nuclear envelope (NE) integrity and DNA damage, improper repair of which leads to genomic aberrations and heterogeneity. We hypothesize that during metastatic progression, expression levels of NE genes are altered, facilitating nuclear deformability and NE fragility, mediating an increase in genetic heterogeneity within the population. In this dissertation, we show a novel bioinformatic analysis of orthogonal RNA-seq data sets from patient samples of metastatic melanoma and benign nevi, revealing several NE proteins upregulated in metastatic disease. Performing a targeted siRNA-based screen using a PDMS confinement device to assay for nuclear fragility, we found reduction of lamin B receptor (LBR) dramatically reduced NE fragility in melanoma cells, and ectopic overexpression of LBR was sufficient to increase NE fragility in benign melanocytes. Utilizing functional protein domain truncations and point mutations in LBR, we found the cholesterol synthase activity of LBR was specifically required for increased NE fragility, independent of LBRs additional roles tethering heterochromatin and lamin B to the NE. Additionally, we found that reduction of LBR in melanoma cells results in a reorganization of cholesterol in the NE. Thus, LBR generated cholesterol in the NE promotes NE fragility. To determine if LBR-mediated NE fragility was correlated with increased nuclear deformability, we assayed NE mechanics with atomic force microscopy. In melanoma cells, we find reduction of LBR results in an increase in nuclear stiffness and a decrease in deformability, while LBR overexpression in benign melanocytes results in an increase in nuclear deformability. These results show for the first time that upregulation of LBR in metastatic melanoma plays dual roles in reducing nuclear deformability and increasing NE rupture, specifically through alterations in cholesterol organization in the NE and open an exciting new direction to the role of cholesterol in NE integrity and mechanics.Item DECIPHERING HOW EGFR-GRB2-SOS1 COMPLEX REGULATES KRAS4B ACTIVATION AND LEADS TO HIPPO SIGNALING THROUGH RASSF5(2021) Liao, Tsung-Jen; Fushman, David; Nussinov, Ruth; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ras is a small GTPase, which regulates cell proliferation and apoptosis. Its bifunctional switch is controlled by the nucleotide state: GTP-bound – switch on; GDP-bound – switch off. Among the three Ras isoforms, HRas, NRas, and KRas (with two splice variants of KRas4A and KRas4B), KRas4B is highly oncogenic, the most frequently mutated in lung, colorectal, and pancreatic cancers. However, Ras was thought to be “undruggable” due to the lack of effective pharmacological inhibitors over the past three decades. Most of the current focus has been directed at inhibiting the activation of Ras signaling. Ras proteins transduce signals between cell surface receptors and multiple intracellular signaling cascades. In response to epidermal growth factor receptor (EGFR) activation, growth factor receptor bound protein 2 (Grb2) establishes the connection between EGFR and Ras-specific nucleotide exchange factor (RasGEF), son of sevenless 1 (SOS1). SOS1 activates Ras by exchanging GDP to GTP. In addition to Ras major effectors and pathways, e.g. MAPK and PI3Kα/Akt, which are cell growth related, GTP-bound Ras associating with RASSF5 activates the Hippo pathway, which acts to suppress cell proliferation. In this serial study, we use NMR measurement and molecular dynamics (MD) simulation to investigate the interactions of Grb2–SOS1, SOS1–KRas4B, KRas4B–RASSF5, and the EGFR effects on the binding of Grb2–SOS1. Our findings successfully uncovered (1) a novel Grb2 binding site PKLPPKTYKREH on SOS1 and the most probable binding mode of Grb2–SOS1, (2) strong SOS1 peptide binders induce a closed conformation of Grb2 nSH3 domain but unchanged conformation of Grb2 cSH3 domain, (3) full length Grb2 performs high affinities for one-site SOS1 peptides, and the EGFR segment may facilitate the binding of Grb2 to the particular two-site SOS1 peptide, (4) KRas4B binding to SOS1 allosteric site induces the conformational changes of catalytic site and accelerate the KRas4B activation cycle, (5) the hypothesized mechanism that RASSF5 is a tumor suppressor in vivo but opposite in vitro, and (6) the dynamic mechanism of RASSF5 auto-inhibition. Our effort in elucidating the mechanism of Ras and Ras effectors results in 8 publications and offers a new venues for future therapeutic strategies.Item Quantifying the Organization and Dynamics of Excitable Signaling Networks(2020) Campanello, Leonard Joseph; Losert, Wolfgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The transmission of extracellular information through intracellular signaling networks is ubiquitous in biology---from single-celled organisms to complex multicellular systems. Via signal-transduction machinery, cells of all types can detect and respond to biological, chemical, and physical stimuli. Although studies of signaling mechanisms and pathways traditionally involve arrays of biochemical assays, detailed quantification of physical information is becoming an increasingly important tool for understanding the complexities of signaling. With the rich datasets currently being collected in biological experiments, understanding the mechanisms that govern intracellular signaling networks is becoming a multidisciplinary problem at the intersection of biology, computer science, physics, and applied mathematics. In this dissertation, I focus on understanding and characterizing the physical behavior of signaling networks. Through analysis of experimental data, statistical modeling, and computational simulations, I explore a characteristic of signaling networks called excitability, and show that an excitable-systems framework is broadly applicable for explaining the connection between intracellular behaviors and cell functions. One way to connect the physical behavior of signaling networks to cell function is through the structural and spatial analyses of signaling proteins. In the first part of this dissertation, I employ an adaptive-immune-cell model with a key activation step that is both promoted and inhibited by a microns-long, filamentous protein complex. I introduce a novel image-based bootstrap-like resampling method and demonstrate that the spatial organization of signaling proteins is an important contributor to immune-cell self regulation. Furthermore, I use the bootstrap-like resampling to demonstrate that the location of contact points between signaling proteins can provide mechanistic insight into how signal regulation is accomplished on the single-cell level. Finally, I probe the excitable dynamics of the system with a Monte Carlo simulation of nucleation-limited growth and degradation. Using the simulations, I show that careful balance between simulation parameters can elicit a tunable response dynamic. The spatiotemporal dynamics of signaling components are also important mediators of cell function. One key readout of the connection between signaling dynamics and cell function is the behavior of the cytoskeleton. In the second part of this dissertation, I use innate-immune-cell and epithelial-cell models to understand how a key cytoskeletal component, actin, is influenced by topographical features in the extracellular environment. Engineered nanotopographic substrates similar in size to typical extracellular-matrix structures have been shown to bias the flow of actin, a concept known as esotaxis. To measure this bias, I introduce a generalizable optical-flow-based-analysis suite that can robustly and systematically quantify the spatiotemporal dynamics of actin in both model systems. Interestingly, despite having wildly different migratory phenotypes and physiological functions, both cell types exhibit quantitatively similar topography-guidance dynamics which suggests that sensing and responding to extracellular textures is an evolutionarily-conserved phenomena. The signaling mechanisms that enable actin responses to the physical environment are poorly understood. Despite experimental evidence for the enhancement of actin-nucleation-promoting factors (NPFs) on extracellular features, connecting texture-induced signaling to overall cell behavior is an ongoing challenge. In the third part of this dissertation, I study the topography-induced guidance of actin in amoeboid cells on nanotopographic textures of different spacings. Using optical-flow analysis and statistical modeling, I demonstrate that topography-induced guidance is strongest when the features are similar in size to typical actin-rich protrusions. To probe this mechanism further, I employ a dendritic-growth simulation of filament assembly and disassembly with realistic biochemical rates, NPFs, and filament-network-severing dynamics. These simulations demonstrate that topography-induced guidance is more likely the result of a redistribution, rather than an enhancement, of NPF components. Overall, this dissertation introduces quantitative tools for the analysis, modeling, and simulations of excitable systems. I use these tools to demonstrate that an excitable-systems framework can provide deep, phenomenological insights into the character, organization, and dynamics of a variety of biological systems.Item Effect of electrostatic interactions on biomolecular self-assembly processes(2018) Xu, Hongcheng; Matysiak, Silvina; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Molecular level self-assembly processes are not only ubiquitous in living cells, but also widely applied in industry to synthesize and fabricate a variety of nanoscale biomaterials. The emergence of ordered aggregates from disordered components typically requires driving forces from electrostatic interactions to hydrophobic-hydrophilic effects. This thesis aims to elucidate the effect of electrostatic interactions, and the intricate balance between electrostatic and hydrophobic interactions in dictating spontaneous self-assembly processes with three case studies covering various types of biomolecules. For the first case study, we have examined the pH-induced polysaccharide hydrogel network formation. The polysaccharide molecule chitosan forms hydrogels composed of water-filled cross-linking polymer chains. The pH-responsive selfassembly behavior of chitosan hydrogel has been utilized in fabricating nanomaterials with a wide range of applications. To investigate the role of electrostatic interactions in the chitosan hydrogel network formation, we have developed a novel coarse-grained (CG) chitosan polymer model that captures the pH-dependent self-assembly behavior. The structural, mechanical, and thermodynamical properties of chitosan polymer hydrogel have been characterized well in the simulations and agree very well with experimental observations. For the second case study, the anticancer peptide folding induced by phospholipid membrane was investigated. Peptide folding in an aqueous environment is a self-assembly process that has been well studied over the years. However, the folding in a membranous environment is complicated by the heterogeneity in phospholipid distributions and membrane-peptide interactions. To provide information about the driving forces behind membrane peptide folding and the effect of lipid composition on folding behavior, my work has combined our recently developed Water-Explicit Polarizable Protein (WEPPRO) and Membrane (WEPMEM) model to explore the driving forces behind model anticancer peptide SVS-1 folding and how they can be affected by changing the membrane composition. For the third case study, we have studied the formation of nanodomains in mixed lipid bilayers. Phospholipid membranes are essential components in animal cells. The heterogeneous distribution of phospholipids on the membrane bilayer plays an important role in cellular structure and function such as signal transduction and membrane fusion. Interactions between a mixture of lipids and different ligands give rise to interesting patterns that are yet to be understood. Model lipid bilayers with a content of anionic lipids have been shown experimentally to be sensitive to the presence of certain ions. Monovalent cation Li+ induces membrane phase transition similarly as Ca2+ and Mg2+, while distinctive from other monovalent cations like Na+ and K+. We have evaluated the role of electrostatics interactions in the sizedependent cation-induced lipid nanodomain formation with binary mixed bilayers composed of zwitterionic and anionic lipids.Item UNDERSTANDING THE MOTILITY OF MOLECULAR MOTORS USING THEORY AND SIMULATIONS(2017) Goldtzvik, Yonathan Yitshak; Thirumalai, Devarajan; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Molecular motors are indispensable machines that are in charge of transporting cargoes within living cells. Despite recent advances in the study of these molecules, there is much that we still do not understand regarding the underlying mechanisms that allow them to efficiently move cargoes along polar cellular filaments. In this thesis, I report my investigation on two motor proteins superfamilies, dyneins and kinesins. Using theoretical modeling, we provide fundamental insight into their function. Dynein is a large motor that transports cargo along microtubules towards their negative pole. Unlike other motors, such as conventional kinesin, the motility of dynein is highly stochastic. We developed a novel theoretical approach, which reproduces a wide variety of its properties, including the unique step size distribution observed in experiments. Furthermore, our model enables us to derive several simple expressions that can be fitted to experiment, thus providing a physical interpretation. A less understood aspect of dynein is the complex set of allosteric transitions in response to ATP binding and hydrolysis, and microtubule binding. The resulting conformational transitions propel the motor forward to the minus end of the microtubule. Furthermore, its activity is regulated by external strain. Using coarse grained Brownian dynamics simulations, we show that a couple of insert loops in the AAA2, a sub domain in the AAA+ ring in the motor domain, play an important role in several of the alllosteric pathways. Kinesins are highly processive motor proteins that transport cargo along microtubules toward their positive poles. Experiments show that the kinesin motor domains propel the motor forward by passing each other in a hand-over-hand motion. However, there is a debate as to whether the motor domains do so in a symmetrical manner or an asymmetrical motion. Using coarse grained Brownian dynamics simulations of the kinesin dimer, we show that the kinesin stepping mechanism is influenced by the size of its cargo. Furthermore, we find that stepping occurs by a combinations of both the symmetric and asymmetric mechanisms. The results I present in this thesis are a testimony that theoretical approaches are invaluable to the study of molecular motors.Item MANIPULATING AND SIMPLIFYING THE INTERMOLECULAR INTERACTIONS IN LIQUID MIXTURES(2017) Gao, Ang; Weeks, John D; Chemical Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Long ranged intermolecular interactions have significant influence on the structure of the liquid and present serious challenges for computer simulations. In particular, the long ranged tail of Coulomb interaction usually needs to be calculated using Ewald summation or related techniques in computer simulation, which can be too time consuming to be carried out for large systems. Local Molecular Field(LMF) theory has been developed to simplify long-ranged Coulomb and Van der Waals interactions for nonuniform liquids by approximating these long ranged interactions as effective static single-particle fields. Despite the success LMF theory made in describing the structure of nonuniform liquids, it is not appropriate to use LMF theory to describe the structure of uniform liquid mixtures, since the dynamically moving unbalanced forces produced in mixture can not be captured by the framework of LMF theory. In this thesis, we propose a new framework which approximates the unbalanced forces produced in a mixture as effective intermolecular interactions. This new framework can simplify the long ranged intermolecular interactions and produce a mimic system with short ranged solvent-solvent interactions, which is much easier to simulate or analyze. Based on this framework and other techniques introduced in this thesis, we have constructed a "Short Solvent Model", which has noticeable advantages compared to the explicit solvent model and implicit solvent model. This framework has also been used to simplify the interactions of phase-separating mixtures. The impact of using this framework on the diffusion dynamics of the solutes has also been discussed. Possible application of this framework and the Short Solvent Model to biopolymers folding problems is briefly discussed.Item Development of Single-Molecule Force and Torque Measurement with Application to Nucleosome Disruption(2014) Chang, Jen-Chien; La Porta, Arthur; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Single-molecule force spectroscopy is a powerful method in biophysical research. The ability of detecting and manipulating single molecule finds applications in studies from DNA to cell, leads to various mechanism-based results. Among many tools in this field, optical traps is suitable for studies involving nucleic acids and its interaction with protein due to the high temporal and spatial resolution. While most experiments characterize force and displacement, the quest for manipulating and detecting torque and angular motion is increasing due to their significant role in many biological processes. This thesis mainly devotes to the development and application of an optical torque wrench which is capable of simultaneously controlling and measuring force, displacement, torque, and angular displacement of a single bio-molecule. First, angular manipulation of oblate polystyrene particles is demonstrated. Moreover, a new method for fabricating birefringent nanocylinders via nanosphere lithography is presented. Both particles are shown to provide stable angular trapping in a home-built optical torque wrench. We then apply them in measuring a single DNA molecule under force and torque. Mechanical stability of nucleosome under tension and torsion is also studied, followed by a theoretical model to elucidate the importance of torsion in such experiments. Finally, the effect of experimental parameters in a surface-based optical traps on measured kinetics of bio-molecule is detailed.