Physics

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Subject: search.filters.subject.Cytoskeleton

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    Collective dynamics of astrocyte and cytoskeletal systems
    (2024) Mennona, Nicholas John; Losert, Wolfgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Advances in imaging and biological sample preparations now allow researchersto study collective behavior in cellular networks with unprecedented detail. Imaging the electrical signaling of neuronal networks at the cellular level has generated exciting insights into the multiscale interactions within the brain. This thesis aims at a complementary view of the general information processing of the brain, focusing on other modes of non-electrical information. The modes discussed are the collective, dynamical characteristics of non-electrically active, non-neuronal brain cells, and mechanical systems. Astrocytes are the studied non-neuronal brain cells, and the cytoskeleton is the studied dynamic, mechanical system consisting of various filamentous networks. The two filamentous networks studied herein are the actin cytoskeleton and the microtubule network. Techniques from calcium imaging and cell mechanics are adapted to measure these often overlooked information channels, which operate at length scales and timescales distinct from electrical information transmission. Structural, astrocyte actin images, microtubule structural image sequences, and the calcium signals of collections of astrocytes are analyzed using computer vision and information theory. Filamentous alignment of actin with nearby boundaries reveals that stellate astrocytes have more perpendicularly oriented actin than undifferentiated astrocytes. Harnessing the larger length scale and slower dynamical time scale of microtubule filaments relative to actin filaments led to the creation of a computer vision tool to measure lateral filamentous fluctuations. Finally, we adapt information theory to the analog calcium (Ca2+) signals within astrocyte networks classified according to subtype. We find that, despite multiple physiological differences between immature and injured astrocytes, stellate (healthy) astrocytes have the same speed of information transport as these other astrocyte subtypes. This uniformity in speed persists when either the cytoskeleton (Latrunculin B) or energy state (ATP) is perturbed. Astrocytes, regardless of physiological subtype, tend to behave similarly when active under normal conditions. However, these healthy astrocytes respond most significantly to energy perturbation, relative to immature and injured astrocytes, as viewed through cross-correlation, mutual information, and partitioned entropy. These results indicate the value of drawing information from structure and dynamics. We developed and adapted tools across scales from nanometer scale alignment of actin filaments to hundreds of microns scale information dynamics in astrocyte networks. Including all potential modalities of information within complex biological systems, such as the collective dynamics of astrocytes and the cytoskeleton in brain networks is a step toward a fuller characterization of brain functioning and cognition.
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    Simulating membrane-bound cytoskeletal dynamics
    (2023) Ni, Haoran; Papoian, Garegin A.; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The cell membrane defines the shape of the cell and plays an indispensable role in bridging intra- and extra-cellular environments. The membrane, consisting of a lipid bilayer and various attaching proteins, mechanochemically interacts with the active cytoskeletal network that dynamically self-organizes, playing a vital role in cellular biomechanics and mechanosensing. Comprehensive simulations of membrane-cytoskeleton dynamics can bring insight in understanding how the cell mechanochemically responds to external signals, but a computational model that captures the complex cytoskeleton-membrane with both refined details and computational efficiency is lacking. To address this, we introduce in this thesis a triangulated membrane model and incorporate it with the active biological matter simulation platform MEDYAN ("Mechanochemical Dynamics of Active Networks"). This model accurately captures the membrane physical properties, showing how the membrane rigidity, the structure of actin networks and local chemical environments regulate the membrane deformations. Then, we present a new method for simulating membrane proteins, using stochastic reaction-diffusion sampling on unstructured membrane meshes. By incorporating a surface potential energy field into the reaction-diffusion sampling, we demonstrate rich membrane protein collective behaviors such as confined diffusion, liquid-liquid phase separation and membrane curvature sensing. Finally, in order to capture stretching, bending, shearing and twisting of actin filaments which are not all available with traditional actomyosin simulations, we introduce new finite-radius filament models based off Cosserat theory of elastic rods, with efficient implementation using finite-dimensional configurational spaces. Using the new filament models, we show that the filaments' torsional compliance can induce chiral symmetry breaking in a cross-linked actin bundle. All the new models are implemented in the MEDYAN platform, shedding light on whole cell simulations, paving way for a better understanding of the membrane-cytoskeleton system and its role in cellular dynamics.
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    Non-equilibrium Thermodynamics of Cytoskeletal Self-organization
    (2021) Floyd, Carlos Shadoan; Papoian, Garegin A; Jarzynski, Christopher; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    The actin-based cytoskeleton is a polymer network that plays an essential role in cell biology. By self-organizing into various local architectures, the cytoskeleton performs physiological functions that allow the cell to physically interact with its environment. It is also an example of biological active matter, consuming chemical free energy at a local scale to produce directed motion and do mechanical work. While it is well-known that cytoskeletal free energy transduction occurs, it has been a challenge to say anything quantitative about this far-from-equilibrium process due to the difficulty of making the necessary experimental measurements. This lack of methodology to quantify cytoskeletal energetics significantly hinders our understanding of the self-organization process underlying the cytoskeleton's physiological functionality. To address this research gap, we develop in this thesis an explicit computational method to quantify chemical and mechanical free energy changes during simulated cytoskeletal self-organization using the software package MEDYAN (Mechanochemical Dynamics of Active Networks). We then apply this tool in several studies to advance our understanding of the self-organization process and its thermodynamic characteristics. For instance, we analyze the thermodynamic efficiency of mechanical stress generation and the network's time-dependent dissipation rates under a range of network conditions. We also investigate the recent experimentally discovered phenomenon of cytoskeletal avalanches, which we identify in simulation as anomalous mechanical dissipation events. Our analysis clarifies the phenomenology and underlying mechanism of these avalanche events, which we propose may play an important role in cellular information processing. The in silico method developed in this thesis provides a new perspective on cytoskeletal self-organization and may be extended to investigate other biological active matter systems.
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    Mechanobiology of T cell activation
    (2015) Hui, King Lam; Upadhyaya, Arpita; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Cells can sense and respond to the physical environment through generation and transmission of mechanical forces from the surroundings to the cell interior and from one cell to another. This dissertation focuses on mechanosensing by T cells, key players in the adaptive immune system, which form a strong line of defense against infections by their ability to recognize foreign molecules and develop an appropriate response. T cells form close contact with an opposing antigen presenting cell upon recognition of protein fragments derived from infecting pathogens (antigens). Recent studies have shown that externally applied forces can trigger biochemical signaling in T cells. How forces are internally generated by T cells, involved in signaling and transmitted at the level of the cell interface, remains unclear. In this thesis, we investigate the molecular mechanisms of force generation by T cells and their response to forces and the stiffness of the opposing surface. We have quantitatively characterized the initial phase of T cell contact with a model of antigen-bearing surfaces. We observe that T cells spread on such substrates and that the kinetics of spreading follows a universal function, with the spreading rate dependent on actin polymerization and myosin II activity. Altering cell-substrate adhesions leads to qualitative changes in cell spreading dynamics and wave-like patterns of actin dynamics. We then used soft elastic substrates with stiffness comparable to that of antigen presenting cells, to measure the forces generated by T cells during activation. Perturbation experiments reveal that these forces are largely due to actin assembly and dynamics, with myosin contractility contributing to the development of traction forces but not its maintenance. We find that Jurkat T-cells are mechanosensitive, with both traction forces and signaling dynamics exhibiting sensitivity to the stiffness of the substrate. We further demonstrate that dynamics of the T cell microtubule cytoskeleton also participates in regulating forces at the cell-substrate interface, through the Rho/ROCK pathway which regulates myosin II light chain phosphorylation. Overall, this work highlights physical force as an essential mediator that connects stiffness sensing to intracellular signaling, which then directs gene expression and eventually the immune response in T cells.
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    THE ROLE OF THE ACTIN CROSSLINKER PALLADIN: FROM RECONSTITUTED NETWORKS TO LIVE CELLS
    (2013) Grooman, Brian; Upadhyaya, Arpita; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Biophysics is a rapidly growing area of research. New discoveries continue to show the importance of mechanical phenomenon in biological processes, even at the cellular and sub-cellular levels. The complexity of living cells, coupled with their small size makes their study particularly difficult. Palladin is an actin-crosslinker that has not yet been studied as much as other actin-crosslinkers. It localizes with alpha-actinin in stress fibers in many adult cell types. Palladin's exact purpose is still unknown. Through in-vitro studies of reconstituted actin networks we gain insight into the mechanical importance of this novel protein, and show that when partnered with α-actinin, palladin efficiently enhances the network stiffness. Pancreatic Stellate Cells are responsible for maintaining organ integrity, and their malignant counterparts are responsible for one of the most deadly forms of cancer. Interestingly, palladin is shown to be up-regulated in tumors derived from these cells. By studying the stiffness of the cells with and without palladin (via genetic manipulation) we investigate the mechanical importance of palladin in vivo. GFP labelled palladin can serve as a useful marker because it naturally localizes into a regular pattern along stress fibers. Combined with image processing, this makes tracking local strain rates within the cell possible. Pancreatic stellate cells will respond to an applied force by actively contracting their stress fibers. The dynamics of these responses are quantified by tracking the spots of palladin. Through various pharmacological manipulations we study possible signaling pathways that lead from an applied force to stress fiber contraction. Overall, this work explores the mechanical importance of palladin and also investigates the mechanical properties of tumor-associated pancreatic stellate cells, neither of which have been previously studied. Our work shows that palladin controls network stiffness in-vitro, but not in-vivo, suggesting a yet undiscovered purpose. We have also shown that pancreatic stellate cells are in the same range of stiffness as other fibroblasts, and can actively respond to external forces. All of these findings contribute to an increased understanding of the complex systems which govern the mechanical properties of living material.