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Item Data for "A tug of war between filament treadmilling and myosin induced contractility generates actin ring"(2022-06-23) Ni, Qin; Wagh, Kaustubh; Pathni, Aashli; Ni, Haoran; Vashisht, Vishavdeep; Upadhyaya, Arpita; Papoian, Garegin A.; Upadhyaya, Arpita; Papoian, Garegin A.In most eukaryotic cells, actin filaments assemble into a shell-like actin cortex under the plasma membrane, controlling cellular morphology, mechanics, and signaling. The actin cortex is highly polymorphic, adopting diverse forms such as the ring-like structures found in podosomes, axonal rings, and immune synapses. The biophysical principles that underlie the formation of actin rings and cortices remain unknown. Using a molecular simulation platform, called MEDYAN, we discovered that varying the filament treadmilling rate and myosin concentration induces a finite size phase transition in actomyosin network structures. We found that actomyosin networks condense into clusters at low treadmilling rates or high myosin concentration but form ring-like or cortex-like structures at high treadmilling rates and low myosin concentration. This mechanism is supported by our corroborating experiments on live T cells, which exhibit ring-like actin networks upon activation by stimulatory antibody. Upon disruption of filament treadmilling or enhancement of myosin activity, the pre-existing actin rings are disrupted into actin clusters or collapse towards the network center respectively. Our analyses suggest that the ring-like actin structure is a preferred state of low mechanical energy, which is, importantly, only reachable at sufficiently high treadmilling rates.Item Integration and Competition in Immune Cell Models(2022) Bull, Abby L; Losert, Wolfgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cell motility plays an integral role in most biological processes. One principle of motility is the protrusions and retractions of cellular membranes called pseudopods. The physical force behind pseudopod formation is actin polymerization. As the physical driver, actin polymerization integrates the cells’ upstream biochemical signaling cascades and turns the signals into action as the combined output and readout of the state of the cell. Actin polymerization not only occurs within pseudopods but propagates throughout a cell in waves that can be understood and modeled as excitable media.This dissertation focuses on actin wave dynamics in the context of directed cell mi- gration using both experimental and numerical techniques. The directional guidance of cell migration is essential in physiological processes including embryonic development, cancer metastasis, and wound healing. In this thesis, I analyze how immune-like cells are guided by external stimuli that are common in wound environments: electric fields, chemical gradients, and surface texture. A focus of this work is on the emergent excitable wave behavior of actin, and the pseudopods they generate, in simplified, in vitro environments. Actin waves have been previously shown to respond to and be guided by the topography of an underlying substrate. Additionally, static quantifications of actin filaments during or after electric field stimulation have shown that filaments become asymmetrically distributed within the cytoplasm. In neutrophils, the combination of cues leads to higher control of cell motility by guiding the internal actin waves. Using an optical flow algorithm, I quantify the actin waves on multiple length scales to ascertain the role of each guidance cue in affecting cell motion. I find that the waves preferentially polymerize near and travel along the nanoridges. Actin waves nucleate preferentially on the cathode side and reorient the cell’s axis of polarity (i.e., the position of the dominant pseudopod). The second example of competing guidance cues involves studying the collective motion of cells in response to cell-cell signal relay in competition with surface topography. I use Dictyostelium discoideum cells as a model system for this work, as they migrate collectively due to signal relay. The signal relay of these cells is similar to many immune cell species. Using a combination of image analysis tools and a coarse-grained stochastic model, I find that guidance by nanoridges overrides the chemical signal relay and forces cells to migrate individually, suppressing streaming behavior. I model both the secretion and propagation of chemical signals using an excitable systems framework. This work highlights that bidirectional signals can be effective at suppressing cell-cell attraction and streaming motion. The response of immune cells to external stimuli in the wound environment is not universal. Macrophages, one of the largest immune cells, are observed to migrate away from the wound upon wound-induced electric field generation. In the third example, I study actin dynamics of M0 (resting) macrophage cells to elucidate how these cells interact with external electric fields. This cell type exhibits oscillatory actin waves at rest. With electric field stimulation, the oscillatory actin waves start to generate protrusions. Often, the protrusions begin with actin-depleted regions, indicating that contractile ele- ments are involved in conjunction with overall cell volume conservation. This thesis highlights the different methods in which actin waves integrate external cues, specifically electric fields, into cell responses that are cell-type specific.Item PHYSICAL FACTORS IN B CELL ACTIN DYNAMICS AND ACTIVATION(2016) Ketchum, Christina; Upadhyaya, Arpita; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Cells adapt to their changing world by sensing environmental cues and responding appropriately. This is made possible by complex cascades of biochemical signals that originate at the cell membrane. In the last decade it has become apparent that the origin of these signals can also arise from physical cues in the environment. Our motivation is to investigate the role of physical factors in the cellular response of the B lymphocyte. B cells patrol the body for signs of invading pathogens in the form of antigen on the surface of antigen presenting cells. Binding of antigen with surface proteins initiates biochemical signaling essential to the immune response. Once contact is made, the B cell spreads on the surface of the antigen presenting cell in order to gather as much antigen as possible. The physical mechanisms that govern this process are unexplored. In this research, we examine the role of the physical parameters of antigen mobility and cell surface topography on B cell spreading and activation. Both physical parameters are biologically relevant as immunogens for vaccine design, which can provide laterally mobile and immobile antigens and topographical surfaces. Another physical parameter that influences B cell response and the formation of the cell-cell junction is surface topography. This is biologically relevant as antigen presenting cells have highly convoluted membranes, resulting in variable topography. We found that B cell activation required the formation of antigen-receptor clusters and their translocation within the attachment plane. We showed that cells which failed to achieve these mobile clusters due to prohibited ligand mobility were much less activation competent. To investigate the effect of topography, we use nano- and micro-patterned substrates, on which B cells were allowed to spread and become activated. We found that B cell spreading, actin dynamics, B cell receptor distribution and calcium signaling are dependent on the topographical patterning of the substrate. A quantitative understanding of cellular response to physical parameters is essential to uncover the fundamental mechanisms that drive B cell activation. The results of this research are highly applicable to the field of vaccine development and therapies for autoimmune diseases. Our studies of the physical aspects of lymphocyte activation will reveal the role these factors play in immunity, thus enabling their optimization for biological function and potentially enabling the production of more effective vaccines.Item The role of actin netoworks in cellular mechanosensing(2015) Azatov, Mikheil; Upadhyaya, Arpita; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis. In addition to stiffness, the local geometry or topography of the surface has been shown to modulate the movement, morphology, and cytoskeletal organization of cells. However, the effect of topography on fluctuations of intracellular structures, which arise from motor driven activity on a viscoelastic actin network are not known. I have used nanofabricated substrates with parallel ridges to show that the cell shape, the actin cytoskeleton and focal adhesions all align along the direction of the ridges, exhibiting a biphasic dependence on the spacing between ridges. I further demonstrated that palladin bands along actin stress fibers undergo a complex diffusive motion with velocities aligned along the direction of ridges. These results provide insight into the mechanisms of cellular mechanosensing of the environment, suggesting a complex interplay between the actin cytoskeleton and cellular adhesions in coordinating cellular response to surface topography. Overall, this work has advanced our understanding of mechanisms that govern cellular responses to their physical environment.