Physics

Permanent URI for this communityhttp://hdl.handle.net/1903/2269

Browse

Search Results

Now showing 1 - 10 of 10

High Resolution Mapping of Intracellular Mechanical Properties during Key Stages of Cancer Progression
(2022) Nikolic, Milos; Scarcelli, Giuliano; Tanner, Kandice; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The mechanical phenotype of the living cell is critical for survival following deformations due to confinement and fluid flow. Furthermore, in recent years mechanical interaction between cells and the cellular environment has been implicated as one of the key regulators of cancer progression and malignant transformation. Due to the need to better understand the mechanical properties of invasive cells and how the mechanical phenotype plays a role in cancer progression, several microrheology techniques have been applied to study cell mechanics in a range of in vitro environments. However, many of these techniques have been limited either to studying cells in only one type of environment (e.g. 2D), with limited resolution, or with invasive probes. To begin to address this question, in this dissertation we aim to quantify the mechanical state of cells in a broader range of different contexts and geometries. To do this we use Brillouin microscopy, a non-contact, label free, non-invasive technique which enables us to probe the mechanical response of cells in a wide range of complex microenvironments. Here we introduce an improved Brillouin microscope with improved signal and acquisition speed which enables us to perform biological studies at the single cell level. Using the improved Brillouin microscopy, we find that individual cells can be softer as function of the invasive potential, but that cells are able to dynamically change their mechanical properties across many different contexts. We validate our results using complementary microrheology methods such as atomic force microscopy and broadband optical tweezer microrheology. We directly observe changes in cell mechanics in key processes relevant for metastatic migration, as well as a function of external and internal parameters like morphology, ECM properties, intracellular factors, and cell-cell cooperativity during multicellular tissue organization. These results support the paradigm that the mechanical state of a cell is a dynamic parameter that varies as a consequence of the microenvironmental and functional context, in addition to the observable changes in cell’s mechanical properties due to malignant transformation.
MODULATION OF INTRACELLULAR EXCITABLE SYSTEMS THROUGH PHYSICAL MICROENVIRONMENTS
(2022) Yang, Qixin; Losert, Wolfgang; Physics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Cells can sense various external cues, such as chemoattractants, electric fields (EFs), and the physical properties of their surroundings. This ability is essential to maintain normal physiological processes, and malfunctions may lead to severe diseases, such as cancer and autoimmune disease. Recent studies have observed that waves of colocalized actin, associated with its upstream signaling molecules, drive cell directional migration. Furthermore, a coupled signal transduction excitable system – cytoskeleton excitable system (STEN-CEN) has been revealed to regulate wave formation in non-neural cell types. The system display hallmarks of excitability, such as refractory periods, all-or-none type responses, and wave behavior. It is traditionally believed that different cells employ different migrational strategies. However, recent studies find that changing the states of STEN-CEN leads to the transition of migrational modes within a single cell type. This dissertation uses molecular experiments, computer-vision quantification techniques, and modeling to understand how cells sense different external cues. Numerous studies have shown that there exist multiple, parallel signal pathways that sense certain external stimuli, which suggests that the external signal is not sensed by a single molecule. Here I investigate the possibility that STEN-CEN waves act as a sensing unit for external cues. To overcome the challenge that wave dynamics are coupled with cell motion in normal-sized cells, I electro-fused tens of Dictyostelium discoideum (D. d) cells together to form giant cells. In giant cells, waves are no longer localized at the cell perimeter. The larger basal membrane area provides the opportunity to study subcellular dynamics. I recorded the dynamics of F-actin and phosphatidylinositol (3,4,5)-triphosphate (PIP3), which are indicative of CEN and STEN, respectively. To decouple STEN and CEN further, I applied a variety of chemical perturbations to suppress/activate STEN and CEN separately. Because the wave properties characterize the stage of STEN-CEN, I developed a series of quantification tools to measure wave area, duration, and speed. I collaborated with theorists to create a reaction-diffusion system model that recreates the experimental results. The dissertation focuses on studying the role of STEN-CEN waves in sensing nanotopography and EF. CEN is found to sense nanotopographical cues directly, forming long-lasting F-actin puncta at ridges in the absence of STEN. At the same time, STEN is essential for long-range wave behaviors for macro-domain nanoridge sensing. STEN and CEN cooperate in the sensing of EF signals. According to quantitative studies of wave properties, nanotopography changes the dimensionality and lifetime of waves, whereas EF can alter the activation thresholds of the intracellular systems. In summary, this thesis shows that the excitable biomechanical and biochemical wave systems act as the sensing units for the physical properties of the extracellular environment. As a result, cells can dynamically sense their surroundings and coordinate intracellular processes to migrate under guidance from external cues.
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.
DISSECTING THE GENE REGULATORY FUNCTION OF THE MYC ONCOGENE WITH SINGLE-MOLECULE IMAGING
(2020) Patange, Simona; Larson, Daniel R; Girvan, Michelle; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The MYC oncogene contributes to an estimated 100,000 cancer-related deaths annually in the United States and is associated with aggressive tumor progression and poor clinical outcome. MYC is a nuclear transcription factor that regulates a myriad of cellular activities and has direct interactions with hundreds of proteins, which has made a unified understanding of its function historically difficult. In recent years, several groups have put forth a new hypothesis that questions the prevailing view of MYC as a gene-specific transcription factor and instead envision it as a global amplifier of gene expression. Instead of being an on/off switch for transcription, MYC is proposed to act as a `volume knob' to amplify and sustain the active gene expression program in a cell. The scope of the amplifier model remains controversial in part because studies of MYC largely consist of cell population-based measurements obtained at fixed timepoints, which makes distinguishing direct from indirect consequences on gene expression difficult. A high-temporal, high-spatial precision viewpoint of how MYC acts in single living cells does not exist. To evaluate the competing hypotheses of MYC function, we developed a single-cell assay for precisely controlling MYC and interrogating the effects on transcription in living cells. We engineered `Pi-MYC,' an optogenetic variant of MYC that is biologically active, can be visualized under the microscope, and can be controlled with light. We combined Pi-MYC with single-molecule imaging methods to obtain the first real-time observations of how MYC affects RNA production and transcription factor mobility in single cells. We show that MYC increases the duration of active periods of genes population-wide, and globally affects the binding dynamics of core transcription factors involved in RNA Polymerase II transcription complex assembly and productive elongation. These findings provide living, single-cell evidence of MYC as a global amplifier of gene expression, and suggests the mechanism is by stabilizing the active period of a gene through interactions with core transcription machinery.
RECEPTOR MOBILITY AND CYTOSKELETAL DYNAMICS AT THE IMMUNE SYNAPSE: THE ROLE OF ACTIN REGULATORY PROTEINS
(2020) Rey Suarez, Ivan Adolfo; Upadhyaya, Arpita; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Spatial and temporal regulation of actin and microtubule dynamics is of utmost importance for many cellular processes at different sub-cellular length scales. This is particularly relevant for cells of the immune system, which must respond rapidly and accurately to protect the host, where B cells and T cells are the main players during the adaptive immune response. An understanding of the biophysical principles underlying cytoskeletal dynamics and regulation of signaling will help elucidate the fundamental mechanisms driving B and T cell immune response. B cell receptor (BCR) diffusivity is modulated by signaling activation, however the factors linking mobility and signaling state are not completely understood. I used single molecule imaging to examine BCR mobility during signaling activation and a novel machine learning based method to classify BCR trajectories into distinct diffusive states. Inhibition of actin dynamics downstream of the actin nucleating factors Arp2/3 and formins resulted decreased BCR mobility. Loss of the Arp2/3 regulator, N-WASP, which is associated with enhanced signaling, leads to a predominance of BCR trajectories with lower diffusivity. Furthermore, loss of N-WASP reduces diffusivity of the stimulatory co-receptor CD19, but not that of unstimulated FcγRIIB, an inhibitory co-receptor. Our results implicate the dynamic actin network in fine-tuning receptor mobility and receptor-ligand interactions, thereby modulating B cell signaling. Activation of T cells leads to the formation of the immunological synapse (IS) with an antigen presenting cell (APC). This requires T cell polarization and coordination between the actomyosin and microtubule cytoskeleton. The interactions between the different cytoskeletal components during T cell activation are not well understood. I use high-resolution fluorescence microscopy to study actin-microtubule crosstalk during IS formation. Microtubules in actin rich zones display more deformed shapes and higher dynamics compared to MTs at the actin-depleted region. Chemical inhibition of formin and myosin activation reduced MT deformations, suggesting that actomyosin contractility plays an important role in defining MT shapes. Interestingly MT growth was slowed by formin inhibition and resulting enrichment of Arp2/3 nucleated actin networks. These observations indicate an important mechanical coupling between the actomyosin and microtubule systems where different actin structures influence microtubule dynamics in distinct ways.
UNDERSTANDING CELL DIFFERENTIATION AND MIGRATION WITH MULTIVARIATE CELL SHAPE QUANTIFICATION
(2018) Chen, Desu; Losert, Wolfgang; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
This thesis focuses on developing multivariate quantification methods of cell shape to facilitate understanding of physiological processes such as cell differentiation and migration. Cell shape reflects complex intracellular and extracellular factors affecting cell function. However, analyses associating cell shape and cell function need to account for challenges of multivariate interpretation, single-cell heterogeneity and reproducibility. Specifically, Human Bone Marrow Stromal Cells (hBMSCs) population in nanofiber scaffolds can develop osteogenic differentiation without chemical cues. I developed a method based on Support Vector Machine (SVM) to train classifiers as boundaries in the shape metric spaces to identify the day 1 cell shape phenotype of hBMSCs population in nanofiber scaffolds. To reduce the effect of single-cell heterogeneity in the population phenotyping, the “supercell” method was introduced to generate average measurements of small groups of cells for SVM training. To overcome the multivariate complexity in biological interpretation, a feature selection process was implemented to select the most significant cell shape metrics. The predictive potential of the achieved classifiers was validated by subsampling. It was found that in nanofiber scaffolds, hBMSCs were narrower with more elongated and dendritic shape and rougher cell boundary. Further, I found that increase in nanofiber density enhanced hBMSCs osteogenic differentiation potential. The pre-trained classifiers successfully predict the modulation of nanofiber density on hBMSCs fates and single-cell shape. While much can be learned from cell shapes alone, it is important to note that shapes can change with time, especially for migrating cells. The second part of my thesis focuses on analysis of shape dynamics. Quantification for cell shape dynamics at the subcellular level was developed to understand the coordination of the subcellular myosin localization and the cell boundary dynamics in neutrophil migration in vivo. The correlation of myosin localization and positive cell boundary curvature was identified as a unique in vivo neutrophil migration phenotype. Correlations of myosin localization and local cell boundary dynamics in vivo were found to be affected by cell motility and polarization. This analysis framework shown here can also be used to study the link between other subcellular features and neutrophil migration and shape dynamics.
Structure and Dynamics of Microtentacles
(2016) Ory, Eleanor Claire-higgins; Losert, Wolfgang; Martin, Stuart; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
While modern cancer diagnostics and treatments are often interpreted through a biomolecular perspective, cancer abounds with many mechanically interesting characteristics and questions. Metastasis, the process by which a primary tumor spreads and forms a second tumor in a distant site is currently responsible for 90% of cancer fatalities [1–3]. One of the key limiting steps in metastasis is extravasa- tion; the process by which a circulating tumor cell (CTC) moves from the blood- stream into surrounding tissue. So far, most in vitro studies in metastasis focus on cell migration and invasiveness with few focused on reattachment of cells to a blood vessel wall, and extravasation. One possible attachment mechanism involves tubulin-based structures called microtentacles, which have been observed to poke into crevices between cells that line blood vessels. Based on biomolecular assays, the current hypothesis is that microtentacles are formed as the result of unbalanced, mechanical interactions between microtubules and actin, allowing microtubules to push the plasma membrane beyond the cell body. The focus of my thesis is to gain insights into the dynamics and mechanical properties of microtentacles and evaluate how microtentacles may be altered by cytoskeletal drugs. In this thesis, I will measure changes in microtubule dynamics using cytoskele- tal drugs to the actomyosin cortex and microtubules. The first study presented examines how drug treatments targeting the actomyosin cortex impact microtubule dynamics for attached cells. The results of the first study demonstrate that weaken- ing the actomyosin cortex allows microtubule end-binding-protein-1 (EB1) to move beyond the cell body boundary. Weakening the actomyosin cortex also results in changes to the speed and straightness of microtubule growth. In the second study, an image analysis framework is presented to quantify microtentacles as well as an eval- uation of the dynamics of microtubules in suspended cells. The study demonstrates a successful image analysis technique that can evaluate microtentacle phenotype for both free-floating and tethered cells as well as dynamics for tethered cells. This second study shows that while microtubule stabilizing drug treatment with Taxol increases total microtentacle phenotype, it also reduces microtentacle dynamics. On the other hand, while microtubule destabilizing drug treatment Colchicine decreases total microtentacle phenotype, Colchicine also reduces microtentacle dynamics. As a summary and outlook, I present a mechanical framework and present hypotheses for 4 different genetic modifications spanning a spectrum of different cytoskeletal states. I also show preliminary, qualitative results for 3 out of the 4 different cell lines. Critical to evaluating microtentacles within this physical framework is a direct mechanical assay; here, I show preliminary work taken at the University of Leipzig on an optical stretcher. Given that microtentacles have demonstrated to be a sufficient prerequisite for reattachment, better understanding of what circumstances lead to microtentacles is a critical basic research question. My work applies a physical perspective to the bal- ance between the actomyosin cortex and microtubules and demonstrates changes in microtubule dynamics. Such work contributes towards the possibility of identifying morphological and dynamics signatures of CTCs with higher metastatic potential.
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.
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.
Molecular basis of the kinematics of the kinesin step
(2012) Zhang, Zhechun; Thirumalai, Devarajan; Biophysics (BIPH); Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
Kinesin is an ATP-dependent cellular transporter that ferries cargos towards the plus-end of a microtubule. Despite significant advances in experiments, which have provided deep insights into the motility of kinesin, the molecular events that occur in a single step have not been fully resolved. In order to provide these details, this thesis develops a structure of the complex between kinesin and microtubule, and devises new simulation methods to probe the stepping kinetics over a wide range of conditions. Hundreds of molecular movies of kinesin walking on the microtubule are generated using coarse-grained simulation methods. Analysis of these movies shows that there are three major stages in the stepping kinetics of kinesin. In addition, an allosteric network within kinesin, responsible for controlling nucleotide release, is identified using microsecond all-atom simulations. These simulations are used to answer two important questions. First, does kinesin move by a "power stroke" or by diffusion? During a single step, the trailing head of the kinesin detaches from the microtubule, passes the microtubule-bound leading head, and attaches to the target binding site 16 nm away. The target binding site, however, is one of eight accessible binding sites on the microtubule. Is it possible that the "power stroke" (a large conformational change) in the leading head, pulls the trailing head into the neighborhood of the target binding site? This remained unclear because the fraction of the 16 nm step associated with the power stroke and diffusion had never been quantified. Second, how does the microtubule accelerate ADP release from kinesin, which is a key step in completing a single step? The ADP binding site of kinesin is more than 1.5 nm away from the microtubule binding surface. Therefore, the microtubule must affect the ADP binding site through an allosteric mechanism. However, the structural basis for transmitting signals through the underlying allosteric network was previously unknown. Analysis of hundreds of kinesin steps generated using coarse-grained simulations showed that the power stroke associated with the docking of the neck linker to the leading head, is responsible for only 4 nm of the 16 nm step, and the remaining 12 nm is covered by diffusion. However, the power stroke in the leading head constrains the diffusion of the trailing head, decreases the probability of side steps, and therefore biases the trailing head, to the target binding site. Additional all-atom simulations of the ADP-kinesin-microtubule complex, revealed a surprisingly simple allosteric network within kinesin that explains the acceleration of ADP release upon microtubule binding. The allosteric network also explains two additional experimental observations on ADP release from kinesin.

Physics

Browse

Filters

Settings

Sort By

Results per page

Search Results