Download or read book Molecular Scale Dynamics of T Cell Immunological Synapse Components by Advanced and Super resolution Fluorescence Microscopy written by George Ashdown and published by . This book was released on 2017 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book Molecular Scale Dynamics of T Cell Immunological Synapse Components by Advanced and Super resolution Flourescence Microscopy written by George W. Ashdown and published by . This book was released on 2017 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Finally, vesicle cargo was shown to correlate with different vesicle populations, based on their membrane order, demonstrating vesicle order may mirror the heterogeneous nature of the PM. These results demonstrate the complex biophysical processes that control the T cell immunological synapse are an amalgamation of cytoskeletal organisation, vesicle dynamics and membrane polarity.
Download or read book The Immune Synapse written by Cosima T. Baldari and published by Springer Nature. This book was released on 2023-04-27 with total page 518 pages. Available in PDF, EPUB and Kindle. Book excerpt: This new collection features the most up-to-date essential protocols that are currently being used to study the immune synapse. Beginning with methods for making biophysical measurements, the volume continues by covering the cell biology of synapses, methods for advanced substrate engineering, mechanobiology topics, new technologies to describe and manipulate synaptic components, as well as methods related to sites of action and immunotherapy. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and fully updated, The Immune Synapse: Methods and Protocols, Second Edition serves as an ideal practical guide for researchers working in this dynamic field. Chapters 5, 11, 18, 27, 30, and 32 are available open access under a Creative Commons Attribution 4.0 International License via link.springer.com.
Download or read book The Immunological Synapse Part B written by and published by Elsevier. This book was released on 2023-07-27 with total page 224 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Immunological Synapse - Part B, Volume 178 in the Methods in Cell Biology series provides state-of-the-art methods for the study of the immunological synapse. This first volume covers various aspects on T cell and natural killer (NK) cell synapses, including imaging polarized granule release using TIRF microscopy, analysis of actin reorganization and centrosome polarization, redirected degranulation, live cell-imaging to quantify cytotoxic and chemotactic dynamics, quantification of interactions between APCs and T cells, assessment of membrane lipid state at the immunological synapse, proteomic analysis and imaging of NK-tumor cell interaction, evaluating natural killer cell effector functions against breast cancer cells derived from human tumor tissue, evaluation of chimeric antigen receptor (CAR)-modified cell immunological synapse quality using the glass-supported planar lipid bilayer, and monitoring potency of therapeutic CAR T cells. It also includes one chapter on quantification of lymphocytic choriomeningitis virus specific T cells and LCMV viral titers, and one chapter on the murine antibody-dependent cellular phagocytosis assay. Covers various methods related to the study of the immunological synapse Includes detailed, point-by-point, methods as well as various important notes Provides the authority and expertise from an international board of leading scientists
Download or read book The Immunological Synapse Part A written by and published by Elsevier. This book was released on 2023-01-16 with total page 208 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Immunological Synapse, Part A, Volume 173 in the Methods in Cell Biology series provides state-of-the-art methods for the study of the immunological synapse. Sections cover Imaging polarized granule release at the cytotoxic T cell immunological synapse using TIRF microscopy: control by polarity regulators, Analysis of centrosomal area actin reorganization and centrosome polarization upon lymphocyte activation at the immunological synapse, P815-based redirected degranulation assay to study human NK cell effector functions, Cytotoxic and Chemotactic Dynamics of Natural Killer Cells Quantified by Live-cell Imaging, Quantification of interaction frequency between antigen-presenting cells and T cells by conjugation assay, and more. Other chapters focus on the Study of the Effects of NK-Tumor Cell Interaction by Proteomic Analysis and Imaging, Quantification of lymphocytic choriomeningitis virus specific T cells and LCMV viral titers, Quantification of lymphocytic choriomeningitis virus specific T cells and LCMV viral titers, An in vitro model to monitor natural killer cell effector functions against primary breast cancer, and Standardized Protocol for the Evaluation of Chimeric Antigen Receptor (CAR)-modified Cell Immunological Synapse Quality using the Glass-supported Planar L. Covers various methods related to the study of the immunological synapse Includes detailed, point-by-point, methods as well as various important notes Provides the authority and expertise from an international board of leading scientists
Download or read book Plasma Membrane Dynamics and Pattern Formation During T Cell Activation written by Adam D. Douglass and published by . This book was released on 2006 with total page 296 pages. Available in PDF, EPUB and Kindle. Book excerpt: T cell activation entails a complex series of signal transduction events that begin with ligation of the T cell receptor by a cognate antigen, displayed on the surface of an antigen presenting cell. During T cell signaling proteins in the cell surface partition from one another into large, stereotyped molecular pattern that is known as the immunological synapse. This thesis attempts to understand the mechanisms by which this segregation occurs, and uses a number of fluorescence imaging techniques---in particular, single molecule microscopy---to this end. Contrary to reports that suggest a role for lipid raft domains in patterning the synapse, we find that protein-protein interactions, and not putative lipid raft associations, are a strong driving force in synapse formation. We also develop a method for reconstituting synapse formation in an immortalized T cell line that allows us to perform single molecule imaging on a fluid substrate of a defined composition. A common finding is that both passive mechanisms, involving diffusional trapping and exclusion, as well as active mechanisms, involving actin-driven transport, can act in concert to shape the immunological synapse.
Download or read book High resolution Imaging of Natural Killer Cell Immunological Synapses written by and published by . This book was released on 2011 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt:
Download or read book The Immune Synapse as a Novel Target for Therapy written by Luis Graca and published by Springer Science & Business Media. This book was released on 2007-12-18 with total page 201 pages. Available in PDF, EPUB and Kindle. Book excerpt: This volume gives an overview on the progress in immune synapse research, from basic science to clinical trials, and the major mechanisms involved. It discusses how interfering with T cell activation may lead to immune tolerance, immune modulation, and the recruitment of regulatory T cells; the role of monoclonal antibodies in tolerance induction; and mechanisms maintaining dominant tolerance.
Download or read book Monitoring Cellular Interactions During T Cell Activation at the Single Molecule Level Using Semiconductor Quantum Dots written by and published by . This book was released on 2005 with total page 8 pages. Available in PDF, EPUB and Kindle. Book excerpt: Enhanced peptide-coated quantum dots (with high brightness and high saturation intensity) were developed. Two high-affinity targeting "velcro-pairs" based on avidin-biotin and fluorescine-antibody interactions were demonstrated and used to specifically target single proteins in membranes of live cells. Single molecule spectroscopy and imaging of individual quantum dot-labeled lipid rafts receptors were performed. Software tools were developed to analyze individual diffusion and trafficking trajectories. These studies provide strong support for the lipid raft hypothesis. Cloning and fusion of avidin to four immune synapse components were achieved. These mutants are being characterized by flow cytometry and fluorescence microscopy. This report provides information on the following new findings: (1) hybrid approach to the synthesis of highly luminescent infrared CdTe/ZnS and CdHgTe/ZnS quantum dots; (2) bioactivation and cell targeting of semiconductor CdSe/ZnS quantum dots with phytochelatin-related peptides; (3) development of bright Cd+ rich peptide-coated quantum dots; (4) comparison of the photophysical and colloidal properties of biocompatible quantum dots using fluorescence correlation spectroscopy (FCS); (5) testing the lipid raft hypothesis by single molecule imaging of targeted peptide-coated quantum dots; and (6) molecular cloning and fusion of avidin to immunological synapse (IS) components.
Download or read book Methods for Imaging Cell Membranes written by Luca Panconi and published by CRC Press. This book was released on 2023-12-21 with total page 137 pages. Available in PDF, EPUB and Kindle. Book excerpt: · Measuring membrane protein distributions using single-molecule localisation microscopy (SMLM) · Measuring membrane protein dynamics and diffusion using fluorescence correlation spectroscopy (FCS) · Mapping membrane lipid backing using environmentally sensitive fluorescence probes · Mapping membrane thickness and rigidity using atomic force microscopy · Mapping membrane proteins and the cytoskeleton using electron microscopy
Download or read book Cellular Imaging Techniques for Neuroscience and Beyond written by Floris G. Wouterlood and published by Academic Press. This book was released on 2012-12-06 with total page 298 pages. Available in PDF, EPUB and Kindle. Book excerpt: The imaging of small cellular components requires powerful instruments, and an entire family of equipment and techniques based on the confocal principle has been developed over the past 30 years. Such methods are commonly used by neuroscience researchers, but the majority of these users do not have a microscopy or a cell biology backgrounds and do can encounter difficulties in obtaining and interpreting results. This volume brings experts in high-resolution optical microscopy applications in neuroscience and cell biology together to document the state of the art. Outlining what is currently possible, the volume also discusses promising developments for the future and aids readers in selecting the most scientifically meaningful approach to solve their questions. Each chapter discusses instrumentation and technology in relationship to application in research. All of the common and cutting edge trends are covered - fluorescence / laser electron / nonlinear microscopy, infrared fluorescence, multiphoton imaging, tomography, FRAP, live imaging, STED, PALM/STORM, etc. Single and multiphoton confocal microscopy, and 4-pi confocal microscopy Obtaining nanoresolution via photoactivation localization microscopy (PALM) Several procedures that correlate observations in optical fluorescence microscopy and electron microscopy Study of morphology and function via high-resolution fluorescence procedures Additional high-resolution microscopic techniques
Download or read book Imaging Initial Events in T cell Activation written by Lawrence Otto Klein and published by Stanford University. This book was released on 2010 with total page 171 pages. Available in PDF, EPUB and Kindle. Book excerpt: This thesis is organized in four chapters. Chapter I is intended to give a general introduction to [alpha][beta] T cells, their role in the immune system, their T cell receptor (TCR), and the specific TCR transgenic system used in this work. In chapter II the TCR signaling pathway is introduced, and a photoactivation method we developed for interrogating proximal events in this pathway is described. We describe experiments using this method that defined delay times between TCR-pMHC binding and initiation of various TCR proximal signaling events. We found delays much shorter than previous measurements suggested, and propose that they may represent a feature of the pathway predicted by the kinetic-proofreading model of TCR signaling. In this chapter we also describe experiments that took advantage of the ability to precisely define a sub-cellular region of TCR stimulation to interrogate the spatial dynamics of TCR signaling. We found that the T cell membrane was compartmentalized such that even rapidly diffusible second-messengers were confined to the local region of stimulation. By stimulating distinct regions of T cells sequentially, we showed that desensitization occurred rapidly in some branches of the TCR signaling pathway but not at all in others. In chapter III we introduce previous research that sought to define properties of the TCR-pMHC interaction that determine stimulatory potency, and explain how these studies have led to interest in measuring kinetic parameters of the TCR-pMHC interaction in a native two-dimensional environment. We describe development of a new method to measure two-dimensional kinetics using a combination of our photoactivation system and direct detection of receptor-ligand binding via FRET. Using this method we showed that the rate of pMHC binding in a T cell contact interface was not influenced by a variety of cellular factors, but was defined by the kinetics of TCR-pMHC binding measured in vitro. We developed a quantitative method for analyzing our data and found that it fit very well to a simple bimolecular binding model, yielding kinetic parameters in clear agreement with 3D in vitro measurements. Our technique allowed direct, bulk measurement of 2D receptor-ligand binding and has the potential to measure kinetics too fast to measure by previous methods. Finally, in chapter IV we discuss earlier work describing molecular movements that occur during formation of the T cell-APC contact, called the immunological synapse. We describe the results of a series of experiments using our combined FRET and photoactivation assay that revealed the dynamics of TCR-pMHC interactions during immunological synapse formation. Our experiments showed that ligand binding was initiated in small clusters that were stable for tens of seconds while being actively transported toward the center of the cell. We describe the interesting observations that TCR-pMHC binding occurred in a distribution more heterogeneous than either the receptor or ligand distribution, and was regulated by cytoskeletal activity. We showed that in naïve cells this distribution was markedly different than in antigen-experienced cells, indicating that these two cell types may search for antigen in different ways. The results in this chapter indicate that molecular interactions in the synapse are actively regulated by cellular processes and are much more complex than would be expected from measurements of molecular distributions.
Download or read book The Immunological Synapse Part B written by and published by Academic Press. This book was released on 2023-07-11 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: The Immunological Synapse - Part B, Volume 178 in the Methods in Cell Biology series provides state-of-the-art methods for the study of the immunological synapse. This first volume covers various aspects on T cell and natural killer (NK) cell synapses, including imaging polarized granule release using TIRF microscopy, analysis of actin reorganization and centrosome polarization, redirected degranulation, live cell-imaging to quantify cytotoxic and chemotactic dynamics, quantification of interactions between APCs and T cells, assessment of membrane lipid state at the immunological synapse, proteomic analysis and imaging of NK-tumor cell interaction, evaluating natural killer cell effector functions against breast cancer cells derived from human tumor tissue, evaluation of chimeric antigen receptor (CAR)-modified cell immunological synapse quality using the glass-supported planar lipid bilayer, and monitoring potency of therapeutic CAR T cells. It also includes one chapter on quantification of lymphocytic choriomeningitis virus specific T cells and LCMV viral titers, and one chapter on the murine antibody-dependent cellular phagocytosis assay.
Download or read book Quantitative Molecular Orientation Imaging of Biological Structures by Polarized Super resolution Fluorescence Microscopy written by Haitham Ahmed Shaban Ahmed and published by . This book was released on 2015 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: .In this thesis we built and optimized quantitative polarized stochastic super-resolution fluorescence microscopy techniques that enabled us to image molecular orientation behaviors in static and dynamic environments at single molecule level and with nano-scale resolution. Using a scheme of stochastic read-out super resolution microscopy in combination with polarized detection, we can reconstruct fluorescence anisotropy images at a spatial resolution of 40 nm. In particular, we have been able to use the techniques to quantify the molecular orientationalorder in cellular and bio-molecular assemblies. For cellular imaging, we could quantify the ability of fluorophore labels to report molecular orientation of actin and microtubules in fixed cells. Furthermore, we used the improvements of resolution and polarization detection to study molecular order of amyloid aggregates at a nanoscopic scale. Also, we studied repair protein RAD51` s interaction with DNA by using dual color polarized fluorescence microscopy, to quantify the orientational order of DNA and RAD51 to understand the homologous recombination of DNA repair mechanism.
Download or read book Polarized Super resolution Fluorescence Microscopy written by César Augusto Valadés Cruz and published by . This book was released on 2014 with total page 253 pages. Available in PDF, EPUB and Kindle. Book excerpt: While super-resolution microscopy has brought a significant improvement in nanoscale imaging of molecular assemblies in biological media, its extension to imaging molecular orientation using fluorescence anisotropy has not yet been fully explored. Providing orientational order information at the nanoscale would be of considerable interest for the understanding of biological functions since they are intrinsically related to structural fundamental processes such as in protein clustering in cell membranes, supra-molecular polymerization or aggregation. In this thesis, we propose a super-resolution polarization-resolved microscopy technique able to image molecular orientation behaviors in static and dynamic environments, in order to report structural information at the single molecule level and at nanometric spatial scale. Using direct Stochastic Optical Reconstruction Microscopy (dSTORM) in combination with polarized detection, fluorescence anisotropy images are reconstructed at a spatial resolution of a few tens of nanometers. We analyze numerically the principle of the method in combination with models for orientational order mechanisms, and provide conditions for which this information can be retrieved with high precision in biological samples based on fibrillar structures. Finally, we propose an alternative technique based on stochastic fluctuations of single molecules: polarized super-resolution optical fluctuation imaging (polar-SOFI), and compare this approach with the previous one. We illustrate both techniques on molecular order imaging in actin stress fibers and tubulin fibers in fixed cells, DNA fibers and insulin amyloid fibrils.
Download or read book Methods for Imaging Cell Membranes written by Luca Panconi and published by . This book was released on 2023 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book will serve as an introduction to microscopy and biomedical imaging methods, with a focus on the study of the distributions and dynamics of molecules on the cell surface. It will provide readers with an in-depth understanding of how modern microscopy methodology can be used to understand the organisation of cell membrane systems and how experiments can be designed around these methodologies. There are numerous methods employed to understand cell membrane organisation, but foremost among them are microscopy methods which can map the distributions of molecules on the cell surface and even map the biophysical properties of membranes themselves. Fluorescence microscopy has been especially widely used due to its specificity and relatively noninvasive nature, allowing live-cell imaging. However, the recent advance of super-resolution fluorescence microscopy has broken the previous resolution limit for this type of microscopy, which has been an important advancement in the field. Atomic force microscopy and electron microscopy have also been deployed to learn about membrane organisation and properties. Each chapter in this volume will be themed around measuring a particular property of cell membranes. In each case, the authors examine the range of methodology applicable to the task, comparing the advantages and disadvantages of each one, and will also provide an overview of important discoveries that have been made using the methodology being discussed. The chapters will cover: •Measuring membrane protein distributions using single-molecule localisation microscopy (SMLM) •Measuring membrane protein dynamics and diffusion using fluorescence correla-tion spectroscopy (FCS) •Mapping membrane lipid backing using environmentally sensitive fluorescence probes •Mapping membrane thickness and rigidity using atomic force microscopy •Mapping membrane proteins and the cytoskeleton using electron microscopy This book will be a valuable resource to graduate and upper-level undergraduate students and industry researchers in the fields of cell biology, microbiology, microscopy, and medical imaging.
Download or read book Regulatory Mechanisms of Early Intracellular Signaling in T Lymphocytes written by Enrique Aguado and published by Frontiers Media SA. This book was released on 2021-05-21 with total page 174 pages. Available in PDF, EPUB and Kindle. Book excerpt:
