Browsing by Subject "Exocytosis"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Electrochemical studies of blood platelet exocytosis.
    (2011-06) Ge, Shencheng
    Platelets play an essential role in hemostasis and thrombosis in the bloodstream. Their critical physiological behavior is intimately related to their cellular function as secretory cells. Platelets contain populous secretory granules and release a large array of chemical messengers ranging from ions to small molecules to large protein-based macromolecules. Until now, the dynamic secretion behavior of platelet secretory granules has been poorly understood, due largely to the lack of tools to probe the small-sized platelets. To address this important biological problem and bridge the current knowledge gap, this thesis work develops and employs state-of-the-art electrochemical techniques to study single platelet exocytosis with a focus on platelet dense-body granules, the prominent secretory granules responsible for releasing clot-promoting small molecule chemical messengers. As a result, the work reveals important insights into the inner-workings of platelet secretory behavior and expands the knowledge basis on platelets for therapeutic purposes. Chapter One reviews state-of-the-art methodologies for single-cell studies of exocytosis. Based on the small-sized platelets, electrochemical methods are uniquely suited to study exocytosis on a platelet-by-platelet basis. Chapters Two and Three develop and employ carbon-fiber microelectrode fast-scan cyclic voltammetry and amperometry methods to measure real-time dense-body granule exocytosis in platelet suspensions and single platelets. The experimental results represent the first dynamic evidence of platelet quantal secretion behavior, i.e. granule content secretion via exocytosis. Chapters Four to Seven systematically examine the fundamental quantal secretion behavior of platelet dense-body granules by combining real-time electrochemical measurement tools and a wide range of chemical and pharmacological manipulation strategies. Chapter Four examines the effects of variations in temperature, extracellular pH and osmolarity on platelet dense-body granule secretion, and reveals common and distinct quantal secretion behavior of platelets among secretory cell types studied to date. Chapter Five investigates the impact of variations in granule size following pharmacological manipulation of granule content on platelet dense-body granule secretion, and reveals the dynamic interplay between granule content and the granule membrane in modulating platelet quantal secretion. Chapter Six examines the variations in natural membrane cholesterol and substituted unnatural epicholesterol content on platelet dense-body granule quantal secretion, and reveals a critical biophysical role for membrane cholesterol in regulating exocytosis. Chapter Seven studies the effects of pharmacologically manipulated cytoskeletal F-actin and microtubule integrity on platelet dense-body granule secretion, and reveals that F-actin, but not microtubule, regulates platelet dense-body granule secretion.
  • Thumbnail Image
    Item
    Exploration of Platelet and Mast Cell Communication: A Study of Membrane Lipids, Asthma, and Inflammation
    (2016-05) Gruba, Sarah
    This work examines how environmental factors such as lipid membrane concentration, opioid agonist exposure, and inflammatory diseases impact cell communication. It explores the use of different cell models, specifically platelets and mast cells, to understand how disease states can impact cellular function. Throughout the thesis, a variety of analytical techniques including electrochemistry, mass spectrometry, dark field imaging, and microfluidics, are used to understand exocytosis, lipid concentration, manufactured inflammatory mediators, adhesion, and shape change in platelets and mast cells. Platelets are cell-like bodies that travel through the bloodstream and are known for their role in hemostasis and diseases like stroke and myocardial infarction. They have also been implicated in inflammatory diseases such as asthma. In addition, their anucleate nature and easy isolation make them an ideal model for studying variations in cell communication upon the modification of their lipid content. Platelets communicate through the exocytosis of their three distinct granule types (δ, α, and lysosome). These granules contain molecules that assist in the transmigration of immune cells to the site of activation and help with additional platelet aggregation and adhesion. In contrast, mast cells are found throughout the body in connective tissue and are one of the immune system’s first lines of defense. They are primarily known for their role in allergies and asthma. Upon detection of antigens that they are sensitized to, the mast cell secretes manufactured chemokines and pre-formed granule mediators, including histamine and serotonin, calling other inflammatory cells to the site of infection. Chapter One reviews single cell analysis techniques with a particular emphasis on the techniques used in this thesis, including electrochemistry and mass spectrometry. Chapter Two through four are focused on understanding how variations in membrane lipids and structure affect platelet function and exocytosis in general. Chapter Two focuses on understanding the variations that the fusion pore undergoes when granules are being exocytosed. Traditionally, a granule release event, monitored using carbon-fiber microelectrode amperometry, has a quick rise in current (spike) and gradual decay. The variations to this spike are classified as different forms of pre- and post-spike features and non-traditional granule secretion events. The role of cholesterol in changing the frequency and duration of these features is also discussed. Chapter Three discusses the role of phosphatidylserine (PS) in cellular communication using a platelet model. In this chapter, we explore how the stereochemistry of the head group and concentration of PS affects various platelet functions including granular content secretion, manufactured lipid release, and adhesion. The cholesterol level change upon addition of PS is also monitored. Finally, Chapter Four aims to understand how natural lipid variations affect cell function by comparing platelets from different species. This chapter highlights the importance of understanding your cell model relative to the actual cells involved in the disease or function being studied. Chapter Five and Six progresses from lipid function into developing a better understanding of how platelets respond to their environment, particularly in the context of inflammatory diseases. Chapter Five’s focus is on platelet response to opioids like those that are used in the treatment of pain due to inflammatory diseases, cancer, or surgery. Specifically, the effects on cell exocytosis as well as the presence of and role that opioid receptors play in platelets are characterized. Chapter Six focuses on studying how platelets respond to allergic asthma, including response to allergens and the chemoattractants (CXCL10 and CCL5) released during an asthma attack. Using bulk and single cell methods in conjunction allows us to obtain in-depth information on both the overall response and the granule fusion pore during exocytosis. Chapter Seven and Eight focus on mouse peritoneal mast cell (MPMC) function in the context of inflammatory diseases including allergic asthma and neurogenic inflammation, respectively. Chapter Seven aims to state the importance of understanding the cell line you are using since variations in response to allergens are noted between commonly used mast cell models (rat basophilic leukocytes cell line and primary culture MPMC). In addition, MPMC response to CXCL10 and CCL5 was monitored. Finally, Chapter Eight explores the role of MPMC in neurogenic inflammation, a process wherein neurons release the neuropeptides substance P and calcitonin gene-related peptide. Mast cell response to these neuropeptides has been highly disputed, and this chapter focuses on the impact of IgE on MPMC bulk granular content secretion. It also aims to understand how these neuropeptides affect the fusion pore opening and closing during exocytosis.
  • Thumbnail Image
    Item
    Exploring the fundamentals of platelet granular storage and secretion at the single cell level
    (2013-01) Koseoglu, Secil
    Platelets are critical cells in hemostasis and thrombosis, but they are also involved in many important physiological events including inflammation, host defense, wound healing and malignancy. Platelets pursue their physiological functions mainly as secretory cells. The three distinct platelets granules, α- and δ- granules and lysosomes, serve as storage units for critical biological mediators. Upon action of a stimulus, platelets release their granules through a conserved mechanism called exocytosis. Because blood platelets are quite small (2-3 µm in diameter) and activate easily, until the recent work in the Haynes group, measurements were limited largely to morphological studies (electron/light/fluorescence microscopy) and ensemble aggregation and release assays, missing fundamental dynamics about chemical messenger delivery. The measurements of platelet δ-granule secretion by using carbon fiber microelectrode amperometry (CFMA) enabled real-time monitoring secretion of serotonin from platelet δ-granules with sub-ms time resolution. The aim of this thesis is to exploit these single cell measurements to study the fundamentals of platelet secretion behavior and advance our current understanding of platelet exocytosis. A brief introduction to the platelet biology and single cell platelet measurements are given in Chapter 1. Since the major step involved in platelet granular secretion is fusion of the granules to either the membrane tubular system known as the open canalicular system (OCS) or the plasma membrane, Chapter 2 investigates the difference in the secretory behavior of the platelets of different species that has OCS or does not have an OCS. CFMA measurements performed on mouse, rabbit and cow platelets indicate that OCS is necessary for an efficient secretory function. Fusion of the granular membrane with the plasma membrane is mediated by both membrane protein and lipid components. While membrane proteins anchor granular membrane with plasma membrane and facilitate fusion, membrane lipids not only regulate the membrane fluidity and curvature they also mediate localization of the fusion proteins on the site of the fusion. Although dynamin-related protein1 (Drp1) is best known as a mediator of membrane fission, recent work showed that it also contributes to granule exocytosis by mediating fusion pore expansion in chromaffin cells. However, there was not any information on the role of Drp1 on platelet granule secretion. To assess whether Drp1 functions in platelet exocytosis, we tested the effect of mdivi-1, a Drp1 inhibitor, on the release of dense granules by using single-cell amperometry (Chapter 3). The results demonstrate the role of Drp-1 in fusion pore dynamics, and indicate that regulation of platelet fusion pore expansion can be used to control thrombus formation in vivo. Phospholipids are the major components of the plasma and granule membrane, and in addition to their structural importance as a cellular barrier that separates the intracellular and extracellular environment, they are also dynamically involved in and regulate many cellular processes. However, there is not much known about how the different phospholipids regulate platelet behavior. Chapter 4 examines the effect of membrane phospholipids on the major platelet functions of aggregation and exocytosis and demonstrates that different phospholipids can act on different aspects of platelet function.Besides their physiological importance, platelets can serve as an ideal model system for studying exocytosis. In the last part of the thesis, we compare the effect of cholesterol on chromaffin cells, a well-studied model cell for neural secretion, and platelets. Chromaffin cell exocytosis at altered cellular cholesterol levels was measured at single cell level and results were compared to the previously published work by Ge et al on cholesterol effects on platelet secretion. This work demonstrated that the effect of cholesterol on each cell type was different which is likely due to the fact that, unlike platelets, chromaffin cells have a nucleus and a significant synthesis capacity that enables them to tightly regulate various cell functions.Overall, the experiments performed herein expand our current understanding of the mechanism of platelet secretion and demonstrate that studying platelet secretion at the molecular level is essential to control platelet function for therapeutic purposes.
  • Thumbnail Image
    Item
    A Study of the Regulation Mechanisms of Platelet Activation
    (2016-01) Finkenstaedt-Quinn, Solaire
    Platelets play an important role in maintaining hemostasis in the body. As they circulate through the blood stream, platelets receive signals from other cells that call them to sites of vascular damage. When platelets reach a damaged area, they go through the processes of activation and aggregation. During activation, platelets begin to change shape and release their granular contents via exocytosis. During exocytosis, a series of small molecules and proteins are released that serve to propagate the platelet activation signal and initiate wound healing. Overall, this work explores the different aspects of platelet activation using microscopy and single cell methods. Chapter One reviews the different components of the cytoskeleton and the current advances in microscopy that are being applied to study it. While light microscopy is a useful technique for studying cellular dynamics, super-resolution imaging allows for more in depth exploration of the many regulatory roles that the cytoskeleton plays in cells. Chapter Two focuses on work performed to investigate the toxicity of mesoporous silica nanoparticles on platelets. In Chapters Three and Four, the role that the cytoskeleton and plasma membrane play in the shape change dynamics of platelet activation is detailed, with a focus on using microscopy to visualize these changes. Chapter Five analyzes platelet activation to characterize fusion pore dynamics during exocytosis. Lastly, Chapter Six provides an overview of three different chemical education projects I have worked on. The first two projects involved the development and implementation of outreach events at a local community center to teach students about the scientific process and the chemistry behind climate change. The third project is in progress and aims to develop labs for use in the high school chemistry classroom that combine literature with general chemistry concepts to promote student engagement and interdisciplinary focus.