University:
Texas A&M University
Course:
BIOL 213 | Molecular Cell Biology
Academic year:

2020
Views:

173

Pages:

90
Author:

Nicole Wang

2. MEMBRANE AND RECEPTORS Objectives Importance of membranes in compartmentalization of cells Overall structure of cellular membranes Methods for studying membranes and cell membrane receptors Properties of cell membrane receptors How Did the Cell Get its Name? Robert Hooke first observed cells in a thin piece of cork (plant). He saw walled boxes that reminded him of the tiny rooms, cellula, occupied by monks in monasteries, and the name "cell“ was coined. Monastery cellula: Cells Cells in a → in an animal plant Hooke’s microscope → Early observations of cells illustrated that there were two types (more primitive and more modern types). A prokaryotic cell has no nucleus and no nuclear membranes around its DNA. they needs oxygen Age of Prokaryotic Eukaryotic cells • Scientists believe that prokaryotic cells (in the form of bacteria) were the first life forms on earth. They are considered “primitive” and originated about 3.5 billion years ago. That's 2 billion years earlier than eukaryotic cells appeared. Prokaryotic cells considered to be 3.5 billion years old Eukaryotic cells are thought to be 1.5 billion years old Single cell living organism The cell theory, developed during the mid 17th century, was made possible by advances in microscopy that allowed scientists to see cells for the first time. “The cell theory” states that cells are: -Components of all living thinks -Unit building blocks of structure and function -Come from existing cells The cell theory is one of the foundations of biology. (Evolution is another fundamental theory of biology). https://www.saylor.org/site/wp-content/uploads/2010/11/Wiki-Cell-Theory.pdf Why is this cell a prokaryote? A prokaryotic cell has no nucleus and no nuclear membranes (e.g., no nuclear envelope) around its DNA. Cell composition of the eukaryote kidney plasma cells Nucleus - nucleoplasm blue with H&E Cytoplasm - rest of cell surrounds nucleus and is red with H&E Cell composition Intestinal absorptive cells Nucleus Nucleoplasm blue with Toluidine blue Cytoplasm Rest of cell surrounds nucleus and is blue with Toluidine blue Cell cytoplasm is composition of the cytosol (gel like fluid that holds the contents inside the cell in place) plus three classes of structures. 1. Membranous organelles - 2. 3. common structures some with metabolic functions: cell membrane, RER, SER, Golgi, mitochondria, lysosomes Non-membranous organelles – cytoskeletal components: microtubules, microfilaments, intermediate filaments, free ribosomes Inclusions - expendables a. nutrients: e.g., glycogen, lipid b. pigments: e.g., melanin granules c. secretory granules: e.g., zymogen granule of pancreas Membranous organelles Non-membranous organelles Inclusions Bright field light microscopy toluidine blue cardiac cells in lung blood vessel Non-membranous organelles Bright-field, light microscopy toluidine blue cardiac cells in lung blood vessel Electron microscopy Inclusions cardiac cells in lung blood vessel Cell Membrane Cell membrane Function: 8.5 - 10 nm thick Cell membrane • Regulate traffic of ions and macromolecules • Possess devices for cell attachment and cell-to-cell communication Cell 1 Cell 2 1 2 Junctions 3 4 Cell Membrane Plasmalemma 8.5 - 10 nm Function: • Contain antigenic molecules, which are the basis of cell recognition and tissue specificity • Regular internal environment - ion pumps Cell Membrane Plasmalemma 8.5 - 10 nm Function: • Possess receptors for hormones • Possess mechanisms for generating messenger molecules that activate the cell’s physiological responses to stimuli Reactions in protein synthesis • Scaler reactions (proteins produced by free ribosomes) A+B=C • Vectorial reactions (requires membranes to partition; RER) A+B = C vectoral secretion Membranes in compartmentalization nuclei inclusions vectorial secretion Membranes in compartmentalization of cells pancreas Drawings of EM stomach Membranes in compartmentalization of cells Parietal cells Bright-field, light microscopy toluidine blue stomach Drawing of EM Cell compartmentalization Membranes: - selective barriers - regulate traffic of ions and macromolecules lipid SER Membranous organelles RER Membranous organelles Membranes Properties: • Self assemble • Self seal Why do they self assemble and self seal? Lipids as membrane components • • • • • Amphipathic molecules - two ends Phospholipid bilayer Fluidity Cholesterol - increase fluidity of membranes Gangliosides - glycolipid with sialic acid residues - Sialic acid gives cells a net negative charge on the plasma membrane What is the basic composition of all membranes? Membranes are composed of phospholipids. Polar = attracted to water Non-polar = repels water Phospholipids spontaneously form structures in water. How does soap work to clean? Micelles occur in soapy water. Dirt, which prefers lipid over water, moves into the micelles of soapy water to be rinsed out with the soapy water. Membranes self assemble and self seal to get the non-polar tails away from direct contact with water. Induction of the PHOSPHOLIPID BILAYER Phospholipid bilayer How do Plasma Membranes work? The plasma membrane is made of a phospholipid bilayer A single Phospholipid H2O H2O 1st layer Hydrophilic (water loving) heads & Hydrophobic (water fearing) tails 2nd layer Outside Cell Bi(2)layer The phospholipid bilayer forms a membrane around the entire cell like a water balloon Inside Cell Cell Membrane Cells are full of fluids and organelles To hold them together and keep them from leaking a cell membrane surrounds the entire cell Like a water balloon, the water inside is the cell’s contents held together by the outer membrane balloon If the plasma membrane were to be damaged (pop!) the cell’s contents would spill out and the cell would die. Cell membrane summary Necessary for life Marks limits of cell (defines cell) Separates cell from its environment Can not be a complete barrier, some items must pass. Membranes • Self assemble • Self seal (due to amphipathic structure of phospholipids where lipid tails move to escape the water environment) Proteins as membrane components • Interpretation of TEM image • Trilaminar structure of membranes • Fluid mosaic model • “meatballs in a sea of fat” • Peripheral vs. Integral • Labeling techniques • Extraction techniques • Transmembrane orientation • Protein domains • Cytoskeletal interactions Trilaminar structure of membranes = two black sheets (appear as lines) outlining the white center sheet Trilaminar structure Fluid mosaic model - “meatballs in a sea of fat” Enzymes of intestinal absorptive cell Several peptidases polypeptides to amino acids Four disaccharidases disaccharides to monosaccharides Lipase neutral fats to glycerol and fatty acid Carbohydratases small amount of amylase SDS Characterization of membranes via SDS-PAGE Biconcave Shape of Erythrocytes SDS-PAGE and fluorography were used to detect binding of different proteins to testosterone. The proteins were identified by molecular weight and the binding to testosterone was detected by fluorography. Fluorography is a method used to visualize substances present in gels, blots, or other biochemical separations by detecting secondary light that was generated by the excitation of a fluorescent screen by a beta particle or a gamma ray Autoradiography vs Fluorography Terminology Autoradiography is the direct exposure of film by beta particles or gamma rays. Fluorography is the exposure of film by secondary light that was generated by the excitation of a fluorescent screen by a beta particle or a gamma ray. Extraction procedures • High salt concentration or pH Removes peripheral proteins • Detergents • Triton x 100: non-ionic detergent Breaks bonds between lipid and proteins • SDS: ionic detergent Extracts all proteins Labeling/digestion techniques • Lactoperoxidase - attaches to proteins outside of membrane that have tyrosine residues • Attach I125 will make it radioactive • Lectins - binds to carbohydrates • Microscopy • Gel electrophoresis • Affinity chromatography • Proteolytic enzymes - reduce sizes of proteins • Alters number of bands and location on the SDS-gel • Immunocytochemistry • Antibodies bind and label specific proteins Immunocytochemistry Microtubules Mitochondria Freeze-fracture Cell 2 Cell 1 Inside the cell P = Protoplasmic side Outside the cell E = Ectoplasmic side Fluidity of membranes illustrated. FLUIDITY Cells have strategies to limit/prevent fluidity of their proteins. Urinary bladder Red blood cells maintain their biconcave shape Specialized cell junctions (tight junctions) isolate the membrane’s fluidity. Receptors 1. Gangliosides - glycolipid with sialic acid residues 2. Glycoproteins - polysaccharide sugars covalently linked to protein Ligand interactions with receptors • Specificity • Saturability Methods of receptor analysis 1. Binding assay 2. Localization 3. Gel electrophoresis - measure molecular weight • Immunoblot - gel to paper, then labeled antibody binding Label, alter MW, or isolate proteins Paper Chromatography Chromatography (named for its first use in separation of colored mixtures) is a method (laboratory technique) for separating complex solubilized mixtures into their components. Mixtures are separated into their components from which they are made in order to identify, purify, or test individual components within the mixture. Paper chromatography uses a solution that induces differential affinity among components of the mixture, that moves up a strip of paper by capillary action, and that separates the components along the strip of paper. Terminology Chromatography is a technique for the separation of complex mixtures that relies on differential affinities of substances in the mixture for a gas or liquid mobile medium and for a stationary adsorbing medium through which these substances pass. Differential means of, relating to, or showing a difference; constructing or making a difference, distinctive. Affinity means a natural attraction or liking between things; an attraction or force between particles that cause then to combine. Mobile medium is the gas or liquid that carries or in which the sample is able to migrate; known as the mobile phase. Stationary adsorbing medium is the part of the apparatus that does not move with the sample; known as the stationary phase. Terminology In other words: Chromatography is a laboratory technique that separates components within a complex mixture, and the separation occurs due to a difference in attraction of each component for the gas or liquid medium (mobile phase) that carries the components in relation to the attraction of each component for the non-moving adsorbing medium (stationary phase) through which these components pass. Paper Chromatography uses liquid (mobile phase) to carry the components of a mixture within a strip of paper, the paper is the stationary adsorbing medium (stationary phase) through which the components pass and separate due to differences in speeds of passage in the stationary phase. Principles of Paper Chromatography Capillary Action – the solvent is able to move up the filter paper because its attraction to itself is stronger than the force of gravity Solubility – solutes will dissolve into solvents that are similar. Likes dissolve likes. This allows solutes (mixture components) to be separated by different combinations of solvents. Separation of mixture components depends on the solubility of each ( must be solubilized to be able to migrate in the mobile phase) in that mobile medium and the differential affinity of the components for both the mobile phase and the stationary phase. Observing the Paper Strips Observe how spots of the same color separated in low concentrations of alcohol compared to higher concentrations. Observe when spots of different colors first started separating in the different concentrations of alcohol. 0% 5% 10% 20% 50% Concentrations of alcohol 100% Uses a material that induces differential affinity among components of the mixture. (A) uses charge, (B) uses pores, and (C) uses covalent bonds to create the differential affinities among the mixture components for the stationary phase. Function of cell receptors 1. 2. 3. 4. Cell adhesion and recognition Cell migration Regulation of metabolic function Down regulation - reduce number of receptors • Reduces the cell’s response to stimuli 5. Up regulation - increase the number of receptors by recycling or from reserves in vesicles • Increases the cell’s response to stimuli Ligand interactions with receptors 1. Specificity 2. Saturability Cell membrane Plasmalemma 8.5 - 10 nm Function: • Possess receptors for hormones • Possess mechanisms for generating messenger molecules that activate the cell’s physiological responses to stimuli Antibodies in immunity Mast cells - allergic response Induced degranulation of mast cells. In summary: Membranes allow cells a high degree of chemical heterogeneity Surfaces & interfaces - sites important for physiological processes Enzymes - catalyze chemical transformations Internal partition of cytoplasm - enzymes, substrates, & products Efficiency of complex chemical reaction amplified area Gradients - by permeability and rate of active transport Regulate cell’s activity Hold reserve a large repertoire of unexpressed biochemical reactions Summary continued: Membranes and receptors Membranes are important in cells for: • • • • Compartmentalization Segregation of products (vectorial reactions) Development of gradients Receptors provide mechanisms for cell’s physiological response to external stimuli

Membrane and Receptors

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