2. MEMBRANE AND RECEPTORS Undergraduate – Graduate Histology Lecture Series Larry Johnson, Professor Veterinary Integrative Biosciences Texas A&M University College Station, TX VIBS 443 and VIBS 602

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

Cell composition Nucleus - nucleoplasm blue with H&E Cytoplasm - rest of cell surrounds nucleus and is red with H&E plasma cells kidney

Cell composition Nucleus Nucleoplasm blue with Toluidine blue Cytoplasm Rest of cell surrounds nucleus and is blue with Toluidine blue Intestinal absorptive cells

Cell composition Three major classes of cytoplasmic structures Membranous organelles - common structures some with metabolic functions: cell membrane, RER, SER, Golgi, mitochondria, lysosomes 2.Non-membranous organelles – cytoskeletal components: microtubules, microfilaments, intermediate filaments, free ribosomes 3. 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 cardiac cells in lung blood vessel Bright field light microscopy toluidine blue

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 Plasmalemma nm Function: Regulate traffic of ions and macromolecules Possess devices for cell attachment and cell-to-cell communication Cell 1 cell 2

Plasmalemma nm Function: Contain antigenic molecules, which are the basis of cell recognition and tissue specificity Regular internal environment - ion pumps Cell Membrane

Plasmalemma nm Function: Possess receptors for hormones Possess mechanisms for generating messenger molecules that activate the cell’s physiological responses to stimuli Cell Membrane

Scaler reactions (proteins produced by free ribosomes) A + B = C Vectoral reactions (requires membranes to partition; RER) A + B = C Reactions in protein synthesis vectoral secretion

nuclei inclusions vectoral secretion Membranes in compartmentalization

Membranes in compartmentalization of cells pancreas stomach Drawings of EM

Membranes in compartmentalization of cells stomach Bright-field, light microscopy toluidine blue Drawing of EM Parietal cells

Cell compartmentalization

Membranes: - selective barriers - regulate traffic of ions and macromolecules

SER RER lipid Membranous organelles

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

Amphipathic molecules - two ends Polar Nonpolar

Phospholipid bilayer

Membranes self assemble and self seal to get the non-polar tails away from contact with water. Induction of the PHOSPHOLIPID BILAYER

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

Characterization of membranes via SDS-PAGE SDS

Biconcave Shape of Erythrocytes

SDS-PAGE and fluorography were used to detect binding of different proteins to testosterone. 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 The proteins were identified by molecular weight and the binding to testosterone was detected by fluorography.

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 I 125 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

Microtubules Mitochondria Immunocytochemistry

Freeze-fracture

Inside the cell Outside the cell P = Protoplasmic side 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.

Voyage inside the Cell: Membrane organelles

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

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. Paper Chromatography

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. Terminology

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.

0% 5% 10% 20% 50% 100% Concentrations of alcohol 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.

(A) uses charge, (B) uses pores, and (C) uses covalent bonds to create the differential affinities among the mixture components for the stationary phase. Uses a material that induces differential affinity among components of the mixture.

Function of cell receptors 1.Cell adhesion and recognition 2.Cell migration 3.Regulation of metabolic function 4.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 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 (vectoral reactions) Development of gradients Receptors provide mechanisms for cell’s physiological response to external stimuli

RER, GOLGI, and SECRETION Next time…

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