2. MEMBRANE AND RECEPTORS
VIBS 443 and VIBS 602
Undergraduate – Graduate
Histology Lecture Series
Larry Johnson, Professor
Veterinary Integrative Biosciences
Texas A&M University
College Station, TX 77843
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
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 composition
Three major classes of
cytoplasmic structures.
--------------------------------------------------------------------------------
1. Membranous organelles - common structures some
with metabolic functions: cell membrane, RER, SER, Golgi,
mitochondria, lysosomes
2. Non-membranous organelles – cytoskeletal
3.
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
Plasmalemma
8.5 - 10 nm
Function:
• Regulate traffic of ions and macromolecules
• Possess devices for cell attachment and cell-to-cell
Cell 1 cell 2
communication
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
• Vectoral reactions
(requires membranes
to partition; RER)
A+B = C
vectoral secretion
Membranes in compartmentalization
nuclei
inclusions
vectoral 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
Polar
Nonpolar
Amphipathic molecules - two ends
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
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
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.
Voyage inside the Cell: Membrane
http://www.youtube.com/watch?v=GW0lqf4Fqpg&NR=1
organelles
https://www.youtube.com/watch?v=yKW4F0Nu-UY
https://www.youtube.com/watch?v=FzcTgrxMzZk
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 (vectoral reactions)
Development of gradients
Receptors provide mechanisms for cell’s
physiological response to external stimuli
Next time…
RER, GOLGI, and SECRETION
Many illustrations in these VIBS Histology YouTube videos were modified from
the following books and sources: Many thanks to original sources!
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Bruce Alberts, et al. 1983. Molecular Biology of the Cell. Garland Publishing, Inc., New York, NY.
Bruce Alberts, et al. 1994. Molecular Biology of the Cell. Garland Publishing, Inc., New York, NY.
William J. Banks, 1981. Applied Veterinary Histology. Williams and Wilkins, Los Angeles, CA.
Hans Elias, et al. 1978. Histology and Human Microanatomy. John Wiley and Sons, New York, NY.
Don W. Fawcett. 1986. Bloom and Fawcett. A textbook of histology. W. B. Saunders Company,
Philadelphia, PA.
Don W. Fawcett. 1994. Bloom and Fawcett. A textbook of histology. Chapman and Hall, New York, NY.
Arthur W. Ham and David H. Cormack. 1979. Histology. J. S. Lippincott Company, Philadelphia, PA.
Luis C. Junqueira, et al. 1983. Basic Histology. Lange Medical Publications, Los Altos, CA.
L. Carlos Junqueira, et al. 1995. Basic Histology. Appleton and Lange, Norwalk, CT.
L.L. Langley, et al. 1974. Dynamic Anatomy and Physiology. McGraw-Hill Book Company, New York,
NY.
W.W. Tuttle and Byron A. Schottelius. 1969. Textbook of Physiology. The C. V. Mosby Company, St.
Louis, MO.
Leon Weiss. 1977. Histology Cell and Tissue Biology. Elsevier Biomedical, New York, NY.
Leon Weiss and Roy O. Greep. 1977. Histology. McGraw-Hill Book Company, New York, NY.
Nature (http://www.nature.com), Vol. 414:88,2001.
A.L. Mescher 2013 Junqueira’s Basis Histology text and atlas, 13th ed. McGraw
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