Cytoskeleton
a microscopic network of protein filaments and tubules in the cytoplasm of many living cells,
giving them shape and coherence. Also is complex, dynamic network of interlinking protein
filaments
present
in
the
cytoplasm
of
all
cells,
including
those
of bacteria and archaea. In eukaryotes, it extends from the cell nucleus to the cell membrane and
is composed of similar proteins in the various organisms. It is composed of three main
components: microfilaments, intermediate filaments, and microtubules, and these are all capable
of rapid growth and or disassembly depending on the cell's requirements.
The structure, function and dynamic behavior of the cytoskeleton can be very different,
depending on organism and cell type. Even within one cell, the cytoskeleton can change through
association with other proteins and the previous history of the network.
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Microfilaments: also known as actin filaments, are composed of linear polymers of Gactin proteins, and generate force when the growing end of the filament pushes against a barrier,
such as the cell membrane, the diameters (6nm). They also act as tracks for the movement
of myosin molecules that affix to the microfilament and "walk" along them. In general, the
major component or protein of microfilaments are actin.
Functions include: Muscle contraction, Cell movement, Intracellular transport/trafficking,
Maintenance of eukaryotic cell shape, Cytokinesis and Cytoplasmic streaming.
Intermediate filaments (Ifs): are a part of the cytoskeleton of many eukaryotic cells, most
commonly known as the support system or "scaffolding" for the cell and nucleus. These
filaments, averaging 10 nanometers in diameter, are more stable (strongly bound) than
microfilaments, and heterogeneous constituents of the cytoskeleton. Like actin filaments.
They function in the maintenance of cell-shape by bearing tension, they organize the internal
tridimensional structure of the cell, anchoring organelles and serving as structural
components of the nuclear lamina. They also participate in some cell-cell and cell-matrix
junctions. Nuclear lamina exist in all animals and all tissues. Keratin intermediate filaments
in epithelial cells provide protection for different mechanical stresses the skin may endure.
They also provide protection for organs against metabolic, oxidative, and chemical stresses.
Strengthening of epithelial cells with these intermediate filaments may prevent onset
of apoptosis or cell death by reducing the probability of stress.
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Microtubules: are hollow cylinders about 23 nm in diameter (lumen diameter of
approximately 15 nm), most commonly comprising 13 protofilaments that, in turn, are
polymers of alpha and beta tubulin. They have a very dynamic behavior, binding GTP for
polymerization. They are commonly organized by the centrosome.
They play key roles in (function)
-intracellular
transport
(associated
with
transport organelles like mitochondria or vesicles).
-the axoneme of cilia and flagella.
-the mitotic spindle.
-synthesis of the cell wall in plants.
-consciousness.
dyneins
and
kinesins,
they
Transmission electron microscope
Transmission electron microscopes (TEM) are microscopes that use a particle beam of
electrons to visualize specimens and generate a highly-magnified image. TEMs can magnify
objects up to 2 million times.
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Transmission electron microscope preparation
-Anesthetization and dissecting the organism
1)Chemical used for fixation: 2-4%glutaraldehyde in 0.1M cacodylate buffer, pH 7.2-7.4, at
4ºC.
Mode of fixation: either immersion (immerse in fixative solution) or perfusion(applying by
intravenous or intracardiac injection while the animal is alive).
2)Postfixation: fixation in osmium tetroxide 1% for 1hr for fixing proteins and staining of
lipoproteins in the membranes.
3)Dehydration by alcohols 50%-100%
4)Clearing in propylene oxide
5)infiltration in plastic mixture. Araldite mixture consists of 4 materials.
6)Embedding(blocking using moulds) in araldite mixture (liquid). This will be polymerized
(solidify) by putting in oven 60ºC for 48 hr.
7) trimming and sectioning. Sectioning using ultramicrotome
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Types of sections according to use and thickness:
a)thick sections (1-2µ) used for light microscopy.
b)semithin sections (0.1-1µm) for light microscopy and high voltage electron microscopy.
c) Ultrathin sections( ˂0.1µm). The best thickness for EM is 600-900ºA(angstrom).
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8) staining according to the types of sections:
Staining thick and semi-thin sections
Pick up the sections from the surface of water (in trough of the knife) by plastic loop on
drops of water in slide
Dry on hot plate (60-70ºC)
Cover the sections by drops of 1% toluidine blue in 1% borax for 50-60sceonds
Wash with distilled water and then dry on hot plate and examine with/ or without using
cover slide
Staining the ultrathin sections
Ultrathin sections which appear silver gold interference in color due to thickness, must be
picked up by electron microscope grids. Copper grids are used because they are cheap.
Staining is done by two heavy metal salts: uranium as uranyl acetate and lead as lead citrate.
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Types of knives used in ultramicrotome:
Glass knives: they are disposable prepared in lab
Diamond knives: they are permanent
Scanning Electron Microscopic Study
The tissues were dissected out, washed repeatedly in 0.2M phosphate buffer and then set at
3% glutaraldehyde. Dehydration was done in acetone grades and was accompanied by
critical point drying. Ultimately dried tissues were mounted on the stub and were
sputtercoated with gold in a gold coating unit (thickness 100A) and were examined and
photographed using JEOL JSM 6360 scanning electron microscope (SEM) Japan.
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