Download Molecular Cell Biology Prof. D. Karunagaran Department of

Molecular Cell Biology
Prof. D. Karunagaran
Department of Biotechnology
Indian Institute of Technology Madras
Module 3
Microtubule, Actin and Filament Based Motile Systems
Lecture 2
Micro and Intermediate Filaments
Micro trabecular lattice
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The active or motile function of the cytoskeleton is mainly due to thin microfilaments.
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Just below the membrane, bundles of actin filaments can be seen.
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They are also in touch with other filaments pervading the cytosol.
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Together they constitute what is called microtrabecular lattice that has channels or
spaces of about 50-100 nm width.
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This may help in rapid diffusion of fluids and metabolites.
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The lattice is in contact with microtubules, vesicles of ER and polysomes.
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However, mitochondria are not part of this lattice.
Microfilaments
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Microfilaments are formed by actin, myosin, tropomyosin, alpha actinin and other
proteins
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Actin has a typical pattern of straight and parallel fibers in close contact with the
plasma membrane
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These are called stress fibers and their arrangement is disrupted by cytochalasin B
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Actin accounts for a large portion of cytoplasmic proteins in many cells
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In developing nerve cells 20% of the cytoplasmic proteins are actins
Actin and Myosin
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Globular actin is called G-actin mol. wt. 42,000
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It can polymerize to form fibrous actin or F-actin
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These proteins are involved in the contraction of skeletal muscle
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However, actin and myosin are also present in non muscle cells where they may help
in cell movement rather than contraction
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Actin filaments of 6 nm can be found in the cytoplasm bound with heavy meromyosin
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This actin-myosin complex has a definite polarity with the arrow-like appearance
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While the assembly of monomers takes place usually at the barbed end the
disassembling is observed at the pointed end of the arrow
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Similar to microtubules this type of polarization is also called treadmilling
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Myosin is relatively low in concentration and is heterogenous in size and composition
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Mol. wt. is usually 500,000 daltons
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Myosin binds reversibly with actin filaments and contains a calcium activated ATPase
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Myosin filaments have 13-22 nm in thickness and 0.7 mm in length
Actin binding proteins
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A group of proteins has been identified that forms crosslinks between actin
filaments to get a more viscous three dimensional network
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This group is called actin binding proteins and there are three types of actin
binding proteins
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Promoting cross linking to produce gelation
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Capping the short filaments that results in diminished viscosity (solation)
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Stabilizing the monomeric form of actin (G-actin) also causing solation
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Filamin, spectrin and alpha actinin have rod shape and are long and flexible
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Spectrin is present in erythrocytes and many other cell types
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Spectrin is present near the plasma membrane within the cytoplasm facilitating its
interaction with the membrane proteins
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As it is basically a calmodulin binding protein, it is sensitive to changes in calcium
concentrations in the cell
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Calcium ions may regulate the no of binding sites or affinity of the cross linking
proteins including spectrin ultimately affecting the gelation
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Villin is a protein present in intestinal microvillae (mol. wt. 95,000 daltons)
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At low calcium concentrations it acts as a cross linker but not at higher concentrations
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Changes in ionic strength, pH, and temperature can affect the formation of dimers and
tetramers of spectrin and filamin
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Since the oligomers only have the crosslinking ability, this can affect the crosslinking
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Cytochalasin B decreases the viscosity by binding to the F-actin causing
depolymerization to G-actin
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Phalloidin on the other hand stabilizes actin microfilaments increasing the viscosity
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Capping proteins generally act by capping at the elongating end thereby interfering
with polymerization
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They can also shorten the actin filaments that results in solation
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Villin in the presence of calcium can cause solation
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Gelsolin can decrease the viscosity mainly by capping to the barbed end of the actin
filaments
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Acumetin also acts in a similar way
Actin Depolymerizing Proteins
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Without changing the length of microfilaments these proteins lower the viscosity of
actin by decreasing their polymerization
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Profilin binds to the monomers preventing them to form polymers
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Thus the changes in viscosity dictate cell motility and this is regulated by the different
regulatory factors by their actions on actin microfilaments
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Increase in viscosity is achieved by increasing the polymerization by crosslinking
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Decrease in viscosity is achieved by decreasing the polymerization
Microfilaments and cell motility
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Microfilaments and actin-myosin interactions are known to be involved in cell motility
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Cytoplasmic streaming or cyclosis
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Amoeboid motion
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Cyclosis is often observed in plants
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Continuous protoplasmic currents characterize cyclosis that results in the reduction of
cytoplasm and displacement of chloroplasts and other cytoplasmic granules
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Chemodynesis is the term used if this is initiated by chemicals
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Photodynesis is the term used if this is initiated by light
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Cyclosis is affected by changes in pH, ions and temperature
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Cyclosis stimulators also include some auxins
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Cyclosis is inhibited by mechnical injuries, electrical shock, some anesthetics
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Cyclosis decreases when the hydrostatic pressure increases and the protoplasm
becomes more fluidic
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Actin-myosin interaction helps in the movement of endoplasm
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In a slime mold Physarum, cortical gel increases the internal pressure by contraction
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Cycles of relaxation and contraction can be noticed with changes in calcium and ATP
Ameboid motion
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Ameboid motion changes the cell shape
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Cytoplasmic projections called pseudopodia emerge out
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Ameboid motion occurs in amoeba as well as in numerous other cell types
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During inflammation for example, the leukocytes come out of the blood vessels by
ameboid motion and move towards the site of infection
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Ameba shows mostly monopodial projections but sometimes it can be polypoidal
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Cylindericallobopodium or filamentous filopodium can be formed
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Anastomosing types of pseudopodia are called reticulopodia
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Fibroblasts can form ruffled membrane projections called lamellipodia with polygonal
shape with sheet-like extensions
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Fine thread like projections are called filopodia
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Ameboid motion can be reversibly inhibited by cytochalasin B
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Ameboid cells do not adhere completely by the bottom surface on a support
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Instead of this they attach with a no of sites of adhesion called adhesion plaques or
focal contacts
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Under in vitro conditions ameboid cells can phagocytose the particles on their way and
create tracks called phagokinetic tracks
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The rate of progression is influenced by temperature and environmental factors
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Chemicals can attract or repel these movements as seen in chemotaxis during
inflammation
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During this motion the actin microfilaments attach with the plasma membrane
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Calcium is needed for this motion
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Mechanical injury, electrical shock or ultraviolet radiation can retract pseudopodia
Interaction of microfilaments with cell membrane and its surface receptors
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Microfilaments assemble and disassemble very quickly even within seconds
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Attachment of actin microfilaments to the plasma membrane has an important role in
ameboid motion
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ATP links and several proteins are involved in this attachment
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The pseudopods of ameboid cells contain a three dimensional network of proteins that
include actin, myosin, α-actinin and other cross linking proteins
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Sliding of actin and myosin filaments helps in generating a force needed for this
movement
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The focal changes can help in the directionality of the motion
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Cell locomotion, receptor redistribution and endocytosis are achieved through this
motion
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In cultured fibroblasts it was observed that actin accumulates followed by vinculin and
tubulin at the leading edge during ruffling
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While the microfilaments are organized on the cytoplasmic side of the membrane, its
surface receptors are bound with clusters of concavalin A (a lectin) and hemocyanin
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As the lectin binds the concentration of actin and myosin increase and the ligand
receptor internalization follows
Focal contacts
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Focal contacts are not only involved in the attachment but also help in movement of
cells
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Actin-containing microfilament bundles/stress fibres get implanted at these focal
contacts
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Similar to sarcomeres in muscle cells, myosin and a-actinin show a regular /periodic
arrangement
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These are also contractile and thus can create the necessary tensile strength between
the cells and the attached surface/substratum
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Vinculin is present at focal contacts
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It is a protein (mol. Wt. 130,000 daltons) that interacts with many actin monomers
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When a cell moves out of a focal site it leaves a footprint of several proteins and
polysaccharides (mostly glycosaminoglycans)
Fibronectin
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Fibronectin is a glycoprotein that participates in cell adhesion in cell types such as
astroglia, fibroblasts, primitive mesenchymal cells and epithelial cells
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It has a mol. Wt of 440,000 daltons with two subunits covalently linked with a disulfide
bond near its C terminal
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This protein is secreted from the attached cells through small vesicles
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It can bind collagen, fibrin, heparin and others as well as to sites in cell membrane
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A soluble form of fibronectin is present in blood plasma
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The soluble form or peptides formed from it can inhibit the binding of cellular
fibronectin to the membrane
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Fibronectin is reduced in cells undergoing transformation
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Fibronectin is needed for the migration of neural crest cells
Laminin
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Laminin is a non collagenous glycoprotein (mol. Wt. 950,000 daltons)
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It is mainly present in the basement membrane of tissues and binds with type IV
collagen and heparan sulfate
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Laminin acts as a major barrier to filtration in kidney and the glomerular basement
membranes in diabetic patients contain increased amounts of laminin
Collagen
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Collagen is the most abundant protein in the animal kingdom and is important in cell
adhesion
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Basement membranes and extracellular space including the reticular fibers contain
collagen
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Although the connective tissues contain the largest amounts of collagen, mesnchymal
and epithelial cells also contain collagen
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Reticular fibers containing collagen are made up of fibrils spaced regularly in a striated
fashion with 67nm repeats
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The fibers give a mechanical support to the tissue and constitute the surface on which
cells may glide over
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Tropocollagen constitutes the molecular unit of collagen consisting of 3 polypeptides
each of mol. wt. 95,000 forming α helix
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The short nonhelical segments are called telopeptides
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Amino acid composition reveals glycine, proline, hydroxyproline (more than 60%
together) and other amino acids.
Five types of collagen are known
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Type I is present in cornea, bone tendons, dermis and dentins
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Type II is present in cartilages
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Type III is present in fetal skin, cardiovascular system, uterus and intestines
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Type IV is present in basal laminae/basement membranes
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Type V is present in blood vessels, cornea and placenta
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Type IV does not have a typical fibrillar structure
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Tropocollagen is thought to be the monomer since by combination it can form different
forms of collagen
Focal contact proteins
Microtubules and microfilaments in cancer
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During transformation there is a disorganization of microtubules and microfilaments
resulting in changes in cell shape
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If normal fibroblasts are transformed by a virus microtubules become short and
microfilaments disappear and the cells show blebs over the surface
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The blebs disappear if cAMP is added and the cells reverse back to typical fibroblastic
shape
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Viral transformation also involves the phosphorylation of vinculin resulting in a more
rounded morphology, cytoskeletal disorganization and loss of adhesion to the
substratum
Intermediate filaments
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Intermediate filaments have thickness (10nm) in between that of microtubules and
microfilaments
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Four types of intermediate filaments are known
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Keratin filaments, neurofilaments, glial filaments and heterogenous filaments
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Intermediate filaments form a network connecting the nucleus with the cell surface
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In fibroblasts they form a cage like structure by surrounding the nucleus in attachment
with the nuclear envelope
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These filaments are continuous with those present in the cytoplasm
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Keratin filaments are also known as tonofilaments or prekeratins
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They are anchored to the cell surface and tend to converge on desmosomes
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Mammalian cytokeratins are a-fibrous proteins synthesized by the epidermis and form
the dead layers or stratum corneum
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They have 3 dimensional structural units of 47 to 58,000 daltons
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Epithelial differentiation typically involves the formation of keratins
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Neurofilaments are present in axons, dendrites and neuronal perikaya together with
microtubules
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They have 3 polypeptides (mol. wt. 68 to 200,000 daltons) forming a three
dimensional lattice in the axoplasm
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Calcium can induce proteolysis of these polypeptides
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Glial filaments are present in the cytoplasm of astrocytes and contain an acidic protein
(mol. wt. 51,000 daltons)
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Heterogenous filaments contain desmin, vimentin and synemin
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Desmin is made up of 2 polypeptides (mol. wt. 50,000 and 55,000 daltons)
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Desmin is present in skeletal and cardiac muscle along with vimentin, synemin and aactinin
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Vimentin filaments are insoluble and bind to the nuclear envelope and centriole
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Sysnemin is a protein of mol. wt. 230,000 daltons
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During mitosis many changes occur in intermediate filaments
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In prophase the filaments are reduced in their thickness from 10 to 2 nm forming
spheroidal aggregates containing cytokeratin and vimentin
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At metaphase and anaphase this continues and in telophase the filamentous shape is
restored.
Study Questions
1. What is meant by micro trabecular lattice?
2. What are the functions of focal contacts?
3. Solation involves
a) no changes in viscosity b) diminished viscosity c) increase in visosity d) increase
in density
4. Match the following
Cyclosis
Cytochalasin B
Laminin
Hydoxyproline
Ameboid motion
Diabetes
Tropocollagen
Auxins
5. Cyclosis initiated by chemicals is called-----------------.