cytoskeletal network

8/14/2019 Cytoskeletal Network

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Cell and Molecular Biology in Medicine 510Cell and Molecular Biology in Medicine 5107

June 11, 2June 11, 20

Preeyanat VongcPreeyanat Vongch


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jectives of topic

Structure and function of cytoskeleton netwBiochemistry of cytoskeleton network

Mechanism of action in cell activities


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CytoskeletonCytoskeleton

nternal network of 3 types of cytosol fibers

rofilament: 7-9 nm in diameterrmediate filament: 10 nm in diameterrotubules: 24 nm in diameter

e: Only 1 and 3 are involve in motility2 structurally brace the inside of a cell


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Intermediate filament

Microfilament

Microtubule

Actin moleculeMinus end

Minus end

Plus end Plus end


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toskeletal fibers built up from protein subunitsnoncovalent bond

eme is the cross-linkage into structure likes bundleodesic-dome-like,or gel-like lattices

d cell takes its shape by bonding plasma membraneese protein supports

Microfilament and microtubules grow by polymerizationMicrofilament and microtubules grow by polymerization

of actin and tubulin subunitsof actin and tubulin subunits

t, Intermediate filament is built with helical subu

Biochemistry of cytoskeletonBiochemistry of cytoskeleton


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MicrofilamentMicrofilament

Microfilaments are also often referred to as actin

filaments.

Long polymerized chains of the molecules areintertwined in a helix, creating a filamentous form of theprotein (F-actin).

All of the subunits that compose a microfilament areconnected in such a way that they have the same orientation. Due to this fact, each microfilament exhibits polarity, the two ends of the filament being distinctly different.

This polarity affects the growth rate of microfilaments,typically extend out from the centrosome of a cell.


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Intermediate-filament proteinsIntermediate-filament proteins

Have a conserved domain structure consisting of avariable globular head domain, a central -helical c

oiled-coil dimerization domain consisting of four coiled coils, based on heptad repeats, interrupted by flexible linker domains and a variable globular tail domain.

The coiled-coil domains are termed 1A, 1B, 2A and2B, respectively. The linker domains are non-


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VimentinVimentin-57 kDa- is theIntermediate

Filament Protein(IFP) ofmesenchymal cells

DesminDesmin (53 kDa)- Exhibits a high

degree of tissuespecificity, itsexpression being

predominantly confinedto all types of musclecells (cardiac, skeletaland smooth muscle).

* Immunoperoxidase staining

skeletal muscle fibers of centralcore disease


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MicrotubulesMicrotubulesMicrotubules are formedfrom molecules of tubulin,each of which is a

heterodimer consisting of two closely related and tightly linked globular polypeptides called -tubulin and -tubulinAlthough tubulin is presentin virtually all eucaryoticcells, the most abundantsource for biochemical studies is the vertebrate brain

Extraction procedures yield10 to 20% of the total solubleprotein in brain as tubulin,reflecting the unusually high

density of microtubules in th


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A microtubule can be regarded as a cylindricalstructure in which the tubulin heterodimers arepacked around a central core, which appears emptyin electron micrographs

More accurately, perhaps, one can view the

structure as being built from linear protofilaments,each composed of alternating - and -tubulin subunits and bundled in parallel to form a cylinder

Since the protofilaments are aligned in parallel with

the same polarity, the microtubule itself is a polarstructure and it is ossible to distin uish a lus fa

Growth of neuron exonGrowth of neuron exon


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Cell divisionCell division


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CColchicineolchicine

An alkaloid extracted from the meadow saffron that

has been used medicinally in the treatment of goutsince ancient Egyptian times

Each molecule of colchicine binds tightly to onetubulin molecule and prevents its polymerization, bu

t it cannot bind to tubulin once the tubulin has polymerized into a microtubule

The exposure of a dividing cell to colchicine, or tothe closely related drug colcemid, causes the rapid

disappearance of the mitotic spindle


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Microtubulin and vesicle transportationMicrotubulin and vesicle transportation

needs motor proteinneeds motor protein

(+) end-direction of microtubule(+) end-direction of microtubule


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KKinesininesin

Consists of two heavy chains (110 and 135 kDand two light chains (60 and 70 kD)

Each head is attached to an -helical neckregion, which forms a coiled-coil dimer

Microtubules bind to the helix indicated, thisinteraction is regulated by the nucleotide bound at the opposite side of the domain

The distance between microtubule binding

sites is 5.5 nm


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DyneinsDyneins

- Large, multimeric proteins, with molecularweights exceeding 1,000 kD.

- They are composed of two or three heavychains (470-540 kD) complexed with a poorlydetermined number of intermediate and light chains

-Dyneins are divided into two functional classes:Cytosolic dynein: involved in the

movement of vesicleschromosomes

Axonemaldynein: responsible for the

Some microtubule-directed movements,such as retrograde axonal transport or

the transit of endocytotic vesicles of the

plasma membrane to lysosomes, are in the (-)end direction movements


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Model showing the attachment of the outer dynein arm to

the A tubule of one doublet and the cross-bridges to the B tubule of an adjacent doublet.

The attachment to the A tubule is stable. In the presence ofATP, the successive formation and breakage of cross-bridgesto the adjacent B tubule leads to movement of one doublet r

elative to the other.

DyneinDynein


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Actin filamentActin filament

The thin actin filament is a dimeric polymer of G-actin sub-units arrangedlike two strings of beads twisted together.

Attached to the actin chain of the thin filament, are the proteins troponin(Tn) and tropomyosin.

A tropomyosin molecule runs along each actin chain, bound to the actin.Each tropomyosin sub-unit covers about 7 G-actin sub-units.

The troponin molecule has three sub-units: TnT that binds to tropomyosinnear the ends of the tropomyosin sub-units; TnI that binds to the actin; and TnC that binds to the TnI and TnT sub-units, and which also has a strong

affinity for Ca2+ at four binding sites


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Most single eukaryotic cells (like these platelets)spread and stick to substrates they encounter

All eukaryotic cells contain actin


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Actin monomer

Two display modes, has subdomains designated 1-4

A simplified cartoon is above right. ATP binds, along with Mg++, withina deep cleftbetween subdomains 2 and 4

Actin can hydrolyze its bound ATP to ADP + Pi, releasing Pi.

The actin monomer can exchange bound ADP for ATP

The conformation of actin is different, depending on whether

there is ATP or ADP in the nucleotide-bindings


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G-actin (globular actin) with boundATP can polymerize, to form F-acti

n (filamentous actin).F-actin may hydrolyze its boundATP to ADP + Pi and release Pi.

ADP release from the filament doesnot occur because the cleft openingis blocked.

ADP/ATP exchange:G-actin can

release ADP and bind ATP, which isusually present in the cytosol at higher concentration than ADP.


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Structure of actin monomer is complexStructure of actin monomer is complex


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Where actin can be found?Where actin can be found?

Why is it important to cells?Why is it important to cells?


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MicrovilliMicrovilli

projections of the plasma membrane forced out byactin;

increase plasma membrane surface area incells such as those lining in:

-the small intestine-the walls of kidney tubules


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StereociliaStereocilia:

microvilli in our earsthat are used to detectsound and generate anaction potential


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In the erythrocyte cytoskeleton, SpectrinSpectrin filamentsare bound together by short filaments ofactinactin thatalso attach to membrane proteins (Glycophorin)through a linker, peripheral membrane protein,

band 4.1 proteinband 4.1 protein.

In addition, AnkyrinAnkyrin, a peripheral membraneprotein, connects the centre of the spectrinfilament to another membrane protein, band 3band 3

proteinprotein, an anion transporter.

Cytoskeleton in erythrocytesCytoskeleton in erythrocytes


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A similar molecule to spectrin is Dystrophin. In muscle cells thisprotein links the extensive actin cytoskeleton to the membranecomplex. It is not the major skeletal component as inerythrocytes.This also has an actin-binding domain and a filament made of-sheet repeats. This protein is found in skeletal muscle andlinks actin filaments to a glycoprotein complex in themembrane:

The complex then binds to laminin and agrin in ECM


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Platelets have two networks of actin.Platelets have two networks of actin.In an inactive platelet a cortical network of actin filamentsbound together by a non-erythroid isoform of spectrin. AnkyrinAnkyrinlinks this network to the membrane sodium-potassium ATPase

molecule.A second network of actin filaments exists in the cytoplasm ofthe platelet which is bound together (or organized by) filaminfilamininto a gel. Filamin also anchors this network to the membrane

glycoprotein complex Gp1b-IXGp1b-IX


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Epithelial cellsEpithelial cellsHere EzrinEzrin is the actin-binding protein that links

through EBP50 to the cystic fibrosistransmembrane conductance receptor (CFTR). Inan inactive state Ezrin folds up and disconnectsfrom the actin filament.


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Functions and roles of cytoskeletonFunctions and roles of cytoskeleton


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Cell lomotion: motile, movement, migrate

Control of cell shape

Contraction of muscle cellElongation of nerve axons

Formation of cell surface protrusions(microvilli and filopodia)

Constriction of a dividing cell during mitosis

Separation of chromosomes

Streaming of cytosolTransportation of membrane vesicles

Cytoskeletal network and the cellCytoskeletal network and the cell


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Cytoskeleton andCytoskeleton and

changes in cell shapechanges in cell shape


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Polarity of actin patches and cables throughout the yeast cell cycle.Filamentous actin structures in the yeast cell, include actin patchesactin patches andactiacti

n cablesn cables

(A) In a mother cell, most of the patches are clustered at one end. Thecables are lined up and point toward the cluster of patches, which is thesite where the bud will emerge

(B) As the small bud grows, most patches remain within it. Cables in

themother cell continue to point toward this site of new cell wall growth

(C) Patches are almost uniformly distributed over the surface of a full-sized bud. Cables in the mother cell remain polarized.

(D) Immediately after cell division, mother and daughter cells form new

patches, which are concentrated near the division site,although both cells have randomly oriented cables


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Cell motility involves 2 mechanismsCell motility involves 2 mechanisms

1. Motor proteins:Use motor proteins to generate energy from ATP to walk,slide or carry proteins along microfilament/ microtubules

2. Cytoskelaton rearrangement:

Use the changes in the shape of cell, which results frompolymerization of tubulin and actin and their assemblyinto bundles and network

Note: Few movement needs both mechanisms

M d l f t i f ll (l lli di )M d l f t i f ll (l lli di )


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Model for protrusion of cell (lamellipodia)Model for protrusion of cell (lamellipodia)

Nucleation is mediated by the ARP complex at the front. Newly nucleated actinfilaments are attached to the sides of preexisting filaments, primarily at a 70 angle.

Filaments elongate, pushing the plasma membrane forward because of some sort

of anchorage of the array behind. At a steady rate, actin filament plus ends become capped.

After newly polymerized actin subunits hydrolyze their bound ATP in the filamentlattice, the filaments become susceptible to depolymerization by cofilin.

This cycle causes a spatial separation between net filament assembly at the frontand net filament disassembly at the rear, so that the actin filament network as a

whole can move forward, even though the individual filaments within it remain stationary with respect to the substratum.


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Actin filament involves in cell movement


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Actin involves in cell movement

Stress fiber

El ti fEl ti f


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(A) Scanning electron micrograph of two growth cones at

the end of a neurite, put out by a chick sympatheticneuron in cultureHere, a previously single growth cone has recentlysplit into two. Note the many filopodia and the largelamellipodia.

The taut appearance of the neurite is due to tensiongenerated by the forward movement of the growth cones, which are often the only firm points of attachment of the axon to the substratum

(B) Scanning electron micrograph of the growth cone of

a sensory neuron crawling over the inner surface of t

Elongation of nerve exonElongation of nerve exon


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Myosin or thick filament


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Myosin: the actin motor protein

In addition to actin polymerisation for cellmovement, many cell movements depend on the interaction of actin with an associated MOTOR protein myosin.

It is called a motor protein due to its ability to runalong the actin filament through a series of conformation changes and bindings

The conformational changes require energy.Myosin is the motor, actin the tracks


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Mechanism of muscle contractionMechanism of muscle contraction

A i f i fil i l iA ti f ti fil t i l t ti


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Action of actin filament in mucle contractionAction of actin filament in mucle contraction


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The Sliding Filament HypothesisThe Sliding Filament Hypothesis


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Thin and thick filaments in sarcomeres


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Actin cross-linking proteins consists of 3 groupsActin cross-linking proteins consists of 3 groups

1.1. Group-1 proteinsGroup-1 proteins- have unique actin-binding domain

2.2. Group II proteinsGroup II proteins

- have a 7 kD actin-binding domain

3.3. Group III proteinsGroup III proteins- have pairs of a 26 kD actin-binding domain

Proteins MW Location


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Proteins MW Location

Group I

Group III

Group II

30 kD

EF-1a

Fascin

Scruin

Vilin

Dematin

Fimbrin

-actinSpectrin

Dystrophin

ABP120

Filamin

33

50

55

102

92

48

68

102

:280 :246-275427

92

280

Filopodia, lamellipodia, stress fibers

Pseudopodia

Filopodia, lamellipodia, stress fibers,Microvilli, acrosomal process

Acrosomal process

Intestinal and kidney brush border microvilli

Microvilli, sterocilia, adhesion plaques,yeast actin cables

Cortical networks

Muscle cortical networks

Pseudopodia

Filopodia, pseudopodia, stress fibers

Erythrocyte cortical networks

Filopodia, lamellipodia, stress fibers,

adhesion plaques


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ReferencesReferences

1. Lodish H., et al. Molecular cell biology 3rd

.Edition2. http://cellbiology.med.unsw.edu.au/units/science/science.htm

cytoskeletal network

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