Cytoskeletal Network

Cell and Molecular Biology in Medicine 5107 June 11, 20 Preeyanat Vongch

bjectives of topic

Structure and function of cytoskeleton netwo Biochemistry of cytoskeleton network Mechanism of action in cell activities

Cytoskeleton

internal network of 3 types of cytosol fibers

rofilament: 7-9 nm in diameter ermediate filament: 10 nm in diameter rotubules: 24 nm in diameter

te: Only 1 and 3 are involve in motility 2 structurally brace the inside of a cell

Actin molecule Minus end

Microfilam ent Minus end Microtubul e

Plus end

Plus end

Intermedia te filament

Biochemistry of cytoskeleton

ytoskeletal fibers built up from protein subunits y noncovalent bond

heme is the cross-linkage into structure likes bundles eodesic-dome-like,or gel-like lattices

nd cell takes its shape by bonding plasma membrane hese protein supports Microfilament and microtubules grow by polymerization of actin and tubulin subunits

ut, Intermediate filament is built with α –helical subun

Microfilament

Microfilaments are also often referred to as actin filaments. Long polymerized chains of the molecules are intertwined in a helix, creating a filamentous form of the protein (F-actin). All of the subunits that compose a microfilament are connected in such a way that they have the same orienta tion. Due to this fact, each microfilament exhibits polarit y, 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.

Intermediate-filament proteins Have a conserved domain structure consisting of a variable globular head domain, a central -helical c oiled-coil dimerization domain consisting of four coi led coils, based on heptad repeats, interrupted by f lexible linker domains and a variable globular tail d omain. The coiled-coil domains are termed 1A, 1B, 2A and 2B, respectively. The linker domains are non-

Vimentin -57 kDa - is the Intermediate Filament Protein (IFP) of mesenchymal cells

Desmin (53 kDa) - Exhibits a high degree of tissue specificity, its expression being predominantly confined to all types of muscle cells (cardiac, skeletal and smooth muscle). * Immunoperoxidase staining skeletal muscle fibers of central core disease

Microtubules are formed Microtubules

from molecules of tubulin, each of which is a heterodimer consisting of tw o closely related and tightly l inked globular polypeptides c alled α -tubulin and β -tubulin Although tubulin is present in virtually all eucaryotic cells, the most abundant source for biochemical studie s is the vertebrate brain Extraction procedures yield 10 to 20% of the total soluble protein in brain as tubulin, reflecting the unusually high density of microtubules in th

Growth of neuron exon

A microtubule can be regarded as a cylindrical structure in which the tubulin heterodimers are packed around a central core, which appears empty in electron micrographs More accurately, perhaps, one can view the structure as being built from linear protofilaments, each composed of alternating α - and β -tubulin sub units and bundled in parallel to form a cylinder Since the protofilaments are aligned in parallel with the same polarity, the microtubule itself is a polar structure, and it is possible to distinguish a plus (fa

Cell division

Colchicine An alkaloid extracted from the meadow saffron that has been used medicinally in the treatment of gout since ancient Egyptian times Each molecule of colchicine binds tightly to one tubulin molecule and prevents its polymerization, bu t it cannot bind to tubulin once the tubulin has poly merized into a microtubule The exposure of a dividing cell to colchicine, or to the closely related drug colcemid, causes the rapid disappearance of the mitotic spindle

Microtubulin and vesicle transportation needs motor protein

(+) end-direction of microtubule

Kinesin Consists of two heavy chains (110 and 135 kD and two light chains (60 and 70 kD) Each head is attached to an α -helical neck region, which forms a coiled-coil dimer Microtubules bind to the helix indicated, this interaction is regulated by the nucleotide bou nd at the opposite side of the domain The distance between microtubule binding sites is 5.5 nm

Some microtubule-directed movements, such as retrograde axonal transport or the transit of endocytotic vesicles of the plasma membrane to lysosomes, are in t Dyneins he (-)end direction movements Large, multimeric proteins, with molecular weights exceeding 1,000 kD. They are composed of two or three heavy chains (470-540 kD) complexed with a poorly determined number of intermediate and light ch ains -Dyneins are divided into two functional classes: Cytosolic dynein: involved in the movement of vesicles chromosomes Axonemal dynein: responsible for the

Dynein

Model showing the attachment of the outer dynein arm to the A tubule of one doublet and the cross-bridges to the B tu bule of an adjacent doublet. The attachment to the A tubule is stable. In the presence of ATP, the successive formation and breakage of cross-bridges to the adjacent B tubule leads to movement of one doublet r elative to the other.

Actin filament

The thin actin filament is a dimeric polymer of G-actin sub-units arranged like 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 tropomyosin near the ends of the tropomyosin sub-units;  TnI that binds to the actin; an d TnC that binds to the TnI and TnT sub-units, and which also has a strong affinity for Ca2+ at four binding sites

All eukaryotic cells contain actin

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

Actin monomer Two display modes, has subdomains designated 1-4 A simplified cartoon is above right. ATP binds, along with Mg++, within a deep cleft between 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

G-actin (globular actin) with bound ATP can polymerize, to form F-acti n (filamentous actin). F-actin may hydrolyze its bound ATP to ADP + Pi and release Pi. ADP release from the filament does not occur because the cleft opening is blocked. ADP/ATP exchange: G-actin can release ADP and bind ATP, which is usually present in the cytosol at hig her concentration than ADP.

Structure of actin monomer is complex

Where actin can be found? Why is it important to cells?

Microvilli projections of the plasma membrane forced out by actin; increase plasma membrane surface area in cells such as those lining in: -the small intestine -the walls of kidney tubules

Stereocilia: Stereocilia microvilli in our ears that are used to detect sound and generate an action potential

Cytoskeleton in erythrocytes In the erythrocyte cytoskeleton, Spectrin filaments are bound together by short filaments of actin that also attach to membrane proteins (Glycophorin) through a linker, peripheral membrane protein, band 4.1 protein. protein In addition, Ankyrin, Ankyrin a peripheral membrane protein, connects the centre of the spectrin filament to another membrane protein, band 3 protein, protein an anion transporter.

A similar molecule to spectrin is Dystrophin. In muscle cells this protein links the extensive actin cytoskeleton to the membrane complex. It is not the major skeletal component as in erythrocytes.   This also has an actin-binding domain and a filament made of β-sheet repeats. This protein is found in skeletal muscle and links actin filaments to a glycoprotein complex in the membrane: The complex then binds to laminin and agrin in ECM

Platelets have two networks of actin.   In an inactive platelet a cortical network of actin filaments bound together by a non-erythroid isoform of spectrin. Ankyrin links this network to the membrane sodium-potassium ATPase molecule.   A second network of actin filaments exists in the cytoplasm of the platelet which is bound together (or organized by) filamin into a gel. Filamin also anchors this network to the membrane glycoprotein complex Gp1b-IX

Epithelial cells   Here Ezrin is the actin-binding protein that links through EBP50 to the cystic fibrosis transmembrane conductance receptor (CFTR). In an inactive state Ezrin folds up and disconnects from the actin filament.

Functions and roles of cytoskeleton

Cytoskeletal network and the cell Cell lomotion: motile, movement, migrate Control of cell shape Contraction of muscle cell Elongation of nerve axons Formation of cell surface protrusions (microvilli and filopodia) Constriction of a dividing cell during mitosis Separation of chromosomes Streaming of cytosol Transportation of membrane vesicles

Cytoskeleton and changes in cell shape

Polarity of actin patches and cables throughout the yeast cell cycle. Filamentous actin structures in the yeast cell, include actin patches and acti n cables (A) In a mother cell, most of the patches are clustered at one end. The cables are lined up and point toward the cluster of patches, which is the site where the bud will emerge (B) As the small bud grows, most patches remain within it. Cables in the mother cell continue to point toward this site of new cell wall growth (C) sized (D)

Patches are almost uniformly distributed over the surface of a fullbud. Cables in the mother cell remain polarized.

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

Cell 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 from polymerization of tubulin and actin and their assembly into bundles and network

Note: Few movement needs both mechanisms

Model for protrusion of cell (lamellipodia)

Nucleation is mediated by the ARP complex at the front. Newly nucleated actin filaments are attached to the sides of preexisting filaments, primarily at a 70° ang le. 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 beco me capped. After newly polymerized actin subunits hydrolyze their bound ATP in the filament lattice, the filaments become susceptible to depolymerization by cofilin. This cycle causes a spatial separation between net filament assembly at the front and 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 sta tionary with respect to the substratum.

Actin filament involves in cell movement

Stress fiber

Actin involves in cell movement

Elongation of nerve exon

(A) Scanning electron micrograph of two growth cones at the end of a neurite, put out by a chick sympathetic neuron in culture Here, a previously single growth cone has recently split into two. Note the many filopodia and the large lamellipodia. The taut appearance of the neurite is due to tension generated by the forward movement of the growth c ones, which are often the only firm points of attachm ent 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

   

Myosin or thick filament

Myosin: the actin motor protein   In addition to actin polymerisation for cell movement, many cell movements depend on the int eraction of actin with an associated MOTOR protei n – myosin. It is called a motor protein due to its ability to run along the actin filament through a series of conform ation changes and bindings The conformational changes require energy. Myosin is the motor, actin the tracks

Mechanism of muscle contraction

Action of actin filament in mucle contraction

The Sliding Filament Hypothesis

Thin and thick filaments in sarcomeres

Actin cross-linking proteins consists of 3 groups

1. Group-1 proteins - have unique actin-binding domain 2. Group II proteins - have a 7 kD actin-binding domain 3. Group III proteins - have pairs of a 26 kD actin-binding domain

Proteins

MW

Location

30 kD

33

Filopodia, lamellipodia, stress fibers

EF-1a

50

Pseudopodia

Fascin

55

Filopodia, lamellipodia, stress fibers, Microvilli, acrosomal process

Scruin

102

Acrosomal process

Vilin

92

Intestinal and kidney brush border microvilli

Dematin

48

Erythrocyte cortical networks

Fimbrin

68

α -actin

102

Microvilli, sterocilia, adhesion plaques, yeast actin cables Filopodia, lamellipodia, stress fibers, adhesion plaques

Spectrin Dystrophin

α :280 β :246-275 427

Muscle cortical networks

ABP120

92

Pseudopodia

Filamin

280

Filopodia, pseudopodia, stress fibers

Group I

Group II

Group III

Cortical networks

References 1. Lodish H., et al. Molecular cell biology 3rd .Edition 2. http://cellbiology.med.unsw.edu.au/units/science/ science.htm