cytology cytoskeleton

8/6/2019 Cytology Cytoskeleton

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Department of Natural Sciences

University of St. La Salle

Bacolod City


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The cytoskeleton is a network of connectedfilaments and tubules extending from thenucleus to the plasma membrane.

It maintains the shape of the cell, anchororganelles, move the cell and control internalmovement of structures.

Motility of cells is determined by special

organelles for locomotion Internal movements (cytoplasmic streaming

or cyclosis) by cytoskeleton components.

CYTOSKELETON


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Three types of cytoskeletal elementsare found in eukaryotic cells:


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1. Actin microfilaments are long, thin fibers approximatelyseven nm in diameter, occurring in bundles or meshlike

networks. They move by interacting with myosin.2. Microtubules are cylinders that form centrioles, cilia and

flagella3. Intermediate filaments support the nuclear envelope,

plasma membrane and form cell-to-cell junctions.


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All types form ashelical assemblies of

subunits that self-associate usinga combination ofend-to-end and side-

to-side proteincontacts. Both microtubules

and microfilamentsgrow fastest from theplus end than the minus end of the assembly.

Removal of monomers at the () end and additionof monomers at the (+) end leaves the filamentsat the same overall length (treadmilling)


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The treadmilling of a microfilament or microtubule, madepossible by the NTP hydrolysis that follows subunit addition

(A) Subunits with bound NTPs polymerize at both ends of a growing filament,& then undergo nucleotide hydrolysis in the filament lattice. As the filament

grows, elongation is faster than hydrolysis at the plus end, & the terminalsubunits at this end are therefore always in the T form. However, hydrolysis is

faster than elongation at the minus end, & so terminal subunits at this endare in the D form. (B) Treadmilling occurs at intermediate concentrations of

free subunits (i.e., the plus end grows while the minus end shrinks)


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Mechanical properties of actin,tubulin, and intermediate

filament polymers.Networks composed of microtubules,

actin filaments, or an intermediatefilament called vimentin, all at equal

concentration, were exposed to ashear force in a viscometer, & the

resulting degree of stretch wasmeasured. The results show that

microtubule networks are strong, rigidhollow tubes that are easily deformed

but rupture (indicated by redstarburst) and begin to flow without

limit when stretched beyond 150% oftheir original length. Actin filament

networks are much more rigid, butthey also rupture easily. Intermediatefilament networks, by contrast, are

not only easily deformed, but theywithstand large stresses and strains

without rupture; they are thereby well

suited to maintain cell integrity.


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F-actin is polymerized through addition of globularactin or G-actin monomers at the growing (+) end,bearing a stabilizing ATP cap.


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They form a dense complex web just under the plasmamembrane; in microvilli of intestinal cells, they act to shorten

the cell In plant cells, actin filaments form tracts along whichchloroplasts circulate.

Involved in cell rigidity, tensile strength and resilience,cellular movement (pseudopodia and mesenchyme cellmigration, platelet activation)

Pseudopodia are associated with actin near the moving edgeof the cell. Actin filaments move by interacting with myosinchanging the configuration to pull the actin filament forward.

Similar action accounts for pinching off cells during celldivision and for amoeboid movement.

Other arrangements of microfilaments in association withaccessory proteins are possible. Ex: contractile rings of celldivision; parallel bundles are found in stress fibers offibroblasts, filopodia and other cell projections; gels of shortrandomly oriented filaments are found in egg cortical regions.


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Microfilaments in a cell. A crawling cell with 3 areas showing thearrangement of actin filaments. The actin filaments are shown in red,

with arrowheads pointing toward the plus end. Stress fibers arecontractile and exert tension. Filopodia are spike-like projections of the

plasma membrane that allow a cell to explore its environments. Thecortex underlies the plasma membrane.


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A model of how forces generated inthe actin-rich cortex move a cell

forward.The actin-polymerization-

dependent protrusion and firmattachment of a lamellipodium at

the leading edge of the cell movesthe edge forward (green arrows at

front) and stretches the actincortex. Contraction at the rear of

the cell propels the body of the cellforward (green arrow at back) torelax some of the tension (traction).New focal contacts are made at the

front, and old ones aredisassembled at the back as the cellcrawls forward. The same cycle can

be repeated, moving the cellforward in a stepwise fashion.

Alternatively, all steps can be tightlycoordinated, moving the cell

forward smoothly. The newlypolymerized cortical actin is shown

in red.


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Platelet activation. (A) Platelet activation is a controlled sequence of actinfilament severing, uncapping, elongation, recapping, and cross-linking that createsa dramatic shape change in the platelet. (B) SEM of platelets prior to activation. (C)An activated platelet with its large spread lamellipodium. (D) An activated platelet

at a later stage than the one shown in C, after myosin II-mediated contraction.


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Several actin-binding proteins influence deployment offilaments in the cytoplasm:

Profilin binds to G-actinmonomers to regulatepolymerization

Capping protein limits

length increase by bindingto the end of actin filament Fimbrin binds adjacent actin

filaments to form bundles Filamin stabilizes filament 3-D network by

intersecting with microfilaments Gelsolin breaks filament into shorter segments by

inserting between subunits Vinculin & actinin mediate binding of actin to cell

membrane at intercellular junctions and cell base.


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The modular structures of four actin-cross-linking proteinsEach of the proteins shown has two actin-binding sites (red) that are

related in sequence. Fimbrin has two directly adjacent actin-binding sites,so that it holds its two actin filaments very close together (14 nm apart),

aligned with the same polarity. The two actin-binding sites in

-actinin areseparated by a spacer around 30 nm long, so that it forms more looselypacked actin bundles. Filamin has two actin-binding sites with a Vshapedlinkage between them, so that it cross-links actin filaments into a networkwith the filaments oriented almost at right angles to one another. Spectrinis a tetramer of two and two subunits, and the tetramer has two actin-

binding sites spaced about 200

nm apart


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F

ilamin cross-links actin filaments into a three-dimensionalnetwork with the physical properties of a gel(A) Each filamin homodimer is about160 nm long when fullyextended and forms a flexible, high-angle link between two

adjacent actin filaments. (B) A set of actin filaments cross-linkedby filamin forms a mechanically strong web or gel.


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A family of globular heterodimer proteins composedofE and F subunits; slender tubules are about 25 nmin diameter, running in straight course in thecytoplasm.

The wall is composed of13 protofilaments of the

protein tubulin, and later bind to microtubule-associated proteins (MAPs).

MICROTUBULES


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The structure of a microtubule and its subunit. (A) The subunit of eachprotofilament is a tubulin heterodimer, formed from a very tightly linked

pair of a- and b-tubulin monomers. The GTP molecule in the b-tubulinmonomer is less tightly bound and has an important role in filamentdynamics. Both nucleotides are shown in red. (B) One tubulin subunit (a-b

heterodimer) and one protofilament consist of many adjacent subunitswith the same orientation. (C) The microtubule is a stiff hollow tube

formed from 13 protofilaments aligned in parallel. (D) A short segment ofa microtubule viewed in an EM. (E) EM of a cross section of a microtubule


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Microtubules are non-contractile polarizedstructures with a (-) end

anchored to thecentrosome, and a free (+)end at which tubulinmonomers are added orremoved.

Capping proteins located inparticular parts of the cellcortex bind at the (-) endsof microtubules, stabilizingthem and controlling the

shape and polarity of cells. Conversion of bound GTP

to GDP at the growing (+)end of the F subunit causedepolymerization.


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Model for the structural consequences of GTP hydrolysis in themicrotubule lattice.Hydrolysis of GTP after assembly changes the conformation of the

subunits & tends to force the protofilament into a shape that is less ableto pack into the microtubule wall. (C) Loss of the GTP cap allows the

GDP-containing protofilaments to relax into their more curved

conformation. This leads to a progressive disruption of the microtubule.


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Dynamic instability due to the structural differences between agrowing and a shrinking microtubule end

(A) A growing microtubule has GTP-containing subunits at its end,forming a GTP cap. If nucleotide hydrolysis proceeds more rapidly thansubunit addition, the cap is lost and the microtubule begins to shrink, anevent called a "catastrophe." But GTP-containing subunits may still addto the shrinking end, and if enough add to form a new cap, microtubule

growth resumes, an event called "rescue."


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Effect of the drug taxol on microtubule organization.(A) Molecular structure of taxol. Recently, organic chemists have

succeeded in synthesizing this complex molecule, which is widely used forcancer treatment. (B) Immunofluorescence micrograph showing themicrotubule organization in a liver epithelial cell before the addition oftaxol. (C) Microtubule organization in the same type of cell after taxol

treatment. Note the thick circumferential bundles of microtubules aroundthe periphery of the cell. (D) A Pacific yew tree, the natural source of taxol.


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Functions of Microtubules

Serve as cytoskeleton to maintain cell shape Involved in changes in cell shape, & serve as a

"temporary scaffolding" for other organelles. They function as diffusion channels for water

and metabolites and even macromolecules, thus

aiding intracellular transport. In mitosis, microtubules form the mitotic spindle

along which chromosomes move. After administration of drugs like colchicine

(binds to monomeric tubulin and preventpolymerization) and vinblastine, microtubulesdisappear and mitosis is arrested because ofinadequate formation of the mitotic spindle.


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MOLECULAR MOTORS

MOTOR PROTEINS- specialized motility structures ineucaryotic cells consisting of highly ordered arrays of

motor proteins that move on stabilized filament tracks. They use the energy of ATP hydrolysis to move alongmicrotubules or actin filaments.

They mediate the sliding of filaments relative to oneanother and the transport of membrane-enclosed

organelles along filament tracks.


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All known motor proteins that move onactin filaments are members of the myosin

superfamily; the motor proteins that move onmicrotubules are members of either thekinesin superfamily or the dynein family.

The myosin and kinesin superfamilies arediverse, with about40 genes encoding each

type of protein in humans. The only structural element shared among all members of

each superfamily is the motor "head" domain. These headscan be attached to a wide variety of "tails," which attach todifferent types of cargo and enable the various family

members to perform different functions in the cell.


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Kinesins move towards the (+) ends of tubules,while dyneins move towards the () ends.

Kinesin is responsible for movement of vesiclesand organelles in the cytoplasm, dynein regulates2-way traffic and dynamin serves as a motor forsliding movements during microtubule elongation.


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Cycle ofstructuralchangesused by

myosin towalk along

an actinfilament.


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Summary of the coupling between ATP hydrolysis and conformationalchanges for myosin II. Myosin begins its cycle tightly bound to the actin

filament, with no associated nucleotide, the so-called "rigor" state. ATPbinding releases the head from the filament. ATP hydrolysis occurs while

the myosin head is detached from the filament, causing the head toassume a cocked conformation, although both ADP and inorganic

phosphate remain tightly bound to the head. When the head rebinds to thefilament, the release of phosphate, followed by the release of ADP, triggerthe power stroke that moves the filament relative to the motor protein. ATPbinding releases the head to allow the cycle to begin again. In the myosincycle, the head remains bound to the actin filament for only about5% ofthe entire cycle time, allowing many myosins to work together to move a

single actin filament.


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Kinesin and kinesin-related proteins. (A) Structures of four kinesinsuperfamily members. Conventional kinesin has the motor domain at the

N-terminus of the heavy chain. The middle domain forms a long coiledcoil, mediating dimerization. The C-terminal domain forms a tail thatattaches to cargo, such as a membrane-enclosed organelle. These

kinesins generally travel toward the minus end instead of the plus end ofa microtubule. (B) Freeze-etch EM of a kinesin molecule with the head

domains on the left.


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Summary of the coupling between ATP hydrolysis and conformationalchanges for kinesin. At the start of the cycle, one of the two kinesin

heads, the front or leading head (dark green) is bound to the microtubule,with the rear or trailing head (light green) detached. Binding of ATP to thefront head causes the rear head to be thrown forward, past the binding

site of the attached head, to another binding site further toward the plusend of the microtubule. Release of ADP from the second head (now in thefront) and hydrolysis of ATP on the first head (now in the rear) brings the

dimer back to the original state, but the two heads have switched theirrelative positions, and the motor protein has moved one step along the

microtubule protofilament. In this cycle, each head spends about50% of

its time attached to the microtubule and 50% of its time detached.


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Are structurally similar but biochemically distinct,with diameters intermediate between microtubulesand microfilaments (about10 nm).

They associate with polypeptidesfillagrin (binds tokeratin), plectin (links vimentin), and synamin (also

links vimentin, but found in muscle). 5 types are:

1. Glial filaments found in non-neural cells of theCNS: astrocytes, oligodendrocytes, microglia.

2. Keratin filaments characteristic of epithelialcells; called tonofilaments are often associatedwith desmosomes at the cell surface. Theyparticipate in the formation of keratin inkeratinizing epithelia.

INTERMEDIATE FILAMENTS


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3.Desmin characteristic of smooth, striated & cardiacmuscle; keep sarcomeres of neighboring myofibrils inregister across the width of the fiber; link Z-bands of

peripheral myofibrils to the sarcolemma; ensures uniformdistribution of tensile strength throughout the muscle cell.

4.Vimentin abundant in fibroblasts and mesenchymalderivatives, in bundles or randomly oriented in a networkthroughout the cytoplasm.

5.Neurofilaments present in nerve cell processes with acytoskeletal function; helps to maintain the gel state of theaxoplasm; involved in intracellular metabolite transport.


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A model of intermediatefilament constructionThe monomer shown in (A)

pairs with an identicalmonomer to form a dimer (B)in which the conservedcentral rod domains arealigned in parallel andwound together into a coiledcoil. (C) Two dimers then lineup side by side to form thetetramer soluble subunit ofintermediate filaments. (D)Within each tetramer, the 2

dimers are offset withrespect to one another, thereby allowing it to associate withanother tetramer. (E) In the final 10-nm rope-like filament,tetramers are packed together in a helical array, which has 16dimers in cross-section. Half of these dimers are pointing in eachdirection.


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Keratin filaments inepithelial cells

Immunofluorescencemicrograph of the networkof keratin filaments (green)in a sheet of epithelial

cells in culture. The filaments in each cellare indirectly connected tothose of its neighbors bydesmosomes.

A 2nd protein (blue) hasbeen stained to reveal thelocation of the cellboundaries.


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Two types of intermediate filaments in cells of the nervous system.(A) Freeze-etch EM image of neurofilaments in a nerve cell axon, showingthe extensive cross-linking through protein cross-bridges an arrangement

believed to give this long cell process great tensile strength. The cross-bridges are formed by the long, nonhelical extensions at the C-terminus ofthe largest neurofilament protein (NF-H). (B) Freeze-etch image of glialfilaments in glial cells, showing that these intermediate filaments aresmooth and have few cross-bridges. (C) Conventional EM of a crosssection of an axon showing the regular side-to-side spacing of the

neurofilaments, which greatly outnumber the microtubules.


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Also called the centrosphere or cell center, whichrefers to a specialized zone of cytoplasm containingthe centrioles and a variable number of small densebodies called centriolar satellites.

Considered to be a center of activities associatedwith cell division, usually adjacent to the nucleus. The Golgi apparatus often partially surrounds the

centrosome on the side away from the nucleus. Thecentrosome is located in the cytoplasm next to the

nucleus It consists of an amorphous matrix of protein

containing the g-tubulin ring complexes that nucleatemicrotubule growth

CENTROSOME


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They serve as basal bodies and sites of anchorfor epithelial cilia.

Plant and fungal cells have a structure equivalentto a centrosome, although they do not containcentrioles

The matrix of the centrosome is organized by a

pair ofcentrioles.An electron micrograph of a

thick section of a centrosomeshowing an end-on view of a

centriole. The ring of modifiedmicrotubules of the centriole is

visible, surrounded by thefibrous centrosome matrix.


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Centrioles are self-duplicating organelles thatexhibit continuity from one cell generation to thenext. They double in number immediately before celldivision but they do not undergo transverse fission.

After cell division, each cell acquires 2 centrioles,one from the parent cell, and one which arose as aprocentriole.

Paired centrioles are called diplosome. The longaxes of the two centrioles are usually perpendicularto each other.

Centrioles become prominent in mitosis. In prophasethey separate and a new procentriole developsadjacent to each.

Microtubule organizing centers become nucleationsites around each centriole to form the fibers of theaster and the mitotic spindle.


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A centrosome with

attachedmicrotubules. Theminus end of each

microtubule isembedded in the

centrosome, havinggrown from a -

tubulin ringcomplex, whereas

the plus end ofeach microtubule is

free in thecytoplasm.


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In EM, each centrioleis found to be a hollow

cylinder closed at oneend and open at theother.

The central cavity is

occupied by smalldense granules. In transverse section,

its wall is composed of9 evenly spaced triplet microtubules (9x3).

Each triplet (A, B and C) is set at an angle of about400 to its respective tangent.

Subunit A is nearest to the centriole axis; short fibersconnect it to subunit C of the adjacent triplet.


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Characteristic 9 x 2

arrangement ofmicrotubules

Tubulin forms doubletscomposed of subunit A, acomplete microtubule

with 13 protofilaments,joined to a C-shapedsubunit B with only 10.

Lateral arms composed ofthe MAP axonemal dynein

project from subunit A tosubunit B of the next.

Major motor portion of theflagellum is called theaxoneme.

CILIA & FLAGELLA


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Ciliary dynein is a large motor protein assembly composed of 9-12polypeptide chains (A) The heavy chains form the major portion of the

globular head & stem domains, & many of the smaller chains areclustered around the base of the stem. The base of the molecule binds

tightly to an A microtubule in an ATP-independent manner, while the

large globular heads have an ATP-dependent binding site for a Bmicrotubule. When the heads hydrolyze their bound ATP, they movetoward the minus end of the B microtubule, thereby producing a sliding

force between the adjacent microtubule doublets in a cilium orflagellum. (B) Freeze-etch EM of a cilium showing the dynein arms

projecting at regular intervals from the doublet microtubules


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The bending of an axoneme.(A) When axonemes are exposed to the proteolytic enzyme trypsin, the

linkages holding neighboring doublet microtubules together are broken.Addition of ATP allows the motor action of the dynein heads to slide one

pair of doublet microtubules against the other pair. (B) In an intactaxoneme (such as in a sperm), sliding of the doublet microtubules is

prevented by flexible protein links. The motor action therefore causes abending motion, creating waves or beating motions


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The contrasting motions of flagellaand cilia. (A) The wavelike motion of

the flagellum of a sperm cell. Wavesof constant amplitude movecontinuously from the base to the tip

of the flagellum. (B) The beat of acilium, which resembles the breast

stroke in swimming.


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cytology cytoskeleton

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