Department of Natural Sciences University of St.

La Salle Bacolod City

CYTOSKELETON
 The cytoskeleton is a network of connected filaments and tubules extending from the nucleus to the plasma membrane.  It maintains the shape of the cell, anchor organelles, move the cell and control internal movement of structures.  Motility of cells is determined by special organelles for locomotion  Internal movements (cytoplasmic streaming or cyclosis) by cytoskeleton components.

Three types of cytoskeletal elements are found in eukaryotic cells:

1. Actin microfilaments are long, thin fibers approximately seven nm in diameter, occurring in bundles or meshlike networks. They move by interacting with myosin. 2. Microtubules are cylinders that form centrioles, cilia and flagella 3. Intermediate filaments support the nuclear envelope, plasma membrane and form cell-to-cell junctions.

 All types form as helical assemblies of subunits that selfassociate using a combination of end-to-end and sideto-side protein contacts.  Both microtubules and microfilaments grow fastest from the plus end than the minus end of the assembly.  Removal of monomers at the () end and addition of monomers at the (+) end leaves the filaments at the same overall length (treadmilling)

The treadmilling of a microfilament or microtubule, made possible 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 terminal subunits 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 end are in the D form. (B) Treadmilling occurs at intermediate concentrations of free subunits (i.e., the plus end grows while the minus end shrinks)

Mechanical properties of actin, tubulin, and intermediate filament polymers. Networks composed of microtubules, actin filaments, or an intermediate filament called vimentin, all at equal concentration, were exposed to a shear force in a viscometer, & the resulting degree of stretch was measured. The results show that microtubule networks are strong, rigid hollow tubes that are easily deformed but rupture (indicated by red starburst) and begin to flow without limit when stretched beyond 150% of their original length. Actin filament networks are much more rigid, but they also rupture easily. Intermediate filament networks, by contrast, are not only easily deformed, but they withstand large stresses and strains without rupture; they are thereby well suited to maintain cell integrity.

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

 They form a dense complex web just under the plasma membrane; in microvilli of intestinal cells, they act to shorten the cell  In plant cells, actin filaments form tracts along which chloroplasts circulate.  Involved in cell rigidity, tensile strength and resilience, cellular movement (pseudopodia and mesenchyme cell migration, platelet activation)  Pseudopodia are associated with actin near the moving edge of the cell. Actin filaments move by interacting with myosin changing the configuration to pull the actin filament forward.  Similar action accounts for pinching off cells during cell division and for amoeboid movement.  Other arrangements of microfilaments in association with accessory proteins are possible. Ex: contractile rings of cell division; parallel bundles are found in stress fibers of fibroblasts, filopodia and other cell projections; gels of short randomly oriented filaments are found in egg cortical regions.

Microfilaments in a cell. A crawling cell with 3 areas showing the arrangement of actin filaments. The actin filaments are shown in red, with arrowheads pointing toward the plus end. Stress fibers are contractile and exert tension. Filopodia are spike-like projections of the plasma membrane that allow a cell to explore its environments. The cortex underlies the plasma membrane.

A model of how forces generated in the actin-rich cortex move a cell forward. The actin-polymerizationdependent protrusion and firm attachment of a lamellipodium at the leading edge of the cell moves the edge forward (green arrows at front) and stretches the actin cortex. Contraction at the rear of the cell propels the body of the cell forward (green arrow at back) to relax some of the tension (traction). New focal contacts are made at the front, and old ones are disassembled at the back as the cell crawls forward. The same cycle can be repeated, moving the cell forward in a stepwise fashion. Alternatively, all steps can be tightly coordinated, moving the cell forward smoothly. The newly polymerized cortical actin is shown in red.

Platelet activation. (A) Platelet activation is a controlled sequence of actin

filament severing, uncapping, elongation, recapping, and cross-linking that creates a 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.

Several actin-binding proteins influence deployment of filaments in the cytoplasm:  Profilin binds to G-actin monomers to regulate polymerization  Capping protein limits length increase by binding to 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.

The modular structures of four actin-cross-linking proteins Each 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 are separated by a spacer around 30 nm long, so that it forms more loosely packed actin bundles. Filamin has two actin-binding sites with a Vshaped linkage between them, so that it cross-links actin filaments into a network with the filaments oriented almost at right angles to one another. Spectrin is a tetramer of two and two subunits, and the tetramer has two actinbinding sites spaced about 200 nm apart

Twisting of an actin filament induced by cofilin. (A) Three dimensional reconstruction from cryo-EM of filaments made of pure actin. The bracket shows the span of two turns of the actin helix. (B) Reconstruction of an actin filament coated with cofilin, which binds in a 1:1stoichiometry to actin subunits all along the filament. Cofilin is a small protein (14 kilodaltons) compared to actin (43 kilodaltons), and so the filament appears only slightly thicker. The energy of cofilin binding serves to deform the actin filament lattice, twisting it more tightly so that the distance spanned by two turns of the helix is reduced.

Filamin cross-links actin filaments into a three-dimensional network with the physical properties of a gel (A) Each filamin homodimer is about 160 nm long when fully extended and forms a flexible, high-angle link between two adjacent actin filaments. (B) A set of actin filaments cross-linked by filamin forms a mechanically strong web or gel.

MICROTUBULES
 A family of globular heterodimer proteins composed of E and F subunits; slender tubules are about 25 nm in diameter, running in straight course in the cytoplasm.  The wall is composed of 13 protofilaments of the protein tubulin, and later bind to microtubuleassociated proteins (MAPs).

The structure of a microtubule and its subunit. (A) The subunit of each protofilament is a tubulin heterodimer, formed from a very tightly linked pair of a- and b-tubulin monomers. The GTP molecule in the b-tubulin monomer is less tightly bound and has an important role in filament dynamics. Both nucleotides are shown in red. (B) One tubulin subunit (a-b heterodimer) and one protofilament consist of many adjacent subunits with the same orientation. (C) The microtubule is a stiff hollow tube formed from 13 protofilaments aligned in parallel. (D) A short segment of a microtubule viewed in an EM. (E) EM of a cross section of a microtubule

 Microtubules are noncontractile polarized structures with a (-) end anchored to the centrosome, and a free (+) end at which tubulin monomers are added or removed.  Capping proteins located in particular parts of the cell cortex bind at the (-) ends of microtubules, stabilizing them and controlling the shape and polarity of cells.  Conversion of bound GTP to GDP at the growing (+) end of the F subunit cause depolymerization.

Model for the structural consequences of GTP hydrolysis in the microtubule lattice.
Hydrolysis of GTP after assembly changes the conformation of the subunits & tends to force the protofilament into a shape that is less able to 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.

Dynamic instability due to the structural differences between a growing 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 than subunit addition, the cap is lost and the microtubule begins to shrink, an event called a "catastrophe." But GTP-containing subunits may still add to the shrinking end, and if enough add to form a new cap, microtubule growth resumes, an event called "rescue."

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 for cancer treatment. (B) Immunofluorescence micrograph showing the microtubule organization in a liver epithelial cell before the addition of taxol. (C) Microtubule organization in the same type of cell after taxol treatment. Note the thick circumferential bundles of microtubules around the periphery of the cell. (D) A Pacific yew tree, the natural source of taxol.

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 prevent polymerization) and vinblastine, microtubules disappear and mitosis is arrested because of inadequate formation of the mitotic spindle.

MOLECULAR MOTORS

 MOTOR PROTEINS- specialized motility structures in eucaryotic cells consisting of highly ordered arrays of motor proteins that move on stabilized filament tracks.  They use the energy of ATP hydrolysis to move along microtubules or actin filaments.  They mediate the sliding of filaments relative to one another and the transport of membrane-enclosed organelles along filament tracks.

 All known motor proteins that move on actin filaments are members of the myosin superfamily; the motor proteins that move on microtubules are members of either the kinesin superfamily or the dynein family.  The myosin and kinesin superfamilies are diverse, with about 40 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 heads can be attached to a wide variety of "tails," which attach to different types of cargo and enable the various family members to perform different functions in the cell.

 Kinesins move towards the (+) ends of tubules, while dyneins move towards the () ends.  Kinesin is responsible for movement of vesicles and organelles in the cytoplasm, dynein regulates 2-way traffic and dynamin serves as a motor for sliding movements during microtubule elongation.

Cycle of structural changes used by myosin to walk along an actin filament.

Summary of the coupling between ATP hydrolysis and conformational changes for myosin II. Myosin begins its cycle tightly bound to the actin filament, with no associated nucleotide, the so-called "rigor" state. ATP binding releases the head from the filament. ATP hydrolysis occurs while the myosin head is detached from the filament, causing the head to assume a cocked conformation, although both ADP and inorganic phosphate remain tightly bound to the head. When the head rebinds to the filament, the release of phosphate, followed by the release of ADP, trigger the power stroke that moves the filament relative to the motor protein. ATP binding releases the head to allow the cycle to begin again. In the myosin cycle, the head remains bound to the actin filament for only about 5% of the entire cycle time, allowing many myosins to work together to move a single actin filament.

Kinesin and kinesin-related proteins. (A) Structures of four kinesin superfamily members. Conventional kinesin has the motor domain at the N-terminus of the heavy chain. The middle domain forms a long coiled coil, mediating dimerization. The C-terminal domain forms a tail that attaches to cargo, such as a membrane-enclosed organelle. These kinesins generally travel toward the minus end instead of the plus end of a microtubule. (B) Freeze-etch EM of a kinesin molecule with the head domains on the left.

Summary of the coupling between ATP hydrolysis and conformational changes 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 the front head causes the rear head to be thrown forward, past the binding site of the attached head, to another binding site further toward the plus end of the microtubule. Release of ADP from the second head (now in the front) 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 their relative positions, and the motor protein has moved one step along the microtubule protofilament. In this cycle, each head spends about 50% of its time attached to the microtubule and 50% of its time detached.

INTERMEDIATE FILAMENTS
 Are structurally similar but biochemically distinct, with diameters intermediate between microtubules and microfilaments (about 10 nm).  They associate with polypeptides fillagrin (binds to keratin), plectin (links vimentin), and synamin (also links vimentin, but found in muscle).  5 types are: 1. Glial filaments found in non-neural cells of the CNS: astrocytes, oligodendrocytes, microglia. 2. Keratin filaments characteristic of epithelial cells; called tonofilaments are often associated with desmosomes at the cell surface. They participate in the formation of keratin in keratinizing epithelia.

3.Desmin characteristic of smooth, striated & cardiac muscle; keep sarcomeres of neighboring myofibrils in register across the width of the fiber; link Z-bands of peripheral myofibrils to the sarcolemma; ensures uniform distribution of tensile strength throughout the muscle cell. 4.Vimentin abundant in fibroblasts and mesenchymal derivatives, in bundles or randomly oriented in a network throughout the cytoplasm. 5.Neurofilaments present in nerve cell processes with a cytoskeletal function; helps to maintain the gel state of the axoplasm; involved in intracellular metabolite transport.

A model of intermediate filament construction The monomer shown in (A) pairs with an identical monomer to form a dimer (B) in which the conserved central rod domains are aligned in parallel and wound together into a coiled coil. (C) Two dimers then line up side by side to form the tetramer soluble subunit of intermediate filaments. (D) Within each tetramer, the 2 dimers are offset with respect to one another, thereby allowing it to associate with another tetramer. (E) In the final 10-nm rope-like filament, tetramers are packed together in a helical array, which has 16 dimers in cross-section. Half of these dimers are pointing in each direction.

Keratin filaments in epithelial cells

Immunofluorescence micrograph of the network of keratin filaments (green) in a sheet of epithelial cells in culture. The filaments in each cell are indirectly connected to those of its neighbors by desmosomes. A 2nd protein (blue) has been stained to reveal the location of the cell boundaries.

Blistering of the skin caused by a mutant keratin gene.

A mutant gene encoding a keratin protein was expressed in a transgenic mouse. The defective protein assembles with the normal keratins and thereby disrupts the keratin filament network in the basal cells of the skin. LM of cross sections of normal (A) and mutant (B) skin show that the blistering results from the rupturing of cells in the basal layer of the mutant epidermis (small red arrows). (C) Cells in the basal layer of the mutant epidermis, as observed by EM. As indicated by the red arrow, the cells rupture between the nucleus & the hemidesmosomes, which connect the keratin filaments to the underlying basal lamina.

Two types of intermediate filaments in cells of the nervous system.

(A) Freeze-etch EM image of neurofilaments in a nerve cell axon, showing the extensive cross-linking through protein cross-bridges an arrangement believed to give this long cell process great tensile strength. The crossbridges are formed by the long, nonhelical extensions at the C-terminus of the largest neurofilament protein (NF-H). (B) Freeze-etch image of glial filaments in glial cells, showing that these intermediate filaments are smooth and have few cross-bridges. (C) Conventional EM of a cross section of an axon showing the regular side-to-side spacing of the neurofilaments, which greatly outnumber the microtubules.

CENTROSOME
 Also called the centrosphere or cell center, which refers to a specialized zone of cytoplasm containing the centrioles and a variable number of small dense bodies called centriolar satellites.  Considered to be a center of activities associated with cell division, usually adjacent to the nucleus.  The Golgi apparatus often partially surrounds the centrosome on the side away from the nucleus. The centrosome is located in the cytoplasm next to the nucleus  It consists of an amorphous matrix of protein containing the g-tubulin ring complexes that nucleate microtubule growth

 They serve as basal bodies and sites of anchor for epithelial cilia.  Plant and fungal cells have a structure equivalent to a centrosome, although they do not contain centrioles  The matrix of the centrosome is organized by a pair of centrioles.
An electron micrograph of a thick section of a centrosome showing an end-on view of a centriole. The ring of modified microtubules of the centriole is visible, surrounded by the fibrous centrosome matrix.

 Centrioles are self-duplicating organelles that exhibit continuity from one cell generation to the next. They double in number immediately before cell division 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 a procentriole.  Paired centrioles are called diplosome. The long axes of the two centrioles are usually perpendicular to each other.  Centrioles become prominent in mitosis. In prophase they separate and a new procentriole develops adjacent to each.  Microtubule organizing centers become nucleation sites around each centriole to form the fibers of the aster and the mitotic spindle.

A centrosome with attached microtubules. The minus end of each microtubule is embedded in the centrosome, having grown from a tubulin ring complex, whereas the plus end of each microtubule is free in the cytoplasm.

 In EM, each centriole is found to be a hollow cylinder closed at one end and open at the other.  The central cavity is occupied by small dense granules.  In transverse section, its wall is composed of 9 evenly spaced triplet microtubules (9x3).  Each triplet (A, B and C) is set at an angle of about 400 to its respective tangent.  Subunit A is nearest to the centriole axis; short fibers connect it to subunit C of the adjacent triplet.

CILIA & FLAGELLA

 Characteristic 9 x 2 arrangement of microtubules  Tubulin forms doublets composed of subunit A, a complete microtubule with 13 protofilaments, joined to a C-shaped subunit B with only 10.  Lateral arms composed of the MAP axonemal dynein project from subunit A to subunit B of the next.  Major motor portion of the flagellum is called the axoneme.

Ciliary dynein is a large motor protein assembly composed of 9-12 polypeptide chains (A) The heavy chains form the major portion of the globular head & stem domains, & many of the smaller chains are clustered 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 B microtubule. When the heads hydrolyze their bound ATP, they move toward the minus end of the B microtubule, thereby producing a sliding force between the adjacent microtubule doublets in a cilium or flagellum. (B) Freeze-etch EM of a cilium showing the dynein arms projecting at regular intervals from the doublet microtubules

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 intact axoneme (such as in a sperm), sliding of the doublet microtubules is prevented by flexible protein links. The motor action therefore causes a bending motion, creating waves or beating motions

The contrasting motions of flagella and cilia. (A) The wavelike motion of the flagellum of a sperm cell. Waves of constant amplitude move continuously from the base to the tip of the flagellum. (B) The beat of a cilium, which resembles the breast stroke in swimming.