Lecture 16. The Cytoskeleton

T Function and dynamics of the cytoskeleton Actin filaments determine the shape of the cells surface and are necessary for whole-cell locomotion; they also drive the pinching of one cell into two Microtubules determine he position of organelles, direct intracellular transport, and form the mitotic spindle that segregates chromosomes during en division The accessory proteins are essential for the controlled assembly of the cytoskeletal filaments in particular locations, and they include the motor proteins, remarkable molecular machines that convert the energy of ATP hydrolysis into mechanical force that can either move organelles along the filaments or move the filaments themselves Cytoskeletal filaments are dynamic, but can nevertheless form stable structures In a similar way, large-scale cytoskeletal structures can change or persist, according to need, lasting for lengths of time ranging from less than a minute up to the cell's lifetime Regulation of the dynamic behavior and assembly of cytoskeletal filaments allows eukaryotic cells to build an enormous range of structures from the three basic filament systems Actin filaments underlie the plasma membrane of animal lens in a layer called the cell cortex, providing strength and shape to the thin lipid belayer Microtubules, which are frequently found in a cytoplasmic array that extends to the cell periphery, can quickly rearrange themselves to form a bipolar mitotic spindle during cell division They [microtubules] can also form cilia, which function as motile whips or sensory devices on the surface of the cell, or tightly aligned bundles that serve as tracks for the transport of materials down long neuronal axons Intermediate filaments line the inner face of the nuclear envelope, forming A protective cage for the cell's DNA; in the cytosol, they are twisted into strong cables that can hold epithelial cell sheets together or help nerve cells to extend long and robust axons, and they allow us to form tough appendages such as hair and fingernails An important and dramatic example of rapid reorganization of the cytoskeleton occurs during cell division for a fibroblast growing in a tissue-culture dish After the chromosomes have replicated, the interphase microtubule array that spreads throughout the cytoplasm is reconfigured into the bipolar mitotic spindle, which transfers the two copies of each chromosome into separate daughter nuclei. At the same time, the specialized actin structures that enable the fibroblast to crawl across the surface of the dish rearrange so that the cell stops moving and assumes a more spherical shape Actin and its associated motor protein myosin then form a belt around the middle of the cell, the contractile ring, which constricts like a tiny muscle to pinch the cell in two 3 When division is complete, the cytoskeletons of the two daughter fibroblasts recreate their interphase I structures, thereby converting the two rounded-up daughter cells into smaller versions of the flattened, crawling mother cell 2 The cytoskeleton determines cellular organization and polarity In cells that have achieved a stable, differentiated morphology - such as mature neurons or epithelial cells the dynamic elements of the cytoskeleton produce stable, large-scale structures for cellular organization 3 Besides forming stable, specialized cell-surface protrusions, the cytoskeleton is also responsible for large-scale cellular polarity, enabling cells to tell the difference between top and bottom of front and back For example, polarized epithelial cells use organized arrays of microtubules, actin filaments, and intermediate filaments to maintain the critical differences between the apical surface and the basolateral surface Filaments assemble from protein subunits that impart specific physical and dynamic properties The cell builds the filaments by assembling large numbers of the small subunits, like building a skyscraper out of bricks. Because these subunits are small, they can diffuse rapidly in the cytosol, whereas the assembled filaments cannot Actin filaments and microtubules are built from subunits that are compact and globular - actin subunits for actin filaments and tubulin subunits for microtubules - whereas intermediate filaments are made up of subunits that are themselves elongated and fibrous All three major types of cytoskeletal filaments form as polymeric assemblies of subunits that selfassociate, using a combination of end-to-end and side-to-side protein contacts. Whereas covalent linkages between their subunits hold together the backbones of many biological polymers - including DNA, RNA, and proteins - it is weak non-covalent interactions that hold together the three types of cytoskeletal polymers The subunits of actin filaments and microtubules are a symmetrical and bind to one another head-to-tail so that they all point in one direction. This subunit polarity gives the filaments structural polarity along their length and makes the two ends of each polymer behave differently It [NTP hydrolysis] allows the cell to harness the polar and dynamic properties of these filaments to generate force in a specific direction to move the leading edge of a migrating cell forward, for example, or to pull chromosomes apart during cell division In contrast, the subunits of intermediate filaments are symmetrical and thus do not form polarized filaments with two different ends. Intermediate filament subunits also do not catalyze the hydrolysis of ATP or GTP A thousand tubulin subunits lined up end-to-end, for example, would span the diameter of a small eukaryotic cell, but a filament formed in this way would lack the strength to avoid breakage by ambient thermal energy, unless each subunit in the filament was bound extremely tightly to its two neighbors Protofilament: linear string of joined end to end The addition or loss of a subunit at the end of one protofilament makes or breaks a small number of bonds. In contrast, loss of a subunit from the middle of a protofilament requires breaking many more bonds, while breaking it in two requires breaking bonds in multiple protofilaments all at the same time Although made up of only two strands instead of 13, helical actin filament subunits also make both endto-end and side-to-side contacts, knowing for rapid dynamics at filament ends while providing sufficient strength along the length of the filament Accessory proteins and motors act on cytoskeletal filaments Direct covalent modification of the filament subunits regulates some filament properties, but most of the regulation is performed by hundreds of accessory proteins that determine the spatial distribution and the dynamic behavior of the filaments, converting information received through signaling pathways into cytoskeletal action In these processes, the accessory proteins bring cytoskeletal structure under the control of extracellular and intracellular signals, including those that trigger the dramatic transformations of the cytoskeleton that occur during each cell cycle Motor proteins bind to a polarized cytoskeletal filament and use the energy derived from repeated cycles of ATP hydrolysis to move along it Many motor proteins carry membrane-enclosed organelles - such as mitochondria, Golgi stacks, or secretory vesicles - to their appropriate locations in the cell. Other motor proteins cause cytoskeletal filaments to exert tension or to slide against each other, generating the force that drives such phenomena as muscle contraction, ciliary beating, and cell division Molecular motors operate in a cellular environment dominated by brownian motion As introduced earlier, Brownian motion drives the diffusion of all of the molecules inside cells, and it is thereby critical for the rate of biochemical reactions, which require molecular collisions The stopping distance for any object moving through a fluid is determined by the Reynolds's number, which is the ratio of inertial to viscous forces acting on that object Actin Each actin subunit, sometimes called globular or G-actin, is a 375-amino acid polypeptide carrying A tightly associated molecule of ATP or ADP Small variations in actin amino acid sequence can cause significant functional differences: in vertebrates, for example, there are three isoforms of actin, termed alpha, beta, and gamma, that differ slightly in their amino acid sequences and have distinct functions Actin subunits assemble head-to-tail to create flexible, polar filaments Actin subunits assemble head-to-tail to form a tight, right-handed helix, forming a structure about 8 nm wide called filamentous or F-actin Because the asymmetrical actin subunits of a filament all point in the same direction, filaments are polar and have structurally different ends: a slower-growing minus end and a faster-growing plus end The minus end is also referred to as the pointed end and the plus end as the barbed end because of the arrowhead appearance of the complex formed between actin filaments and the motor protein myosin visible in election micrographs The stiffness of a filament can be characterized by its persistence length, the minimum filament length at which random thermal fluctuations are likely to cause it to bend In a living cell, however, accessory proteins frequently cross-link and bundle the filaments together, making large-scale actin structures that are much more rigid than an individual actin filament Nucleation is the rate-limiting step in the formation of actin filaments Actin subunits can spontaneously bind one another, but the association is unstable until subunits assemble into an initial oligomer, or nucleus, that is stabilized by multiple subunit-subunit contacts and can then elongate rapidly by addition of more subunits. This process is called filament nucleation When polymerization is initiated, this results in a log phase during which no filaments are observed. During this lag phase, however, the small, unstable oligomers gradually succeed in making the transition to a more stable form that resembles an actin filament Finally, as the concentration of actin monomers declines, the system approaches a steady state at which the rate of addition of new subunits to the filament ends exactly balances the rate of subunit dissociation The lag phase in filament growth is eliminated if preexisting seeds (such as fragments of actin filaments) are added to the solution at the beginning of the polymerization reaction Actin filaments have two distinct ends that grow at different rates The kinetic rate constants for actin subunit association and dissociation - k on and k off, respectively are much greater at the plus end than at the minus end If filaments are rapidly diluted so that the free subunit concentration drops below the critical concentration, the plus end also depolymerizes faster It is important to note, however, that the two ends of an actin filament have the same net affinity for actin subunits, if all of the subunits are bound to either ATP or ADP. Addition of a subunit to either end of a filament of n subunits results in a filament of n+1 subunits ATP hydrolysis within actin filaments leads to treadmilling at steady state Shortly after ATP hydrolysis occurs, the free phosphate group is released from each subunit, but the ADP remains trapped in the filament structure. Thus, two different types of filament structures can exist, one in the ATP-bound T form and one in the ADP-bound D form If the concentration of actin monomers is greater than the critical concentration for both the T-form and D-form polymers, then subunits will add to the polymers at both ends before the ATP in the previously added subunits is hydrolyzed; as a result, the tips of the actin filament will remain in the T-form At intermediate concentrations of actin subunits, it is possible for the rate of subunit addition to be faster than ATP hydrolysis at the plus end but slower than ATP hydrolysis at the minus end. in this case, the plus end of the filament remains in the T conformation, while the minus end adopts the D conformation The filament then undergoes a net addition of subunits at the plus end while simultaneously losing subunits from the minus end. This leads to the remarkable property of filament treadmilling Actin-binding proteins influence filament dynamics and organization Actin-binding proteins dramatically alter actin filament dynamics through spatial and temporal control of filament nucleation, elongation, and depolymerization Actin nucleation is tightly regulated and generates branched or straight filaments A major reason is that actin filament polymerization is tightly regulated in cells by a large number of proteins that control actin filament nucleation, almost always adjacent to a membrane surface The ARP 2/3 complex nucleates actin filament growth and remains bound to the minus end, allowing rapid elongation at the plus end The ARP 2/3 complex is further stimulated when it attaches to the side of a preexisting actin filament. Thus, ARP 2/3 complex activation generates a branched array of actin filaments adjacent a membrane, building individual filaments into a treelike network Formins: dimeric protein that nucleates the growth of straight, unbranched actin filaments that can be cross-linked by other proteins to form parallel bundles As the newly nucleated filament grows, the formin dimer remains associated with the rapidly growing plus end while still allowing the addition of new subunits at that end In addition to their role in actin nucleation, formin proteins also dramatically accelerate actin filament growth. Formins mediate assembly of a variety of cellular structures that contain pollarded actin cables, including filopodia, stress fibers, and the contractile ring Actin filament nucleation by the ARP 2/3 complex and formin occurs primarily at the plasma membrane, and therefore the highest density of actin filaments in most cells is at the cell periphery within the cell cortex Cell cortex: specialized layer of cytoplasm on the inner face of the plasma membrane Actin filaments in this region determine the shape and movement of the cell surface, allowing the cell to change its shape and stiffness rapidly in response to changes in its external environment Actin filament elongation is regulated by monomer-binding proteins Profilin binds to the face of the actin monomer opposite the ATP-binding cleft, blocking the side of the monomer that would normally associate with the filament minus end, while leaving exposed the site on the monomer that binds to the plus end When the profilin-actin complex binds a free plus end, a conformational change in actin reduces its affinity for profilin and the profilin falls off, leaving the actin filament one subunit longer First, profilin maintains a large pool of action monomers poised for polymerization at filament plus ends. Second, binding sites for profilin are present in many formin proteins, as well as in many NPFs that activate the ARP 2/3 complex Actin filament-binding proteins alter filament dynamics and organization Actin filament behavior is regulated by two major classes of binding proteins: those that bind along the side of a filament and those that bind to the ends In addition to stabilizing and stiffening the filament, the binding of tropomyosin can prevent the actin filament from interacting with other proteins; this aspect of tropomyosin function is important in the control of muscle contraction An actin filament that stops growing and is not specifically stabilized in the cell will depolymerize rapidly, particularly at its plus end, once the actin molecules have hydrolyzed their ATP The binding of plus-end capping protein stabilizes an actin filament at its plus end by rendering it inactive, greatly reducing the rates of filament growth and depolymerization Tropomodulin, best known for its function in the capping of actin filaments in muscle, binds lightly to the minus ends of actin filaments that have been coated and thereby stabilized by tropomyosin Because subunit addition and loss occur exclusively at filament ends, one molecule of an end-binding protein per actin filament can be enough to transform the architecture of an actin filament network The very close packing of actin filaments caused by fimbrin apparently excludes myosin, and thus the parallel actin filaments held together by fibrin are not contraceptive ERM proteins are thought to bind to and organize the cortical actin cytoskeleton in a variety of contexts, thereby affecting the shape and stiffness of the membrane as well as the localization and activity of signaling molecules Severing proteins regulate actin filament depolymerization Another important mechanism of actin filament regulation depends on proteins that break an actin filament into many smaller filaments, thereby generating a large number of new filament ends Under some conditions, newly formed ends nucleate filament elongation, thereby accelerating the assembly of new filament structures. Under other conditions, severing promotes the depolymerization of old filaments, speeding up the depolymerization rate by tenfold or more Activated gelsolin is thought to sever an actin filament when a thermal fluctuation creates a small gap between neighboring subunits, at which point gelsolin inserts itself into the gap to break the filament Mechanical stress induced by cofilin binding weakens the contacts between actin subunits in the filament, making the filament less stable and more easily severed by thermal motions, generating filament e ends that undergo rapid disassembly Because ATP hydrolysis is usually slower than filament assembly, the newest actin filaments in the cell still contain mostly ATP and are resistant to depolymerization by cofilin. Cofilin therefore tends to dismantle the older filaments in the cell Actin at the cell cortex determines cell shape Different structures are initiated by the action of distinct nucleating proteins: the actin filaments of branched networks are nucleated by the ARP 2/3 complex, while bundles are made of the long, straight filaments produced by formins Myosin and Actin A crucial feature of the actin cytoskeleton is that it can form contractile structures that cross-link and slide actin filaments relative to one another through the action of myosin motor proteins Actin-based motor proteins are members of the myosin superfamily The first motor protein to be identified was skeletal muscle myosin, which generates the force for muscle contraction. This protein, now called myosin II, is an elongated protein formed from two heavy chains and two copies of each of two light chains Each heavy chain has a globular head domain at its N-terminus that contains the force-generating machinery, followed by a very long alpha-helical amino acid sequence that forms an extended coiled-coil to mediate heavy-chain dimerization The two light chains bind close to the N-terminal head domain, while the long coiled-coil bundles itself with the tails of other myosin molecules Each myosin head birds and hydrolyzes ATP, using the energy of ATP hydrolysis to walk toward the plus end of an actin filament In skeletal muscle, in which carefully arranged actin filaments are aligned in thin filament arrays surrounding the myosin thick filaments, the ATP-driven siding of actin filaments results in a powerful contraction Myosin generates force by coupling ATP hydrolysis to conformational changes For myosin II, each step of the movement along actin is generated by the swinging of an 8.5-nm -long alpha helix, or lever arm, which is structurally stabilized by the binding of light chains These changes in the conformation of the myosin are coupled to changes in its binding affinity for actor, allowing the myosin head to release its grip on the actin filament at one point and snatch hold of it again at another Sliding of myosin II along actin filaments causes muscles to contract The long, thin muscle fibers of skeletal muscle are actually huge single cells that form during development by the fusion of many separate cells A myofibril is a cylindrical structure 1-2 um in diameter that is often as long as the muscle itself. It consists of a long, repeated chain of tiny contractile units - called sarcomeres, each about 2.2 um long - which give the vertebrate my myofibril its striated appearance The thin filaments are composed of actin and associated proteins, and they are attached at their plus ends to a Z disc at each end of the sarcomere The capped minus ends of the actin filaments extend in toward the middle of the sarcomere, where they overlap with thick filaments, the bipolar assemblies formed from specific muscle isoforms of myosin II The rapid synchronized shortening of the thousands of sarcomeres lying end-to-end in each myofibril enables skeletal muscle to contract rapidly enough for running and flying or for playing the piano A sudden rise in cytosolic Ca 2+ concentration initiates muscle contractions The individual myosin motor heads in each thick filament spend only a small fraction of the ATP cycle time bound to the filament and actively generating force, so many myosin heads can act in rapid succession on the same thin filament without interfering with one another The signal from the nerve triggers an action potential in the muscle cell plasma membrane, and this electrical excitation spreads swiftly into a series of membranous folds - the transverse tubules, or T tubules - that extend inward from the plasma membrane around each myofibril When the incoming action potential activates a Ca 2+ channel in the T tubule membrane, it triggers the opening of a Ca 2+ release channel in the closely associated sarcoplasmic reticulum membrane Thus, muscle contraction depends on two processes that consume enormous amounts of ATP: filament sliding, driven by the ATPase of the myosin motor domain, and Ca 2+ pumping, driven by the Ca 2+ pump In a resting muscle, the troponin I-T complex pulls the tropomyosin out of its normal binding groove into a position along the actin filament that interferes with the binding of myosin heads, thereby preventing any force-generating interaction When the level of Ca 2+ is raised, troponin C - which binds up to four molecules of Ca 2+ - causes troponin I to release its hold on actin Troponin C is closely related to the ubiquitous Ca 2+ - binding protein calmodulin; it can be thought of as a specialized form of calmodulin that has acquired binding sites for troponin I and troponin t, thereby ensuring that the myofibril responds extremely rapidly to an increase in Ca 2+ concentration Microtubules Microtubules are polymers of the protein tubulin. The tubulin subunit Is a heterodimer formed from two closely related globular proteins called alpha-tubulin and beta-tubulin, each comprising 445 - 450 amino acids, which are tightly held together by non-covalent bonds The GTP that is bound to alpha-tubulin is physically trapped at the diner interface and is never hydrolyzed or exchanged; it can therefore be considered to be an integral part of the tubulin heterodimer structure Although the different forms of tubulin are very similar and copolymerize into mixed microtubules in a test tube, they can have distinct locations in cells and tissues and perform subtly different functions Microtubules are hollow tubes made of protofilaments A microtubule is a hollow cylindrical structure built from 13 parallel protofilaments, each composed of alpha-beta tubulin heterodimers stacked head to tail and then folded into a tube Along the longitudinal axis of a protofilament, the "top” of one beta-tubulin molecule forms an interface with the "bottom" of the alpha-tubulin molecule in the adjacent heterodimer A slight stagger in lateral contacts gives rise to the helical microtubule lattice, which holds most of the subunits tightly in place. As a result, the addition and loss of subunits occur almost exclusively at the microtubule ends The average length over which microtubules stay straight (persistence length) is more than 10 times that of actin filaments, making microtubules the stiffest and straightest structural element found in most animal cells The subunits in each protofilament in a microtubule all point in the same direction, and the protofilaments themselves are aligned in parallel, with alpha-tubulins exposed at the minus end and beta-tubulins exposed at the plus end As for actin filaments, the regular, parallel orientation of their subunits gives microtubules both a structural and a dynamic polarity: the microtubules plus end grows and shrinks much more rapidly than its minus end Microtubules undergo a process called dynamic instability GTP hydrolysis, which occurs only within the beta-tubulin subunit of the tubulin dinner, proceeds very slowly in free tubulin subunits and is greatly accelerated when they are incorporated into microtubules Thus, as in the case of actin filaments, two different types of microtubule structures can exist, one in the T form bound to GTP and one in the D form bound to GDP Because some of the energy of phosphate bond hydrolysis is stored as elastic strain in the polymer lattice, the free-energy change for dissociation of a subunit from the D form polymer is more favorable (negative) than the free-energy change for dissociation of a submit from the T form polymer Whether the tubulin subunits at the very end of a microtubule are in the T or the D form depends on the relative rates of tubulin addition and GTP hydrolysis If the rate of subunit addition is high - and thus the filament is growing rapidly - the it is likely that a new subunit Will be added to the end of the polymer before the GTP in the previously added subunit has been hydrolyzed Often GTP-tubulin subunits will assemble at the end of the microtubule at a rate similar to the rate of GTP hydrolysis, and in this case hydrolysis will sometimes "catch up" with the rate of subunit addition and transform the end to a D form On a single microtubule, an end might grow for a certain length of time in a T form, but then suddenly change to the D form and begin to shrink rapidly This rapid interconversion between a growing and shrinking state, at a uniform free tubulin concentration, is called dynamic instability. The change from growth to shrinkage is called a catastrophe, while the change from shrinkage is called a rescue On the basis of observations of the ends of growing and shrinking microtubules in vitro, one model proposed that tubulin subunits in the T form, with GTP bound to the beta monomer, produce straight protofilaments that make strong and regular lateral contacts with one another, and that the hydrolysis of GTP to GDP makes these protofilaments curve Microtubule functions are inhibited by both polymer-stabilizing and polymer-destabilizing drugs Both microtubule-depolymerizing drugs and microtubule-polymerizing drugs preferentially kill dividing cells, because microtubule dynamics are crucial for correct function of the Nilotic spindle A protein complex containing gamma-tubulin nucleates microtubules While alpha and beta tubulins are the regular building blocks of microtubules, another type of tubulin, called gamma-tubulin, is present in much smaller amounts than alpha and beta-tubulin and is involved in the nucleation of microtubule growth in organisms ranging from yeasts to humans Within this complex [gamma-tubulin ring complex], two accessory proteins bind directly to the gammatubulin, along with several other proteins that help create a spiral ring of gamma-tubulin molecules, which serves as a template that creates a microtubule with 13 protofilaments The centrism is a prominent microtubule nucleation site Many animal cells possess a single, well-defined MTOC called the cento some, which is located adjacent to the nucleus and from which microtubules are nucleated at their minus ends, so the plus ends point outward and continually grow and shrink, probing the entire three-dimensional volume of the cell Thus, the majority of gamma-TuRC is found in the cytoplasm, and centrosomes are not absolutely required for microtubule nucleation, as destroying with them a laser pulse does not prevent microtubule nucleation elsewhere in the cell Embedded in the centrosome are the centrioles, a pair of cylindrical structures arranged at right angles to each other in an L-shaped configuration Together with a large number of accessory proteins, the centrioles recruit the pericentriolar material, where microtubule nucleation takes place The centrosome duplicates before mitosis, forming a pair of centrosomes that each contain a centriole pair. When mitosis begins, the two centrosomes more a apart to form the poles of the mitotic spindle Microtubule organization varies widely among cell types In budding yeast, microtubules are nucleated at an MTOC that is embedded in the nuclear envelope as a small, multilayered structure called the spindle pole body, also found in other fungi and diatoms Higher-plant cells lack centrosomes and nucleate microtubules at sites distributed all around the nuclear envelope and at the cell cortex Cultured fibroblasts contain an aster-like configuration of microtubules, with dynamic plus ends pointing outward toward the cell periphery and stable minus ends gathered near the nucleus In axons, all the microtubules are oriented with their minus ends pointing back toward the cell body and their plus ends pointing toward the axon terminals These aligned microtubule tracks act as a highway to transport specific proteins, protein-containing vesicles, and mRNAs to the axon terminals, where synapses are constructed and maintained Microtubule-binding proteins modulate filament dynamics and organization Microtubules in cells exhibit a much higher polymerization rate (typically 10 - 15 um/min, relative to about 1.5 um/min with purified tubulin at similar concentrations), a greater catastrophe frequency, and extended pauses in microtubule growth, a dynamic behavior rarely observed in pure tubulin solutions Because the short (~20 amino acid) C-terminal tails of both alpha and beta-tubulin that protrude from the microtubule are enriched in glutamic and aspartic acids, the surface of the microtubule possesses a net negative charge Cells over-expressing MAP2, which has a long projecting domain, form bundles of stable microtubules that are kept widely spaced, while cells over-expressing tau, a MAP with a much shorter projecting domain, form bundles of more closely packed microtubules MAPs are the targets of several protein kinases, and phosphorylation of a MAP can control both its activity and localization inside cells by disrupting its electrostatic interaction with microtubules Because they lack centrosomes, plant cells rely extensively on augmin-dependent microtubule branching nucleation to organize the microtubule cytoskeleton Microtubule plus end-binding proteins modulate microtubule dynamics and attachments While the minus ends of microtubules in many cells are usually stabilized and inert, plus ends, in contrast, efficiently explore and probe the entire volume of the cell These proteins can influence the rate at which microtubule switches from a growing to a shrinking state (the frequency of catastrophes) Or from a shrinking to a growing state (the frequency of rescues) By attaching to the plus end, these factors control microtubule dynamics and also allow the cell to harness the energy of microtubule polymerization to generate pushing forces that can be used for positioning the spindle, chromosomes, or organelles Tubulin-sequestering and microtubule-severing proteins modulate microtubule dynamics As it does with actin monomers, the cell sequesters unpolymerized tubulin subunits to maintain a pool of active subunits at a level near the critical concentration One molecule of the small protein stathmin binds two tubulin hetero timers and prevents their addition to the ends of microtubules. Stathmin thus decreases the effective concentration of tubulin subunits that are available for polymerization and enhances the likelihood that a growing microtubule Will switch to the shrinking state Severing is another mechanism employed by the cell to destabilize microtubules. To sever a microtubule, 13 longitudinal bonds most be broken, one for each protofilament Paradoxically, the loss of microtubule-severing protein activity in many cell types leads to a decrease rather than an increase in microtubules If a sufficient number of GTP-tubulin subunits are incorporated before the severing is complete, the new plus end of the cut microtubule will possess a stabilizingGTP-tubulin cap and will therefore polymerize Alternatively, incomplete severing could lead to an island of GTP-tubulin in the microtubule lattice that could promote a future rescue event when this site is exposed after catastrophe Two types of motor proteins move along microtubules There are two major classes of microtubule-based motors, kinesins and dyneins, which perform three major functions First, they more cargo such as organelles and macromolecules within the cell. Unlike actin-based transport, however, microtubule-based motors are used to transport cargo over long distances Second, motors can slide microtubules relative to one another, thereby generating specific arrangements of microtubules, as in neurons and epithelial cells Third, a subset of microtubule-based motors regulates microtubule dynamics Kinesin-1 is similar to myosin II in having two heavy chains per active motor; these form two globular head motor domains that are held together by an elongated coiled-coil that mediates heavy-chain dimerization In kinesin-1, small movements at the ATP - binding site regulate the docking and undocking of the motor head domain to a long linker region The ATP - hydrolysis cycles in the two heads are closely coordinated, so that this cycle of linker docking and undocking allows the two-headed motor to move in a hand-over-hand stepwise manner The dyneins are a family of minus end-directed microtubule motors unrelated to the kinesins. They are composed of one, two, Or three heavy chains and a large and variable number of associated intermediate, light-intermediate - and light chains Although structurally unrelated to myosins and kinesins, dyneins follow the general rule of coupling ATP hydrolysis to microtubule binding and unbinding as well as to a force-generating conformational change Microtubules and motors move organelles and vesicles Kinesin was originally identified as the protein responsible for fast anterograde axonal transport, the rapid movement of mitochondria, secretory vesicle precursors, and various synapse components down the microtubule highways of the axon to the distant nerve terminals A typical microtubule array in an interphase cell is oriented with the minus ends near the center of the cell at the centrosome and the plus ends extending to the cell periphery Thus, centripetal movements of organelles or vesicles toward the cell center require the action of minus end-directed cytoplasmic dynein motors, whereas centrifugal movements toward the periphery require plus end-directed kinesin motors In vitro, kinesins can tether ER-derived membranes to preformed microtubule tracks and walk toward the microtubule plus ends, dragging the ER membranes out into tubular protrusions and forming a membranous web that looks very much like the ER in cells Conversely, dyneins are required for positioning the Golgi apparatus near the cell center of animal cells; they do this by moving Golgi vesicles along microtubule tracks toward the microtubules' minus ends at the centrosome Membrane-associated motor receptors that are sorted to specific membrane-enclosed compartments interact directly or indirectly with the tails of the appropriate kinesin family members A number of other proteins also contribute to dynein cargo and binding and motor regulation, and their function is especially important in neurons, where defects in microtubule-based transport have been linked to neurological diseases The cell can regulate the activity of motor proteins and thereby cause either a change in the positioning of its membrane - enclosed organelles or whole-cell movements Motive cilia and flagella are built from microtubules and dyneins Just as myofibrils are highly specialized and efficient motility machines built from actin and myosin filaments, cilia and flagella are highly specialized and efficient mobility structures built from microtubules and dynein Intermediate filaments and other cytoskeletal polymers Intermediate filaments are particularly prominent in the cytoplasm of cells that are subject to mechanical stress and are generally not found in animals that have rigid exoskeletons, such as arthropods and echinoderms Cytoplasmic intermediate filaments are closely related to their ancestors, the much more prevalent nuclear lamins, which are found in many eukaryotes but missing from unicellular organisms The nuclear lamins form a meshwork lining the inner membrane of the nuclear envelope, where they provide anchorage sites for chromosomes and nuclear pores In contrast to the highly conserved actins and tubulin isoforms that are encoded by a handful of genes, different families of intermediate filaments are much more diverse and are encoded by 70 different humans genes with distinct, cell type-specific functions Intermediate filament structure depends on the lateral bundling and twisting of coiled-coils Although their amino and carboxyl-terminal domains differ, all intermediate filament family members are elongated proteins with a conserved central alpha-helical domain containing 40 or so heptad repeat motifs that form an extended coiled - call structure with another monomer Furthermore, because the tetrameric subunit is made up of two dimers pointing in opposite directions, its two ends are the same. The assembled intermediate filament therefore lacks the overall structural polarity that is critical for actin filaments and microtubules They can be easily bent with a persistence length of less than 1 um (compared to several millimeters for microtubules and about 10 um for actin), but they are extremely difficult to break and can be stretched to more than three times their length In pure protein solutions, intermediate filaments are extremely stable due to tight association of subunits, but some types of intermediate filaments, including vimentin, form highly dynamic structures in cells such as fibroblasts Intermediate filaments impart mechanical stability to animal cells Keratins are the most diverse intermediate filament family: there are about 20 found in different types of human epithelial cells and about 10 more that are specific to hair and nails; analysis of the human genome sequence has revealed that there are 54 distinct keratins Cross-linked keratin networks held together by disulfide bonds can survive even the death of their cells, forming tough coverings for animals, as in the outer layer of skin and in hair, nails, claws, and scales Keratin filaments impart mechanical strength to epithelial tissues in part by anchoring the intermediate filaments at sites of cell-cell contact, called desmosomes, or cell-matrix contact, called hemidesmosomes Other types of blistering diseases, including disorders of the mouth, esophageal lining, and the corned of the eye, are caused by mutations in the different keratins whose expression is specific to those tissues Members of another family of intermediate filaments, called neurofilaments, are found in high concentrations along the axons of vertebrate neurons During axonal growth, new neurofilament subunits are incorporated all along the axon in a dynamic process that involves the addition of subunits along the filament length and at the ends The neurodegenerative disease amyotrophic lateral sclerosis (ALS, Or Lou Gehrig's disease) is associated with an accumulation and abnormal assembly of neurofilaments in motor neuron cell bodies and in the axon, aberrations that may interfere with normal axonal transport In humans, mutations in desmin are associated with various forms of muscular dystrophy and cardiac myopathy, illustrating the important role of desmin in stabilizing muscle fibers In addition, it is becoming increasingly evident that one class of lamins, the A-type, together with many proteins of the nuclear envelope, are scaffolds for proteins that control myriad cellular processes including transcription, chromatin organization, and signal transduction Linker proteins connect cytoskeletal filaments and bridge the nuclear envelope The intermediate filament network is linked to the rest of the cytoskeleton by members of a family of proteins called plakins. Plakins are large and modular, containing multiple domains that connect cytoskeletal filaments to each other and to functional complexes In addition to funding intermediate filaments, it links the intermediate filaments to microtubules, actin filament bundles, and filaments of the motor protein myosin II; it also helps attach intermediate filament bundles to adhesive structures at the plasma membrane Thus, although plectin may not be necessary for the initial formation and assembly of intermediate filaments, its cross-linking action is required to provide cells with the strength they need to withstand the mechanical stresses inherent to vertebrate life Septins form filaments that contribute to subcellular organization Septins assemble into non-polar filaments that form rings and cage-like structures, which act as scaffolds to compartmentalize membranes into distinct domains or to recruit and organize the actin and microtubule cytoskeletons At this location, septins form a barrier that restricts lateral diffusion of proteins embedded in the plasma membrane, enabling cell growth to be concentrated preferentially with the bud