The Cytoskeleton and Cell Movement (Lecture 13)

13 The Cytoskeleton and Cell Movement 13 The Cytoskeleton and Cell Movement Structure and Organization of Actin Filaments Myosin Motors Microtubules Microtubule Motors and Movement Intermediate Filaments Introduction The cytoskeleton is a...

13 The Cytoskeleton and Cell Movement 13 The Cytoskeleton and Cell Movement Structure and Organization of Actin Filaments Myosin Motors Microtubules Microtubule Motors and Movement Intermediate Filaments Introduction The cytoskeleton is a network of protein filaments extending throughout the cytoplasm of eukaryotic cells. It provides a structural framework that determines cell shape, positions of organelles, and general organization of the cytoplasm. Introduction The cytoskeleton is also responsible for movement of entire cells, and internal transport of organelles and other structures. It is not rigid, but is a dynamic structure that is continually reorganized as cells move and change shape. Introduction The cytoskeleton is composed of three main types of protein filaments: Actin filaments Microtubules Intermediate filaments Structure and Organization of Actin Filaments Actin polymerizes to form actin filaments (microfilaments). Microfilaments are flexible fibers 7 nm in diameter and several μm in length, organized into structures such as bundles and 3-D networks. Figure 13.1 Actin filaments Structure and Organization of Actin Filaments Actin-binding proteins regulate assembly and disassembly of actin filaments, cross-linking into bundles and networks, and associations with other cell structures. Table 13.1 Examples of Actin-Binding Proteins Structure and Organization of Actin Filaments Actin was first isolated from muscle cells in 1942. It is very abundant (5–10% of total protein) in all eukaryotic cells. Mammals have six actin genes: four are expressed in muscle cells and two in nonmuscle cells. Structure and Organization of Actin Filaments All actins are very similar and have been highly conserved throughout the evolution of eukaryotes. Yeast actin is 90% identical to actins of mammalian cells. Actin-related proteins also play important roles in bacteria. Structure and Organization of Actin Filaments 3-D structure of actin molecules and filaments was determined in 1990. Each actin monomer (globular [G] actin) has tight binding sites that mediate head-to-tail interactions with two other actin monomers, to form filaments (filamentous [F] actin). Figure 13.2 Assembly and structure of actin filaments (Part 1) Figure 13.2 Assembly and structure of actin filaments (Part 2) Figure 13.2 Assembly and structure of actin filaments (Part 3) Structure and Organization of Actin Filaments All the actin monomers are oriented in the same direction, so actin filaments have polarity. This is important in their assembly and in establishing the direction of myosin movement relative to actin. Structure and Organization of Actin Filaments Nucleation is the first step of actin polymerization—dimers and trimers are formed, then monomers are added to either end. Actin polymerization is reversible; the filaments can be broken down when necessary. Structure and Organization of Actin Filaments Treadmilling: The barbed end of a filament grows 5– 10 times faster than the pointed end. Actin bound to ATP associates with the barbed ends, and the ATP is then hydrolyzed to ADP. Structure and Organization of Actin Filaments ADP-actin is less tightly bound than ATP- actin, and dissociates at the pointed end. Treadmilling illustrates the dynamic behavior of actin filaments. It is critical in regulating actin filaments within the cell. Figure 13.3 Treadmilling and the role of ATP in actin filament polymerization Structure and Organization of Actin Filaments Several drugs affect actin polymerization. Cytochalasins bind to barbed ends and block elongation. This can inhibit movements, such as cell division. Phalloidin binds to actin filaments and prevents dissociation. It can be labeled with fluorescent dye to allow visualization of actin filaments. Structure and Organization of Actin Filaments Actin-binding proteins: Formins bind ATP-actin and nucleate initial polymerization of long unbranched actin filaments. Profilin binds actin monomers and stimulates exchange of bound ADP for ATP, increasing the local concentration of ATP-actin. Figure 13.4 Initiation and growth of actin filaments by formin Structure and Organization of Actin Filaments Arp2/3 complex (actin-related proteins): initiate growth of branched actin filaments, important in driving cell movement at the plasma membrane. Figure 13.5 Initiation of actin filament branches Structure and Organization of Actin Filaments Tropomyosins stabilize actin filaments by binding lengthwise along the groove of the filament. Capping proteins stabilize actin by binding to the barbed or pointed ends. Figure 13.6 Stabilization of actin filaments Structure and Organization of Actin Filaments Other actin-binding proteins remodel or modify existing filaments. Cofilin severs filaments, generating new ends which are then available for polymerization or depolymerization. Figure 13.7 Filament severing by cofilin Structure and Organization of Actin Filaments The actin-binding proteins act together to promote rapid turnover of filaments and remodeling of the cytoskeleton needed for cell movements and changes in cell shape. Their activities are controlled by signaling mechanisms in response to environmental signals. Structure and Organization of Actin Filaments Actin filaments are organized into: Actin bundles—filaments are cross- linked into parallel arrays. Actin networks—filaments are cross- linked in arrays that form 3-D meshworks with the properties of semisolid gels. Figure 13.8 Actin bundles and networks Structure and Organization of Actin Filaments Cross-linking proteins have at least two domains that bind actin. Actin-bundling proteins are small, rigid proteins that force filaments to align closely. Proteins that organize actin networks are large, flexible proteins that cross-link perpendicular filaments. Structure and Organization of Actin Filaments Two types of actin bundles: Parallel bundles—closely spaced filaments aligned in parallel, with the same polarity, with barbed ends adjacent to the plasma membrane. Fimbrin is a bundling protein first isolated from intestinal microvilli. Structure and Organization of Actin Filaments Contractile bundles—widely-spaced filaments cross-linked by α-actinin dimers. Increased spacing between filaments allows the motor protein myosin to interact with the actin filaments. Figure 13.9 Actin-bundling proteins Structure and Organization of Actin Filaments In actin networks, proteins such as filamin form flexible cross-links. A filamin dimer is a flexible V-shaped molecule with actin-binding domains at the end of each arm. Figure 13.10 Actin networks and filamin Structure and Organization of Actin Filaments Actin filaments are concentrated at the cell periphery where they form a 3-D network beneath the plasma membrane. This network and associated proteins (the cell cortex) determines cell shape and is involved in activities such as movement. Structure and Organization of Actin Filaments Red blood cells (erythrocytes) are useful for studies of the cortical cytoskeleton. They have no nucleus or organelles, so plasma membranes and associated proteins are easily isolated. They also lack other cytoskeletal components, so the cortical cytoskeleton is the principal determinant of cell shape. Figure 13.11 Morphology of red blood cells Structure and Organization of Actin Filaments Spectrin is a member of the calponin family of actin-binding proteins. It is a tetramer of two polypeptides, α and β. The ends of the tetramers associate with short actin filaments, resulting in the spectrin-actin network. Figure 13.12 Structure of spectrin Structure and Organization of Actin Filaments Ankyrin links the spectrin-actin network and the plasma membrane by binding to spectrin and a transmembrane protein (band 3). Protein 4.1 is another link that binds spectrin-actin junctions and the transmembrane protein glycophorin. Figure 13.13 Association of the erythrocyte cortical cytoskeleton with the plasma membrane Structure and Organization of Actin Filaments Other types of cells have similar linking proteins. Dystrophin (a calponin) in muscle cells links actin filaments to transmembrane proteins in the plasma membrane, which link to the extracellular matrix, helping maintain cell stability during muscle contraction. Structure and Organization of Actin Filaments Muscular dystrophy, an X-linked inherited disease, results in progressive degeneration of skeletal muscle. Dystrophin is absent or abnormal in patients with Duchenne’s or Becker’s muscular dystrophy, respectively. Structure and Organization of Actin Filaments Most cells have specialized plasma membrane regions that form contacts with adjacent cells, the extracellular matrix, or other substrata (such as the surface of a culture dish). These regions are also attachment sites for actin bundles, evident in fibroblasts maintained in tissue culture. Figure 13.14 Stress fibers and focal adhesions Structure and Organization of Actin Filaments Cultured fibroblasts secrete extracellular matrix proteins that stick to the dish. The fibroblasts attach to the matrix via binding of transmembrane proteins (integrins). The sites of attachment (focal adhesions) are also attachment sites for large actin bundles called stress fibers. Structure and Organization of Actin Filaments Stress fibers are contractile bundles, cross-linked by α-actinin and stabilized by tropomyosin. Two other proteins, talin and vinculin are involved in binding stress fibers. Figure 13.15 Attachment of stress fibers to the plasma membrane at focal adhesions Structure and Organization of Actin Filaments In sheets of epithelial cells, cell–cell contacts (adherens junctions) form a continuous adhesion belt around each cell. Contact is mediated by transmembrane proteins called cadherins, which bind to cytoplasmic catenins, anchoring actin filaments to the plasma membrane. Figure 13.16 Attachment of actin filaments to adherens junctions Structure and Organization of Actin Filaments Cell surfaces have a variety of protrusions involved in cell movement, phagocytosis, or functions such as absorption of nutrients. These extensions are based on actin filaments, in relatively permanent or rapidly rearranging bundles or networks. Structure and Organization of Actin Filaments Microvilli are fingerlike extensions; abundant on cells involved in absorption. Microvilli of epithelial cells lining the intestine form a layer on the apical surface (brush border) of about 1000 microvilli per cell. They increases the surface area for absorption by ten to twentyfold. Figure 13.17 Electron micrograph of microvilli Structure and Organization of Actin Filaments Intestinal microvilli contain parallel bundles of 20 to 30 actin filaments. Filaments are cross-linked by fimbrin and villin. Actin bundles are attached to the plasma membrane by the calcum-binding protein calmodulin in association with myosin I. Figure 13.18 Organization of microvilli Structure and Organization of Actin Filaments Other surface protrusions are transient and form in response to environmental stimuli. Pseudopodia are extensions of moderate width, responsible for phagocytosis and the movement of amoebas. Structure and Organization of Actin Filaments Lamellipodia are broad, sheetlike extensions at the leading edge of fibroblasts. Many cells also extend filopodia, thin projections of the plasma membrane supported by actin bundles. Figure 13.19 Examples of cell surface projections involved in phagocytosis and movement Structure and Organization of Actin Filaments Cell movement or extension of cellular processes involves a coordinated series of movements. Structure and Organization of Actin Filaments Movement of a cell across a surface proceeds in three stages: Extension of the leading edge Attachment of leading edge to the substratum Retraction of the rear of the cell into the cell body Figure 13.20 Cell migration Structure and Organization of Actin Filaments Extension of the leading edge involves branching and polymerization of actin filaments. Inhibition of actin polymerization blocks formation of cell surface protrusions. Structure and Organization of Actin Filaments Cells move in response to signals from other cells or the environment. Example: Wound healing—cells at the edge of a cut move across the extracellular matrix to cover the wound. Formation of cell surface protrusions in response to extracellular stimuli is regulated by Rho proteins. Structure and Organization of Actin Filaments Rho proteins activate WASP proteins, which stimulate the Arp2/3 complex and initiate growth of branched actin filaments. Figure 13.21 Actin filament remodeling at the leading edge Structure and Organization of Actin Filaments As new actin filaments extend into the cell process, they provide pathways for vesicles containing lipids and proteins needed for continued extension. Vesicle cargos include actin-bundling proteins and focal adhesion proteins such as talin and vinculin. Structure and Organization of Actin Filaments For slow-moving cells, attachment to the surface involves formation of focal adhesions. Vinculin and talin activate integrins to bind to the extracellular matrix as well as connect integrins to actin filaments. Structure and Organization of Actin Filaments Retraction of the trailing edge involves small GTP-binding proteins of the Arf and Rho families. They regulate breakdown of existing focal adhesions and stimulate endocytosis of the plasma membrane at the trailing edge of the cell. Myosin Motors Myosin is a molecular motor—a protein that converts chemical energy (ATP) to mechanical energy, generating force and movement. Muscle contraction is the model for understanding actin-myosin interactions and the motor activity of myosin. Myosin Motors Skeletal muscles are bundles of muscle fibers, large cells formed by fusion of many cells during development. Most of the cytoplasm consists of myofibrils, bundles of thick myosin filaments and thin actin filaments. Myosin Motors Each myofibril is a chain of contractile units called sarcomeres, which give skeletal and cardiac muscle their striated appearance. Figure 13.22 Structure of muscle cells Myosin Motors Sarcomere regions are discernible by electron microscopy. The bands correspond to presence or absence of myosin filaments. Actin filaments are attached at the barbed ends to the Z disc, which includes the cross-linking protein α-actinin. Figure 13.23 Structure of the sarcomere Myosin Motors The sliding filament model of muscle contraction was proposed in 1954. During contraction, each sarcomere shortens, bringing the Z discs closer together. There is no change in the width of the A band, but the I bands and H zone almost disappear. Myosin Motors The actin and myosin filaments slide past one another so that the actin filaments move into the A band and H zone. The molecular basis for this is the binding of myosin to actin filaments, allowing myosin to function as a motor that drives filament sliding. Figure 13.24 Sliding filament model of muscle contraction Myosin Motors Myosin II (the type in muscle) has two heavy chains and two pairs of light chains. The heavy chains have a globular head region and a long α-helical tail. The tails twist around each other in a coiled-coil. Figure 13.25 Myosin II Myosin Motors Thick filaments: several hundred myosin molecules in a parallel staggered array. The globular heads bind actin, forming cross-bridges between thick and thin filaments. The orientation of myosin and polarity of actin filaments reverses at the M-line. Figure 13.26 Organization of actin and myosin filaments Myosin Motors Myosin heads hydrolyze ATP, providing energy to drive filament sliding. Myosin changes shape during repeated cycles of interaction between myosin heads and actin. The conformational changes in myosin result in movement of myosin heads along actin filaments. Myosin Motors The model of myosin function comes from in vitro studies and determination of the 3-D structure of myosin: Binding of ATP dissociates myosin from actin. ATP hydrolysis induces a conformational change that displaces the myosin head group. Myosin Motors The myosin head binds to a new position on the actin filament and Pi is released. The “power stroke”: myosin head returns to its original conformation, which drives actin filament sliding, and ADP is released. Figure 13.27 Model for myosin action (Part 1) Figure 13.27 Model for myosin action (Part 2) Myosin Motors Muscle contraction is triggered by nerve impulses which stimulate release of Ca2+ from the sarcoplasmic reticulum. The increased Ca2+ concentration in the cytosol affects two actin filament binding proteins: tropomyosin and troponin. Myosin Motors Tropomyosin binds lengthwise along actin filaments, and is also bound to troponins. When Ca2+ is absent, the tropomyosin- troponin complex blocks binding of myosin to actin. Binding of Ca2+ to troponin C shifts the complex, and allows contraction to proceed. Figure 13.28 Association of tropomyosin and troponins with actin filaments (Part 1) Figure 13.28 Association of tropomyosin and troponins with actin filaments (Part 2) Myosin Motors In nonmuscle cells, contractile assemblies are similar to muscle fibers. They also produce contraction by sliding of actin filaments relative to one another. Stress fibers and adhesion belts are examples of contractile assemblies. Figure 13.29 Contractile assemblies in nonmuscle cells Myosin Motors Cytokinesis: division of a cell following mitosis. A contractile ring of actin and myosin II is assembled by membrane-bound myosin just beneath the plasma membrane. Contraction of the ring pinches the cell in two. Figure 13.30 Cytokinesis Myosin Motors In nonmuscle cells and smooth muscle, contraction is regulated primarily by phosphorylation of a myosin light chain. It is catalyzed by myosin light-chain kinase, which is regulated by the Ca2+- binding protein calmodulin. Figure 13.31 Regulation of myosin II by phosphorylation Myosin Motors Unconventional myosins: Nonmuscle myosins that don’t form filaments and are not involved in contraction. They function in a variety of cell movements, such as transport of vesicles and organelles. Myosin Motors Myosin I Globular head groups act as molecular motors. Short tails bind to other structures. Movement of myosin I along an actin filament can transport its attached cargo, such as a vesicle. Figure 13.32 Myosin I Myosin Motors Myosin V: Two-headed dimer that transports vesicles and other cargo along actin filaments; important in neurons. Some unconventional myosins are involved in actin filament reorganization or anchor actin filaments to the plasma membrane. Figure 13.33 Myosin V Microtubules Microtubules are rigid hollow rods. They are dynamic structures that undergo continual assembly and disassembly. They function in cell movements and determining cell shape. Microtubules Tubulin dimers polymerize to form microtubules: 13 protofilaments around a hollow core. Protofilaments are head-to-tail arrays of tubulin dimers arranged in parallel. Microtubules have polarity (plus and minus ends), which determines direction of movement. Microtubules Microtubules are made of the globular protein tubulin. Tubulin dimers consist of α-tubulin and β- tubulin, which are encoded by related genes. γ-tubulin in the centrosome helps in initiating microtubule assembly. Figure 13.34 Structure of microtubules (Part 1) Figure 13.34 Structure of microtubules (Part 2) Microtubules Microtubules can undergo rapid cycles of assembly and disassembly. GTP bound to β-tubulin is hydrolyzed to GDP shortly after polymerization. This weakens binding affinity of tubulin dimers for each other, causing rapid depolymerization and loss of tubulin bound to GDP from the minus end. Figure 13.35 The role of GTP in microtubule polymerization Microtubules In microtubules stabilized at the minus end, rapid GTP hydrolysis results in dynamic instability: alternating between cycles of growth and shrinkage. As long as new GTP-bound tubulin dimers are added more rapidly than GTP is hydrolyzed, a GTP cap remains at the plus end and microtubule growth continues. Microtubules If GTP is hydrolyzed more rapidly than new subunits are added, GDP-bound tubulin at the plus end of the microtubule leads to disassembly and shrinkage. Figure 13.36 Dynamic instability of microtubules Microtubules Rapid turnover of microtubules allows for remodeling of the cytoskeleton that occurs during mitosis. Drugs such as colchicine and colcemid that affect microtubule assembly are useful as experimental tools and in cancer treatments. Microtubules Vincristine and vinblastine are used in cancer chemotherapy because they inhibit microtubule polymerization and thus affect rapidly dividing cells. Taxol stabilizes microtubules, which also blocks cell division. Microtubules Microtubule-associated proteins (MAPs) regulate the dynamic behavior of microtubules. Minus ends are stabilized by proteins that prevent depolymerization. Growth or shrinkage of plus ends is regulated by MAPs. Table 13.2 Microtubule-Associated Proteins (MAPs) Microtubules Polymerase MAPs accelerate growth by increasing incorporation of GTP-bound tubulin. Depolymerase MAPs dissociate GTP- tubulin from the plus end, leading to microtubule shrinkage (catastrophe). Figure 13.37 Roles of microtubule-associated proteins in dynamic instability (Part 1) Figure 13.37 Roles of microtubule-associated proteins in dynamic instability (Part 2) Microtubules CLASP proteins rescue microtubules from catastrophe by stopping disassembly and restarting growth. Other MAPs bind to plus-ends and mediate attachment of microtubules to other structures (e.g., plasma membrane or ER), and regulate microtubule dynamics. Figure 13.37 Roles of microtubule-associated proteins in dynamic instability (Part 3) Microtubules In animal cells, most microtubules extend outward from the centrosome. During mitosis, they extend outward from duplicated centrosomes to form the mitotic spindle. The spindle controls separation and distribution of chromosomes to daughter cells. Figure 13.38 Intracellular organization of microtubules Microtubules The centrosome is a microtubule- organizing center. In fungi, spindle pole bodies have similar function. Plant cells do not have centrosomes; microtubules form an array underlying the plasma membrane and function in synthesis of plant cell walls. Microtubules Centrosomes are initiation sites for microtubule assembly, which grow outward from minus ends anchored in the centrosome. If cells are treated with colcemid, microtubules disassemble. When the drug is removed, new microtubules grow outward from the centrosome. Figure 13.39 Growth of microtubules from the centrosome Microtubules The role of centrosomes is to initiate microtubule growth. γ-tubulinin is associated with other proteins in a ring-shaped structure called the γ-tubulin ring complex. This complex is thought to bypass the rate-limiting nucleation step, speeding microtubule growth. Microtubules Most animal cell centrosomes have a pair of centrioles, oriented perpendicular to each other and surrounded by pericentriolar material. Centrioles are cylindrical, containing nine triplets of microtubules. Figure 13.40 Structure of centrosomes Microtubules Centrioles also form basal bodies of cilia and flagella. But they are not found in plant cells, many unicellular eukaryotes, and most meiotic animal cells. In these cells the pericentriolar material initiates microtubule assembly. Microtubules Microtubule stability is regulated by post- translational modification of tubulin by phosphorylation, acetylation, etc. These modifications affect microtubule behavior by providing sites for binding of specific MAPs. Microtubules Interactions with MAPs allow cells to stabilize microtubules in particular locations and help determine cell shape and polarity. Many MAPs are cell-type specific. The tau protein is a MAP characteristic of lesions found in the brains of Alzheimer’s patients. Microtubules Nerve cells have two types of processes supported by stable microtubules. Axons: microtubules have plus ends towards the tips; associated with tau. Dendrites: microtubules are oriented in both directions; associated with MAP2. Figure 13.41 Organization of microtubules in nerve cells Microtubule Motors and Movement Two families of motor proteins are responsible for powering movements in which microtubules participate. Kinesins: move along microtubules toward the plus end. Dyneins: move toward the minus end. Figure 13.42 Microtubule motor proteins Microtubule Motors and Movement Axonemal dynein was the first to be identified, because it is very abundant in cilia. Other motor proteins are present in lower amounts; isolation required development of in vitro assays by video-enhanced microscopy. Key Experiment, Ch. 13, p. 509 (4) Microtubule Motors and Movement Kinesin I moves along microtubules in one direction—toward the plus end. But in axons, vesicles were also observed moving back towards cells. Cytoplasmic dynein moves along microtubules towards the minus end (previously identified as axonemal dynein from cilia). Microtubule Motors and Movement Kinesin I has two heavy chains and two light chains. The heavy chains have α-helical regions that form coiled-coils. X-ray crystallography shows that kinesin and myosin evolved from a common ancestor and are structurally similar. Microtubule Motors and Movement The kinesins are a family of related motor proteins. Most move along microtubules in the plus-end direction; they have N-terminal motor domains. Minus-end-directed kinesins have C- terminal motor domains. Microtubule Motors and Movement Some kinesins act as microtubule depolymerizing enzymes. Their motor domains are in the middle of the heavy chain (middle motor kinesins). Microtubule Motors and Movement Several types of axonemal dyneins power the beating of cilia. Cytoplasmic dynein is extremely large with 2–3 heavy chains and a variable number of light and intermediate chains. Motor domains of dynein are less well understood than those of kinesins. Microtubule Motors and Movement A major role of microtubules is to transport macromolecules, vesicles, and organelles through the cytoplasm. Different members of the kinesin and dynein families are thought to transport cargo in opposite directions. Figure 13.43 Transport of vesicles along microtubules Microtubule Motors and Movement Cargo selection can be very specific. Several types of molecular motors associate with a given cargo at the same time, allowing precise positioning. A future challenge is to determine how transport is controlled by switching among the different motors. Microtubule Motors and Movement Microtubules and motor proteins also position organelles within the cell. Example: the ER extends to the periphery of the cell in association with microtubules, which involves kinesin I. Drugs that depolymerize microtubules cause the ER to retract toward the cell center. Figure 13.44 Association of the endoplasmic reticulum with microtubules Microtubule Motors and Movement Cytoplasmic dynein has a role in positioning the Golgi apparatus near the centrosome. If microtubules are disrupted, the Golgi breaks up into small vesicles that disperse. When microtubules re-form, the Golgi apparatus also reassembles. Microtubule Motors and Movement Cilia and flagella are microtubule-based projections of the plasma membrane, responsible for movement of many eukaryotic cells. Some bacteria have flagella, but they are protein filaments projecting from the cell surface. Microtubule Motors and Movement Cilia beat in a coordinated back-and- forth motion, which either moves the cell through a fluid or moves fluid over the surface of the cell. Flagella are longer, and have a wavelike pattern of beating. Figure 13.45 Examples of cilia and flagella Microtubule Motors and Movement Structure of cilia and flagella is similar: The axoneme consists of microtubules in a “9 + 2” pattern: a central pair surrounded by nine outer doublets. Each doublet is a complete A tubule fused to an incomplete B tubule. Nexin links the tubules, and two arms of dynein are attached to each A tubule. Figure 13.46 Structure of the axoneme of cilia and flagella Microtubule Motors and Movement The microtubule minus ends are anchored in a basal body, similar in structure to a centriole. It has nine triplets of microtubules. Basal bodies initiate growth of axonemal microtubules and anchor cilia and flagella to the surface of the cell. Figure 13.47 Electron micrographs of basal bodies Microtubule Motors and Movement Movement of cilia and flagella results from sliding of outer microtubule doublets relative to one another, powered by motor activity of axonemal dyneins. Dynein bases bind to A tubules, while the head groups bind to B tubules of adjacent doublets. Figure 13.48 Movement of microtubules in cilia and flagella Microtubule Motors and Movement Microtubules completely reorganize during mitosis; dynamic instability accelerates. The interphase microtubule array disassembles and free tubulin subunits are reassembled into the mitotic spindle. Figure 13.49 Electron micrograph of the mitotic spindle Microtubule Motors and Movement The centrosome is duplicated in interphase. During prophase, the centrosomes migrate to form the two poles of the mitotic spindle. Figure 13.50 Formation of the mitotic spindle (Part 1) Figure 13.50 Formation of the mitotic spindle (Part 2) Figure 13.50 Formation of the mitotic spindle (Part 3) Microtubule Motors and Movement As the cell enters mitosis, the rate of microtubule disassembly increases, resulting in shrinkage of microtubules. But the number of microtubules emanating from the two centrosomes increases. Microtubule Motors and Movement Four types of microtubules make up the mitotic spindle: 1. Kinetochore microtubules attach to the condensed chromosomes at the centromeres, stabilizing them. 2. Chromosomal microtubules connect to chromosome ends via chromokinesin. Microtubule Motors and Movement 3. Polar microtubules are not attached to chromosomes but are stabilized by overlapping with each other in the center of the cell. 4. Astral microtubules extend outward from the centrosomes with the plus ends anchored in the cell cortex. Microtubule Motors and Movement After the centrosomes move to opposite sides of the cell, the duplicated chromosomes attach to kinetochore and chromosomal microtubules, and align on the metaphase plate. Then the links between the sister chromatids are severed and anaphase begins. Microtubule Motors and Movement Chromosome movement occurs in two steps: Anaphase A—chromosomes move toward spindle poles along kinetochore microtubules, driven by kinesins that depolymerize and shorten the tubules. Figure 13.51 Anaphase A chromosome movement (Part 1) Figure 13.51 Anaphase A chromosome movement (Part 2) Microtubule Motors and Movement Anaphase B: spindle poles separate. Overlapping interpolar microtubules elongate and slide against one another to push the spindle poles apart. Plus-end-directed kinesins cross-link interpolar microtubules and move them toward the plus end. Figure 13.52 Spindle pole separation in anaphase B Microtubule Motors and Movement Spindle poles are also pulled apart by the astral microtubules. Cytoplasmic dynein anchored to the cell cortex moves along astral microtubules in the minus-end direction. Simultaneous shrinkage of astral microtubules by depolymerases leads to separation of the spindle poles. Intermediate Filaments Intermediate filaments: diameters intermediate between actin filaments and microtubules. Not directly involved in cell movements, but provide mechanical strength and a scaffold for localization of cell processes. Not found in yeast, plants, and some insects. Intermediate Filaments Intermediate filaments are composed of many types of proteins expressed in different types of cells. Type I and II are keratins, in epithelial cells. Vimentin forms a network extending out from the nucleus toward the cell periphery. Table 13.3 Intermediate Filament Proteins Intermediate Filaments Desmin is expressed in muscle cells where it connects the Z discs of individual contractile elements. Neurofilament (NF) proteins (with α-internexin) are the major intermediate filaments of many neurons; provide support for long axons. Intermediate Filaments Nestins are expressed during embryonic development in some stem cells. Type V are the nuclear lamins, which form a meshwork underlying the nuclear membrane. Intermediate Filaments Intermediate filaments have a central α-helical rod domain which plays a central role in filament assembly. The head and tail domains determine the specific functions. Intermediate Filaments The central rod domains of two polypeptides form a coiled coil. The dimers associate in a staggered antiparallel fashion to form tetramers, which assemble end-to-end to form protofilaments. Eight protofilaments wind together to form a filament. Figure 13.53 Structure and assembly of intermediate filaments Intermediate Filaments Intermediate filaments do not have distinct ends. They are more stable and don’t have the dynamic behavior of actin filaments or microtubules. Phosphorylation can regulate assembly and disassembly (e.g., nuclear lamins are disassembled during mitosis). Intermediate Filaments Intermediate filaments form a cytoplasmic network in most cells, extending from a ring around the nucleus to the plasma membrane. They can also associate with other cytoskeleton elements, providing a scaffold that organizes the internal structure of the cell. Figure 13.54 Intracellular organization of keratin filaments Intermediate Filaments Epithelial cells have specialized cell contacts: Desmosomes—junctions between adjacent cells. Keratin filaments attach to dense protein plaques on the cytoplasmic side. Attachment is mediated by desmoplakin (a plakin family protein). Figure 13.55 Attachment of intermediate filaments to desmosomes and hemidesmosomes (Part 1) Figure 13.55 Attachment of intermediate filaments to desmosomes and hemidesmosomes (Part 2) Intermediate Filaments Hemidesmosomes—junctions between epithelial cells and underlying connective tissue Keratin filaments are linked to integrins by different plakins (plectin). Figure 13.55 Attachment of intermediate filaments to desmosomes and hemidesmosomes (Part 3) Intermediate Filaments Some plakins link intermediate filaments to other elements of the cytoskeleton. Plectin binds actin filaments and microtubules, forming bridges between them and between intermediate filaments. This increases the mechanical stability of the cell. Figure 13.56 Electron micrograph of plectin bridges between intermediate filaments and microtubules Intermediate Filaments Direct evidence for the function of intermediate filaments is recent. Some cells in culture don’t make intermediate filaments. Injection of cultured cells with antibody against vimentin disrupts intermediate filament networks without affecting cell growth or movement. Intermediate Filaments The primary role of intermediate filaments is probably to strengthen the cytoskeleton of cells in the tissues of multicellular organisms. In tissues, cells are subjected to a variety of mechanical stresses that don’t affect cells in a culture dish. Intermediate Filaments The role of intermediate filaments was shown in studies using transgenic mice with a keratin mutation. The mutation disrupted formation of a normal keratin cytoskeleton, resulting in severe skin abnormalities. Figure 13.57 Experimental demonstration of keratin function Key Experiment, Ch. 13, p. 525 (2) Intermediate Filaments These experiments also pointed to the molecular basis of Epidermolysis bullosa simplex (EBS). Patients develop skin blisters from cell lysis after minor trauma. EBS is caused by keratin gene mutations that interfere with normal assembly of keratin filaments.