Lec-5-Cytoskeleton-2024-stud.pdf

2024-09-09 Cytoskeleton 1 Introduction The cytoskeleton is built on a framework of three types of protein filaments: intermediate fil...

2024-09-09 Cytoskeleton 1 Introduction The cytoskeleton is built on a framework of three types of protein filaments: intermediate filaments, microtubules, and actin filaments. Each type of filament has distinct mechanical properties and is formed from a different protein subunit. They are shown here in epithelial cells, but they are all found in almost all animal cells. 2 2 1  2024-09-09 Intermediate Filaments Are Strong and Ropelike Intermediate Filaments Are Strong and Ropelike in which many long strands are twisted together to provide tensile strength An electron micrograph of intermediate filaments. The strands of this cable are made of intermediate filament proteins, fibrous subunits each containing a central elongated rod domain with distinct unstructured domains at either end The rod domain consists of an extended α-helical region that enables pairs of intermediate filament proteins to form stable dimers by wrapping around each other in a coiledcoil configuration. Two of these coiled-coil dimers, running in opposite directions, associate to form a staggered tetramer. 3 3 Intermediate Filaments Are Strong and Ropelike These dimers and tetramers are the soluble subunits of intermediate filaments. The tetramers associate with each other side-by-side and then assemble to generate the final ropelike intermediate filament. 4 4 2  2024-09-09 Intermediate Filaments Strengthen Cells Against Mechanical Stress Intermediate filaments can be grouped into four classes: (1) keratin filaments in epithelial cells; (2) vimentin and vimentin-related filaments in connective-tissue cells, muscle cells, and supporting cells of the nervous system (glial cells); (3) neurofilaments in nerve cells; and (4) nuclear lamins, which strengthen the nuclear envelope. The first three filament types are found in the cytoplasm, whereas the fourth is found in the nucleus. 5 5 The Nuclear Envelope Is Supported by a Meshwork of Intermediate Filaments The nuclear lamina from lamins disassembles and reforms at each cell division, when the nuclear envelope breaks down during mitosis and then re-forms in each daughter cell. Cytoplasmic intermediate filaments also disassemble in mitosis. The disassembly and reassembly of the nuclear lamina are controlled by the phosphorylation and dephosphorylation of the lamins. 6 6 3  2024-09-09 The Nuclear Envelope Is Supported by a Meshwork of Intermediate Filaments When the lamins are phosphorylated by protein kinases, the consequent conformational change weakens the binding between the lamin tetramers and causes the flaments to fall apart. Dephosphorylation by protein phosphatases at the end of mitosis causes the lamins to reassemble. 7 7 Microtubules Are Hollow Tubes with Structurally Distinct Ends Microtubules are built from subunits—molecules of tubulin— each of which is itself a dimer composed of two very similar globular proteins called α-tubulin and β-tubulin, bound tightly together by noncovalent interactions. 8 8 4  2024-09-09 Microtubules Are Hollow Tubes with Structurally Distinct Ends The tubulin dimers stack together, again by noncovalent bonding, to form + the wall of the hollow cylindrical microtubule. This tube like structure is made of 13 parallel protofilaments, each a linear chain of tubulin dimers with α- and β-tubulin alternating along its length. Each protofilament has a structural polarity. One end of the microtubule, thought to be the β- tubulin end, is called its plus end, and the other, the α-tubulin end, its minus end. - 9 9 The Centrosome Is the Major Microtubule-organizing Center in Animal Cells In animal cells, for example, the centrosome—which is typically close to the cell nucleus when the cell is not in mitosis— organizes an array of microtubules that radiates outward through the cytoplasm The centrosome consists of a pair of centrioles, surrounded by a matrix of proteins. The centrosome matrix includes hundreds of ring shaped structures formed from a special type of tubulin, called γ-tubulin, and each γ-tubulin ring complex serves as the starting point, or nucleation site, for the growth of one microtubule. The αβ-tubulin dimers add to each γ-tubulin ring complex in a specific orientation, with the result that the minus end of each microtubule is embedded in the centrosome, and growth occurs only at the plus end that extends into the cytoplasm 10 10 5  2024-09-09 Growing Microtubules Display Dynamic Instability Once a microtubule has been nucleated, it typically grows outward from the organizing center for many minutes by the addition of αβ-tubulin dimers to its plus end. Then, without warning, the microtubule can suddenly undergo a transition that causes it to shrink rapidly inward by losing tubulin dimers from its free plus end It may shrink partially and then, no less suddenly, start growing again, or it may disappear completely, to be replaced by a new microtubule that grows from the same γ-tubulin ring complex. This remarkable behavior—switching back and forth between polymerization and depolymerization—is known as dynamic instability. 11 11 Growing Microtubules Display Dynamic Instability A microtubule growing out from the centrosome can, however, be prevented from disassembling if its plus end is stabilized by attachment to another molecule or cell structure so as to prevent its depolymerization. If stabilized by attachment to a structure in a more distant region of the cell, the microtubule will establish a relatively stable link between that structure and the centrosome 12 12 6  2024-09-09 Dynamic Instability is Driven by GTP Hydrolysis The dynamic instability of microtubules stems from the intrinsic capacity of tubulin dimers to hydrolyze GTP. GTP hydrolysis controls the dynamic instability of microtubules. (A) Tubulin dimers carrying GTP (red) bind more tightly to one another than do tubulin dimers carrying GDP (dark green). Therefore, rapidly growing plus ends of microtubules, which have freshly added tubulin dimers with GTP bound, tend to keep growing. 13 13 Dynamic Instability is Driven by GTP Hydrolysis From time to time, however, especially if microtubule growth is slow, the dimers in this GTP cap will hydrolyze their GTP to GDP before fresh dimers loaded with GTP have time to bind. The GTP cap is thereby lost. Because the GDP-carrying dimers are less tightly bound in the polymer, the protofilaments peel away from the plus end, and the dimers are released, causing the microtubule to shrink. 14 14 7  2024-09-09 Microtubules Organize the Cell Interior Most differentiated animal cells are polarized; that is, one end of the cell is structurally or functionally different from the other. Nerve cells, for example, put out an axon from one end of the cell and dendrites from the other. In the nerve cell, for example, all the microtubules in the axon point in the same direction, with their plus ends toward the axon terminals; along these oriented tracks, the cell is able to transport organelles, membrane vesicles, and macromolecules—either from the cell body to the axon terminals or in the opposite direction 15 15 Motor Proteins Drive Intracellular Transport The motor proteins that move along cytoplasmic microtubules, such as those in the axon of a nerve cell, belong to two families: the kinesins generally move toward the plus end of a microtubule; the dyneins move toward the minus end. Both kinesins and dyneins are generally dimers that have two globular ATP-binding heads and a single tail 16 16 8  2024-09-09 Motor Proteins Drive Intracellular Transport The heads interact with microtubules in a stereospecific manner, so that the motor protein will attach to a microtubule in only one direction. The tail of a motor protein generally binds stably to some cell component, such as a vesicle or an organelle, and thereby determines the type of cargo that the motor protein can transport. 17 17 Cilia and Flagella Contain Stable Microtubules Moved by Dynein Cilia beat in a whiplike fashion, either to move fluid over the surface of a cell or to propel single cells through a fluid. A cilium beats by performing a repetitive cycle of movements, consisting of a power stroke followed by a recovery stroke. In the fast power stroke, the cilium is fully extended and fluid is driven over the surface of the cell; in the slower recovery stroke, the cilium curls back into position with minimal disturbance to the surrounding fluid. Each cycle typically requires 0.1–0.2 second and generates a force parallel to the cell surface. 18 18 9  2024-09-09 Cilia and Flagella Contain Stable Microtubules Moved by Dynein The flagella that propel sperm and many protozoa are much like cilia in their internal structure but are usually very much longer. They are designed to move the entire cell, rather than moving fluid across the cell surface. Flagella propagate regular waves along their length, propelling the attached cell along. The movement of a single flagellum on an invertebrate sperm is seen in a series of images captured by stroboscopic illumination at 400 flashes per second. 19 19 Cilia and Flagella Contain Stable Microtubules Moved by Dynein The microtubules in cilia and flagella are slightly different from cytoplasmic microtubules; they are arranged in a curious and distinctive pattern. A cross section through a cilium shows nine doublet microtubules arranged in a ring around a pair of single microtubules. This “9 + 2” array is characteristic of almost all eukaryotic cilia and flagella—from those of protozoa to those in humans 20 20 10  2024-09-09 Cilia and Flagella Contain Stable Microtubules Moved by Dynein The movement of a cilium or a flagellum is produced by the bending of its core as the microtubules slide against each other. The microtubules are associated with numerous accessory proteins, which project at regular positions along the length of the microtubule bundle. Some of these proteins serve as cross- links to hold the bundle of microtubules together; others generate the force that causes the cilium to bend. Here you see a Diagram of the flagellum in cross section. The nine outer microtubules carry two rows of dynein molecules. The heads of each dynein molecule appear in this view like arms reaching toward the adjacent doublet microtubule. 21 21 Cilia and Flagella Contain Stable Microtubules Moved by Dynein Ciliary dynein is attached by its tail to one microtubule, while its two heads interact with an adjacent microtubule to generate a sliding force between the two microtubules. Because of the multiple links that hold the adjacent microtubule doublets together, the sliding force between adjacent microtubules is converted to a bending motion in the cilium (A) If the outer doublet microtubules and their associated dynein molecules are freed from other components of a sperm flagellum and then exposed to ATP , the doublets slide against each other, telescope- fashion, due to the repetitive action of their associated dyneins. (B) In an intact flagellum, however, the doublets are tied to each other by flexible protein links so that the action of the system produces bending rather than sliding. 22 22 11  2024-09-09 Introduction Actin filaments interact with a large number of actin-binding proteins that enable the filaments to serve a variety of functions in cells. Depending on which of these proteins they associate with, actin filaments can form stiff and stable structures, such as the microvilli on the epithelial cells lining the intestine or the small contractile bundles that can contract and act like tiny muscles in most animal cells (B). They can also form temporary structures, such as the dynamic protrusions formed at the leading edge of a crawling cell (C) or the contractile ring that pinches the cytoplasm in two when an animal cell divides (D). Actin-dependent movements usually require actin’s association with a motor protein called myosin. 23 23 Actin and Tubulin Polymerize by Similar Mechanisms A naked actin filament, like a microtubule without associated proteins, is inherently unstable, and it can disassemble from both ends. If the concentration of free actin monomers is very high, an actin filament will grow rapidly, adding monomers at both ends. At intermediate concentrations of free actin, however, something interesting takes place. Actin monomers add to the plus end at a rate faster than the bound ATP can be hydrolyzed, so the plus end grows. At the minus end, by contrast, ATP is hydrolyzed faster than new monomers can be added; because ADP-actin destabilizes the structure, the filament loses subunits from its minus end at the same time as it adds them to the plus end. Inasmuch as an individual monomer moves through the filament from the plus to the minus end, this behavior is called treadmilling. Both the treadmilling of actin filaments and the dynamic instability of microtubules rely on the hydrolysis of a bound nucleoside triphosphate to regulate the length of the polymer. 24 24 12  2024-09-09 A Cortex Rich in Actin Filaments Underlies the Plasma Membrane of Most Eukaryotic Cells In human red blood cells, a simple and regular network of fibrous proteins—including actin and spectrin filaments— attaches to the plasma membrane, providing the support necessary for the cells to maintain their simple discoid shape. 25 25 Cell Crawling Depends on Cortical Actin The molecular mechanisms of these and other forms of cell crawling entail coordinated changes of many molecules in different regions of the cell. Three interrelated processes are known to be essential: 1.the cell pushes out protrusions at its “front,” or leading edge; 2.these protrusions adhere to the surface over which the cell is crawling; and 3.the rest of the cell drags itself forward by traction on these anchorage points. Actin polymerization at the leading edge of the cell pushes the plasma membrane forward and forms new regions of actin cortex, shown here in red. New points of anchorage are made between the bottom of the cell and the surface (substratum) on which the cell is crawling (attachment). Contraction at the rear of the cell— mediated by myosin motor proteins moving along actin filaments—then draws the body of the cell forward. New anchorage points are established at the front, and old ones are released at the back, as the cell crawls forward. The same cycle is repeated over and over again, moving the cell forward in a stepwise 26 fashion. 26 13  2024-09-09 Cell Crawling Depends on Cortical Actin The formation and growth of actin filaments at the leading edge of a cell are assisted by various actin-binding proteins. With the aid of additional actin-binding proteins, this web undergoes continual assembly at the leading edge and disassembly further back, pushing the lamellipodium forward. 27 27 Actin Associates with Myosin to Form Contractile Structures Myosin-I is present in all types of cells. Myosin-I molecules have a head domain and a tail. The head domain binds to an actin filament and has the ATP-hydrolyzing motor activity that enables it to move along the filament in a repetitive cycle of binding, detachment, and rebinding. (B) This arrangement allows the head domain to move a vesicle relative to an actin filament, which in this case is anchored to the plasma membrane. (C) Myosin-I can also bind to an actin filament in the cell cortex, ultimately pulling the plasma membrane into a new shape. The head group always walks toward the plus end of the actin filament. 28 28 14  2024-09-09 Muscle Contraction Depends on Interacting Filaments of Actin and Myosin Muscle myosin belongs to the myosin-II subfamily of myosins, all of which are dimers, With two globular ATPase heads at one end and a single coiled-coil tail at the other. Clusters of myosin-II molecules bind to each other through their coiled-coil tails, forming a bipolar myosin filament from which the heads project. 29 29 Muscle Contraction Depends on Interacting Filaments of Actin and Myosin The myosin filament is like a double-headed arrow, with the two sets of myosin heads pointing in opposite directions, away from the middle. One set binds to actin filaments in one orientation and moves the filaments one way; the other set binds to other actin filaments in the opposite orientation and moves the filaments in the opposite direction. As a result, a myosin filament slides sets of oppositely oriented actin filaments past one another. 30 30 15  2024-09-09 Actin Filaments Slide Against Myosin Filaments During Muscle Contraction Skeletal muscle fibers are huge, multinucleated individual cells formed by the fusion of many separate smaller cells. The nuclei of the contributing cells are retained in the muscle fiber and lie just beneath the plasma membrane. The bulk of the cytoplasm is made up of myofibrils, the contractile elements of the muscle cell. A myofibril consists of a chain of identical tiny contractile units, or sarcomeres. Each sarcomere is about 2.5 μm long, and the repeating pattern of sarcomeres gives the vertebrate myofibril a striped appearance. 31 31 Actin Filaments Slide Against Myosin Filaments During Muscle Contraction Sarcomeres are highly organized assemblies of two types of filaments— actin filaments and myosin filaments composed of a muscle specific form of myosin-II. The myosin filaments (the thick filaments) are centrally positioned in each sarcomere, whereas the more slender actin filaments (the thin filaments) extend inward from each end of the sarcomere, where they are anchored by their plus ends to a structure known as the Z disc. The minus ends of the actin filaments overlap the ends of the myosin filaments. 32 32 16  2024-09-09 Actin Filaments Slide Against Myosin Filaments During Muscle Contraction Detail of the electron micrograph showing two myofibrils; the length of one sarcomere and the region where the actin and myosin filaments overlap are indicated. (B) Schematic diagram of a single sarcomere showing the origin of the light and dark bands seen in the microscope. Z discs at either end of the sarcomere are attachment points for the plus ends of actin filaments. The centrally located thick filaments (green) are each composed of many myosin-II molecules. 33 33 Actin Filaments Slide Against Myosin Filaments During Muscle Contraction The contraction of a muscle cell is caused by a simultaneous shortening of all the cell’s sarcomeres, which is caused by the actin filaments sliding past the myosin filaments, with no change in the length of either type of filament. The sliding motion is generated by myosin heads that project from the sides of the myosin filament and interact with adjacent actin filaments. When a muscle is stimulated to contract, the myosin heads start to walk along the actin filament in repeated cycles of attachment and detachment. 34 34 17  2024-09-09 Muscle Contraction Is Triggered by a Sudden Rise in Cytosolic Ca2+ The force-generating molecular interaction between myosin and actin filaments takes place only when the skeletal muscle receives a signal from a motor nerve. The neurotransmitter released from the nerve terminal triggers an action potential in the muscle cell plasma membrane. (B) Electron micrograph showing a cross section of two T tubules and their adjacent sarcoplasmic reticulum compartments. This electrical excitation spreads in a matter of milliseconds into a series of membranous tubes, called transverse (or T) tubules that extend inward from the plasma membrane around each myofibril. The electrical signal is then relayed to the sarcoplasmic reticulum, an adjacent sheath of interconnected flattened vesicles that surrounds each myofibril like a net stocking. The sarcoplasmic reticulum is a specialized region of the endoplasmic reticulum in muscle cells. 35 35 Muscle Contraction Is Triggered by a Sudden Rise in Cytosolic Ca2+ The sarcoplasmic reticulum contains a very high concentration of calcium [Ca2+] and in response to the incoming electrical excitation, much of this Ca2+ is released into the cytosol through a specialized set of ion channels that open in the sarcoplasmic reticulum membrane in response to the change in voltage across the plasma membrane and T tubules. This schematic diagram shows how a Ca2+-release channel in the sarcoplasmic reticulum membrane is thought to be opened by activation of a voltage-gated Ca2+ channel in the T-tubule membrane. Skeletal muscle contraction is triggered by the release of Ca2+ from the sarcoplasmic reticulum into the cytosol. 36 36 18  2024-09-09 Muscle Contraction Is Triggered by a Sudden Rise in Cytosolic Ca2+ In muscle, the rise in cytosolic Ca2+ concentration activates a molecular switch made of specialized accessory proteins closely associated with the actin filaments. One of these proteins is tropomyosin, a rigid, rod-shaped molecule that binds in the groove of the actin helix, where it prevents the myosin heads from associating with the actin filament. The other is troponin, a protein complex that includes a Ca2+-sensitive protein associated with the end of a tropomyosin molecule. 37 37 Muscle Contraction Is Triggered by a Sudden Rise in Cytosolic Ca2+ When the concentration of Ca2+ rises in the cytosol, Ca2+ binds to troponin and induces a change in the shape of the troponin complex. This in turn causes the tropomyosin molecules to shift their positions slightly, allowing myosin heads to bind to the actin filaments, initiating contraction 38 38 19  2024-09-09 Muscle Contraction Is Triggered by a Sudden Rise in Cytosolic Ca2+ Because the signal from the plasma membrane is passed within milliseconds to every sarcomere in the cell, all the myofibrils in the cell contract at the same time. The increase in Ca2+ in the cytosol is transient because, when the nerve signal terminates, the Ca2+ is rapidly pumped back into the sarcoplasmic reticulum by abundant Ca2+-pumps in its membrane. As soon as the Ca2+ concentration returns to the resting level, troponin and tropomyosin molecules move back to their original positions. This reconfiguration once again blocks myosin binding to actin filaments, thereby ending the contraction. 39 39 Different Types of Muscle Cells Perform Different Functions A similar activation mechanism operates in smooth muscle, which is present in the walls of the stomach, intestine, uterus, and arteries, and in many other structures that undergo slow and sustained involuntary contractions. This mode of myosin activation is relatively slow, because time is needed for enzyme molecules to diffuse to the myosin heads and carry out the phosphorylation and subsequent dephosphorylation. However, this mechanism has the advantage that—unlike the mechanism used by skeletal muscle cells—it can be activated by a variety of extracellular signals: thus smooth muscle, for example, is triggered to contract by adrenaline, serotonin, prostaglandins, and several other signal molecules. 40 40 20  2024-09-09 Essential Cell Biology-4e Chapter 17 Cytoskeleton 565 Intermediate Filaments 567 Intermediate Filaments Are Strong and Ropelike Intermediate Filaments Strengthen Cells Against Mechanical Stress The Nuclear Envelope Is Supported by a Meshwork of Intermediate Filaments Microtubules 571 Microtubules Are Hollow Tubes with Structurally Distinct Ends The Centrosome Is the Major Microtubule organizing Center in Animal Cells Growing Microtubules Display Dynamic Instability Dynamic Instability is Driven by GTP Hydrolysis Microtubule Dynamics Can be Modified by Drugs Microtubules Organize the Cell Interior Motor Proteins Drive Intracellular Transport Microtubules and Motor Proteins Position Organelles in the Cytoplasm Cilia and Flagella Contain Stable Microtubules Moved by Dynein Actin Filaments 583 Actin Filaments Are Thin and Flexible Actin and Tubulin Polymerize by Similar Mechanisms Many Proteins Bind to Actin and Modify Its Properties A Cortex Rich in Actin Filaments Underlies the Plasma Membrane of Most Eukaryotic Cells Cell Crawling Depends on Cortical Actin Actin Associates with Myosin to Form Contractile Structures Muscle Contraction 592 Muscle Contraction Depends on Interacting Filaments of Actin and Myosin Actin Filaments Slide Against Myosin Filaments During Muscle Contraction Muscle Contraction Is Triggered by a Sudden Rise in Cytosolic Ca2+ Different Types of Muscle Cells Perform Different Functions 41 41 21