Cytoskeleton and Cellular Motility PDF

Cytoskeleton and Cellular Motility Radhika Bhardwaj, PhD Department of Biotechnology, AIU  The cytoskeleton A network of protein filaments extending throughout the cytoplasm of all eukaryotic cells.  Cytoskeleton: The Role Provides a structural...

Cytoskeleton and Cellular Motility Radhika Bhardwaj, PhD Department of Biotechnology, AIU  The cytoskeleton A network of protein filaments extending throughout the cytoplasm of all eukaryotic cells.  Cytoskeleton: The Role Provides a structural framework for the cell, serving as a scaffold that determines cell shape, the positions of organelles, and the general organization of the cytoplasm In addition, the cytoskeleton is responsible for cell movements The cytoskeleton is composed of three principal types of protein filaments: 1. Actin filaments 2. Intermediate filaments, and 3. Microtubules which are held together and linked to subcellular organelles and the plasma membrane by a variety of accessory proteins. Outlines: STRUCTURE AND ORGANIZATION OF ACTIN FILAMENTS Assembly and Disassembly of Actin Filaments Organization of Actin Filaments Association of Actin Filaments with the Plasma Membrane Protrusions of the Cell Surface ACTIN, MYOSIN, AND CELL MOVEMENT Muscle Contraction Contractile Assemblies of Actin and Myosin in Nonmuscle Cells Nonmuscle Myosins Formation of Protrusions and Cell Movement Outlines: INTERMEDIATE FILAMENTS Intermediate Filament Proteins Assembly of Intermediate Filaments Intracellular Organization of Intermediate Filaments Functions of Keratins and Neurofilaments Diseases of the Skin and Nervous System Outlines: MICROTUBULES Structure and Dynamic Organization of Micro tubules Assembly of Microtubules Organization of Micro tubules within Cells MICROTUBULE MOTORS AND MOVEMENT Identification of Microtubule Motor Proteins Cargo Transport and Intracellular Organization Cilia and Flagella Chromosome Movement Actin Filaments  Structure and Organization of Actin Filaments The major cytoskeletal protein of most cells is actin, which polymerizes to form actin filaments- thin, flexible fibers approximately 7 nm in diameter and up to several micrometers in length. Actin filaments (also called microfilaments) are abundant beneath the plasma membrane where they form a network that provides: -mechanical support, -determines cell shape, and -allows movement of the cell surface, Thereby enabling cells to migrate, engulf particles, and divide.  Assembly and Disassembly of Actin Filaments (A) Actin monomers (G actin) polymerize to form actin filaments (F actin). (B) Structure of an actin The first step is the formation monomer. of dimers and trimers, which then grow by the addition of monomers to both ends.  Assembly and Disassembly of Actin Filaments Individual actin molecules are globular proteins of 375 amino acids (43 kd). Each actin monomer (globular [G] actin) has tight binding sites that mediate head-to-tail interactions with two other actin monomers, so actin monomers polymerize to form filaments (filamentous [F] actin). Each monomer is rotated by 166° in the filaments, which therefore have the appearance of a double-stranded helix. Because all the actin monomers are oriented in the same direction, actin filaments have a distinct polarity and their ends (called barbed or plus ends and pointed or minus ends) are distinguishable from one another.  Organization of Actin Filaments Individual actin filaments are assembled into two general types of structures called Actin bundles Actin networks In bundles, the actin filaments are In networks, the actin filaments are cross-linked into closely packed crosslinked in orthogonal arrays that parallel arrays. form three-dimensional meshworks with the properties of semisolid gels. The proteins that cross-link actin In contrast, the proteins that organize actin filaments into bundles (called filaments into networks tend to be large flexible actin-bundling proteins) usually proteins that can cross-link perpendicular are small rigid proteins that force filaments. the filaments to align closely with one another Actin cross-linking proteins are modular proteins consisting of related structural units.  Actin bundles and networks (A) Electron micrograph of actin bundles (arrowheads) projecting from the actin network (arrows) underlying the plasma membrane of a macrophage. The bundles support cell surface projections called filopodia. (B) Schematic organization of bundles and networks. Actin filaments in bundles are cross-linked into parallel arrays by small proteins that align the filaments closely with one another. In contrast, networks are formed by large flexible proteins that cross-link orthogonal filaments.  Actin-bundling proteins Actin filaments are associated into two types of bundles by different actin-bundling proteins. Fimbrin has two adjacent actin-binding In contrast, the two separated actin-binding domains of α-actinin domains (ABO) and cross-links actin filaments dimers cross-link filaments into more loosely spaced contractile into closely packed parallel bundles in which bundles in which the filaments are separated by 40 nm. the filaments are approximately 14 nm apart. Both fimbrin and α-actinin contain two related Ca2+-binding domains, and α -actinin contains four repeated a-helical spacer domains Actin networks and filamin Filamin is a dimer of two large (280 kd) subunits, forming a flexible V-shaped molecule that crosslinks actin filaments into orthogonal networks. The carboxy-terminal dimerization domain is separated from the amino- terminal actin-binding domain by repeated β-sheet spacer domains.  Association of Actin Filaments with the Plasma Membrane Actin filaments are highly concentrated at the periphery of the cell where they form a three-dimensional network beneath the plasma membrane. This network of actin filaments and associated actin-binding proteins (called the cell cortex) determines cell shape and is involved in a variety of cell surface activities, including movement. The association of the actin cytoskeleton with the plasma membrane is thus central to cell structure  Protrusions of the Cell Surface The surfaces of most cells have a variety of protrusions or extensions that are involved in cell movement, phagocytosis, or specialized functions, such as absorption of nutrients. Most of these cell surface extensions are based on actin filaments, which are organized into either relatively permanent or rapidly rearranging bundles or networks.  The best-characterized of these actin-based cell surface protrusions are microvilli, finger like extensions of the plasma membrane that are particularly abundant on the surfaces of cells involved in absorption, such as the epithelial cells lining the intestine However, the major actin-bundling protein in intestinal microvilli is villin, a 95 kd protein present in microvilli of only a few specialized types of cells, such as those lining the intestine and kidney tubules. The microvilli of intestinal epithelial cells are fingerlike projections of the plasma membrane. They are supported by actin bundles anchored in a dense region of the cortex called the terminal web.  Organization of microvilli The core actin filaments of microvilli are cross-linked into closely packed bundles by fimbrin and villin. They are attached to the plasma membrane along their length by lateral arms, consisting of myosin I and calmodulin. The barbed ends of the actin filaments are embedded in a cap of unidentified proteins at the tip of the microvillus. In contrast to microvilli many surface protrusions are transient structures that form in response to environmental stimuli. Pseudopodia are extensions of moderate width, based Several types of these structures extend from the leading edge of a moving cell and are involved in cell locomotion Lamellipodia are broad, sheetlike extensions at the leading edge of fibroblasts, which similarly contain a network of actin filaments. Many cells also extend microspikes or filopodia, thin projections of the plasma membrane supported by actin bundles. Cell surface projections involved in phagocytosis and movement (A)Pseudopodia of a macrophage engulfing a tumor cell during phagocytosis. (B) An amoeba with several extended pseudopodia. (C) A tissue culture cell illustrating lamellipodia {L) and filopodia (arrow).  Formation of Protrusions and Cell Movement The movement of cells across a surface represents a basic form of cell locomotion employed by a wide variety of different kinds of cells. Examples include o the crawling of amoebas, o the migration of embryonic cells during development, o the invasion of tissues by white blood cells to fight infection, o the migration of cells involved in wound healing, and o the spread of cancer cells during the metastasis of malignant tumors. Similar types of movement are also responsible for phagocytosis and for the extension of nerve cell processes during development of the nervous system. All of these movements are based on local specializations and extensions of the plasma membrane driven by the dynamic properties of the actin cytoskeleton.  Actin, Myosin, and Cell Movement Actin filaments, often in association with myosin, are responsible for many types of cell movements. Myosin is the prototype of a molecular motor-a protein that converts chemical energy in the form of ATP to mechanical energy, thus generating force and movement. The most striking variety of such movement is muscle contraction  Actin, Myosin, and Cell Movement However, interactions of actin and myosin are responsible not only for muscle contraction but also for a variety of movements of nonmuscle cells, including cell division, so these interactions play a central role in cell biology.  Muscle Contraction Muscle cells are highly specialized for a single task-contraction and it is this specialization in structure and function that has made muscle the prototype for studying movement at the cellular and molecular level There are three distinct types of muscle cells in vertebrates: Skeletal muscle: which is responsible for all voluntary movements; Cardiac muscle: which pumps blood from the heart; and Smooth muscle: responsible for involuntary movements of organs such as the stomach, intestine, uterus, and blood vessels.  Skeletal muscles are bundles of muscle fibers Muscle fibers are single large cells (approximately 50 pm in diameter and up to several centimeters in length) formed by the fusion of many individual cells during development. Most of the cytoplasm consists of myofibrils, which are cylindrical bundles of two types of filaments: thick filaments of myosin (about 15 run in diameter) and thin filaments of actin (about 7 nm in diameter). Each myofibril is organized as a chain of contractile units called sarcomeres, which are responsible for the striated appearance of skeletal and cardiac muscle.  Structure of muscle cells Muscles are composed of bundles of single large cells (called muscle fibers) that form by cell fusion and contain multiple nuclei. Each muscle fiber contains many myofibrils, which are bundles of actin and myosin filaments organized into a chain of repeating units called sarcomeres.  Structure of the sarcomere (A)Electron micrograph of a sarcomere. (B) Diagram showing the organization of actin (thin) and myosin (thick) filaments in the indicated regions. Titin and Nebulin Molecules of titin extend from the Z disc to the M line and act as springs to keep myosin filaments centered in the sarcomere. Molecules of nebulin extend from the Z disc and are thought to determine the length of associated actin filaments. The I bands contain only thin (actin) filaments, whereas the A bands contain thick (myosin) filaments. The myosin and actin filaments overlap in peripheral regions of the A band, whereas a middle region (called the H zone) contains only myosin. The actin filaments are attached at their barbed ends to the Z disc, which includes the cross-linking protein α-actinin. The myosin filaments are anchored at the M line in the middle of the sarcomere. Two additional proteins (titin and nebulin) also contribute to sarcomere structure and stability Titin is an extremely large protein (3000 kd), and single titin molecules extend from the M line to the Z disc. These long molecules of titin are thought to act like springs that keep the myosin filaments centered in the sarcomere and maintain the resting tension that allows a muscle to snap back if overextended. Nebulin filaments are associated with actin and are thought to regulate the assembly of actin filaments by acting as rulers that determine their length. Muscle contraction thus results from an interaction between the actin and myosin filaments that generates their movement relative to one another. The molecular basis for this interaction is the binding of myosin to actin filaments, allowing myosin to function as a motor that drives filament sliding.  Sliding-filament model of muscle contraction The actin filaments slide past the myosin filaments toward the middle of the sarcomere. The result is shortening of the sarcomere without any change in filament length. Contractile Assemblies of Actin and Myosin in Nonmuscle Cells The most dramatic example of actin-myosin contraction in nonmuscle cells, however, is provided by cytokinesis- the division of a cell into two cells following mitosis Contractile Assemblies of Actin and Myosin in Nonmuscle Cells In addition to myosin II (the two-headed myosins found in muscle cells), several other types of myosin are found in nonmuscle cells. These myosins do not have tails able to form coiled-coils, so they do not form filaments and are not involved in contraction. However, they are important in a variety of cell movements, such as the transport of membrane vesicles and organelles along actin filaments, phagocytosis, and extension of pseudopods in amoebae 3. Muscle contraction detail Concept Cell Biology - YouTube Intermediate Filaments Intermediate filaments have diameters between 8 and 11 nm, which is intermediate between the diameters of the two other principal elements of the cytoskeleton, actin filaments (about 7 nm) and microtubules (about 25 nm). In contrast to actin filaments and microtubules, the intermediate filaments are not directly involved in cell movements. Instead, they appear to play basically a structural role by providing mechanical strength to cells and tissues. Whereas actin filaments and microtubules are polymers of single types of proteins (actin and tubulin, respectively), intermediate filaments are composed of a variety of proteins that are expressed in different types of cells. More than 65 different intermediate filament proteins have been identified and classified into six groups based on similarities between their amino acid sequences Intermediate Filament Proteins  Structure of intermediate filament proteins Contain a central α-helical rod domain of approximately 310 amino acids (350 amino acids in the nuclear lamins). The N-terminal head and C-terminal tail domains vary in size and shape.  Assembly of Intermediate Filaments The central rod domains of two polypeptides wind around each other in a coiled-coil structure to form dimers. Dimers then associate in a staggered antiparallel fashion to form tetramers. Tetramers associate end-to-end to form protofilaments and laterally to form filaments. Each filament contains approximately eight protofilaments wound around each other in a ropelike structure.  Intracellular Organization of Intermediate Filaments Intermediate filaments form an elaborate network in the cytoplasm of most cells, extending from a ring surrounding the nucleus to the plasma membrane  Intracellular organization of keratin filaments Micrograph of epithelial cells stained with fluorescent antibodies to keratin (green). Nuclei are stained blue. The keratin filaments extend from a ring surrounding the nucleus to the plasma membrane. Intermediate filaments are generally more stable than actin filaments or microtubules and do not exhibit the dynamic behavior associated with these other elements of the cytoskeleton However, intermediate filament proteins are frequently modified by phosphorylation, which can regulate their assembly and disassembly within the cell. One example is phosphorylation of the nuclear lamins, which results in disassembly of the nuclear lamina and breakdown of the nuclear envelope during mitosis. Cytoplasmic intermediate filaments, such as vimentin, are also phosphorylated, which can lead to their disassembly and reorganization in dividing or migrating cells Both keratin and vimentin filaments attach to the nuclear envelope, apparently serving to position and anchor the nucleus within the cell. In addition, intermediate filaments can associate not only with the plasma membrane but also with the other elements of the cytoskeleton, actin filaments and microtubules.  Intracellular Organization of Intermediate Filaments Intermediate filaments form a network extending from a ring surrounding the nucleus, to the plasma membrane of most cell types. In epithelial cells, intermediate filaments are anchored to the plasma membrane at regions of specialized cell contacts (desmosomes and hemidesmosomes). Intermediate filaments also play specialized roles in muscle and nerve cells. Microtubules  Microtubules, the third principal component of the cytoskeleton, are rigid hollow rods approximately 25 nm in diameter. Like actin filaments, microtubules are dynamic structures that undergo continual assembly and disassembly within the cell. They function both to determine cell shape and in a variety of cell movements, including some forms of cell locomotion, the intracellular transport of organelles, and the separation of chromosomes during mitosis.  Assembly of Microtubules In animal cells: Most microtubules extend outward from the centrosome, which is located adjacent to the nucleus near the center of interphase (nondividing) cells  Structure and Dynamic Organization of Microtubules Microtubules: Composed of a single type of globular protein called tubulin Structure of microtubule The building blocks of microtubules are tubulin dimers consisting of two closely related 55 kd polypeptides: α tubulin and β-tubulin. Dimers of α tubulin and β-tubulin polymerize to form microtubules, which are composed of 13 protofilaments assembled around a hollow core. In addition, a third type of tubulin (γ-tubulin) is concentrated in the centrosome where it plays a critical role in initiating microtubule assembly The protofilaments, which are composed of head-to-tail arrays of tubulin dimers, are arranged in parallel. Microtubules (like actin filaments) are polar structures with two distinct ends: a fast-growing plus end and a slow growing minus end. Polarity determines the direction of movement along microtubules, just as the polarity of actin filaments defines the direction of myosin movement. Intracellular organization of microtubules The minus ends of microtubules are anchored in the centrosome. In interphase cells, the centrosome is located near the nucleus and microtubules extend outward to the cell periphery. During mitosis, duplicated centrosomes separate, and microtubules reorganize to form the mitotic spindle.  Microtubule Motors and Movement Microtubules are responsible for a variety of cell movements, including the intracellular transport and positioning of membrane vesicles and organelles, the separation of chromosomes at mitosis, and the beating of cilia and flagella. Movement along microtubules is based on the action of motor proteins that utilize energy derived from ATP hydrolysis to produce force and movement.  Cargo Transport and Intracellular Organization One of the major roles of microtubules is to transport macromolecules, membrane vesicles, and organelles through the cytoplasm of eukaryotic cells.  Reorganization of Microtubules during Mitosis Microtubules reorganize at the beginning of mitosis to form the mitotic spindle, which is responsible for chromosome separation. Chromosome Movement The duplicated chromosomes align on the metaphase plate. During anaphase of mitosis, daughter chromosomes separate and move to opposite poles of the mitotic spindle. Chromosome separation results from several types of movements in which different classes of spindle microtubules and motor proteins participate.  Microtubules - YouTube Intermediate Filaments - YouTube