CellBio_L3_L4_ Cytoskeleton.pdf

 Cytoskeleton-crisscrossed by a network of protein fibers that supports the shape of the cell and anchors organelles to fixed locations. Dynamic- Constantly assembling and disassembling The cytoskeleton is a cellular scaffolding or skeleton contained within a cells cytoplasm present in...

 Cytoskeleton-crisscrossed by a network of protein fibers that supports the shape of the cell and anchors organelles to fixed locations. Dynamic- Constantly assembling and disassembling The cytoskeleton is a cellular scaffolding or skeleton contained within a cells cytoplasm present in all cells Eukaryotes and Prokaryotes The eukaryotic cytoskeleton. Actin-red, Eukaryotes- actin filaments (Microfilaments), microtubules, Microtubules- Green Nuclei-blue. intermediate filaments Prokaryotes- FtsZ, Mre B and ParM, Crescentin Movers and Shapers of Cytoskeleton Shapers: Actin (Microfilaments)- Ex-Muscles Intermediate filaments- Ex-Keratin, Vimentin Nerve cells-neurofilaments Microtubules Ex- α-tubulin, β-tubulin Movers: Myosin Kinesin Dynenin Actin (MICROFILAMENTS) Microfilaments- the thinnest class of the cytoskeletal fibers, are solid rods of the globular protein known as actin. An actin microfilament consists of a twisted double chain of actin subunits. Microfilaments are designed to resist tension. With other proteins, they form a three-dimensional network just inside the plasma membrane. Actin cytoskeleton of mouse embryo fibroblasts, stained with phalloidin. Actin Filaments Actin was first isolated from muscle cells in 1942, Constitutes approximately 20% of total cell protein of muscle cell Extremely abundant protein (5 to 10% of total protein) in all types of eukaryotic cells. Monomers of Actin protein 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. First step in actin polymerization (nucleation) is the formation of a small aggregate consisting of three actin monomers. Actin polymerization is reversible, filaments can depolymerize by the dissociation of actin subunits, allowing actin filaments to be broken down when necessary Depending on Amino acid sequence there are different types of actin Examples are α actin (muscle cells), β actin and γ actin (non muscle cells) Assembly of Actin filaments barbed + end Fast growing G-actin Dimer Trimer Nucleation- It is the first step in actin polymerization. This step start the formation of complex of three actin monomers known as an actin nucleus, from which an actin filament may elongate. Pointed (-)end Slow growing Assembly of Actin filaments barbed + end Fast growing G-actin Dimer Trimer ADP ATP Pointed (-)end Slow growing Treadmilling in the ATP polymerization A treadmilling state is occurs when the concentration of G-actin monomers is lower at the minus end, causing disassembly, and higher at the plus end, leading to polymerization. Cofilin binds to actin filaments and increases the rate of dissociation of actin monomers (bound to ADP) from the minus end. Cofilin remains bound to the ADP-actin monomers, preventing their reassembly into filaments. Another protein profilin can stimulate the exchange of bound ADP for ATP, resulting in the formation of ATP-actin monomers that can be repolymerized into filaments, including new filaments nucleated by the Arp2/3 proteins. Assembly of Actin filaments barbed end Pointed end Actin filaments can be stabilized by actin binding proteins Role Examples Filament initiation Arp2/3, formin Stabilization Nebulin, tropomysin Cross Linking α-actinin, filamin, fascin, villin End Capping CapZ, tropomodulin, Severing ADF/cofilin, gelsolin, thymosin Monomer binding Profilin, twinfilin Actin filament linkage α-catenin, dystrophin, spectrin, talin, viculin Overview of the main families of actin-binding proteins. Actin-binding proteins are divided into seven groups according to their specific functions on actin polymers and/or monomers. They can (1) promote actin nucleation; (2) regulate G-actin polymerization/F-actin depolymerization; (3) cap or sever F-actin filaments; (4) bundle or crosslink F- actin filaments; (5) anchor F-actin filaments to the plasma membrane; (6) generate force; (7) stabilize F-actin filaments. Representative proteins are indicated for each group Comparison of the mechanism of Actin binding proteins Spontaneous actin assembly from purified monomers is shown for comparison. Note that actin dimers and trimers are highly unstable species that rapidly dissociate. The three known cellular nucleators of actin assembly each overcome these kinetic barriers by a different mechanism. Formins stabilize actin polymerization intermediates, likely short-pitch dimers. The Arp2/3 complex, associated with WASp actin, is thought to mimic an actin trimer. Spire recruits and organizes up to four actin monomers into a stable prenucleation complex. In each panel, the dotted line (with arrow ) points to the barbed end of the polymerized filament. The two halves of the formin FH2 dimer are green, connected by flexible linkers ( black ). The two actin-like subunits of Arp2/3 complex (Arp2 and Arp3) are pink. The four WH2 domains of Spire ( purple ) each are capable of binding one actin monomer. Organization of Actin Filaments Formation of these structures is governed by a variety of actin-binding proteins which contain at least two domains In bundles- (actin-bundling proteins) usually are small rigid proteins In networks- tend to be large flexible proteins nature of the association between these filaments is then determined by the size and shape of the cross-linking proteins parallel arrays orthogonal arrays Functions of Actin Filaments 1. To form the dynamic cytoskeleton, which gives structural support to cells and links the interior of the cell with its surroundings. Forces acting on the actin cytoskeleton are translated and transmitted by signaling pathways to convey information about the external environment. 2. To allow cell motility. For example, through the formation and function of Filopodia or Lamellipodia. 3. During mitosis, intracellular organelles are transported by motor proteins to the daughter cells along actin cables 4. In muscle cells, actin filaments are aligned and myosin proteins generate forces on the filaments to support muscle contraction. These complexes are known as ‘thin filaments’. 5. In non-muscle cells, actin filaments form a track system for cargo transport that is powered by non- conventional myosins such as myosin V and VI. Non-conventional myosins use the energy from ATP hydrolysis to transport cargo (such as vesicles and organelles) at rates much faster than diffusion. Actin and Myosin 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. Functions- phagocytosis and cell movement muscle contraction Globular head cell division Essential light chain Regulatory light chain -Myosin are motor proteins and contains a globular head and tail Heavy chains Coil of two α helical chain (tail) -Myosin head have ATPase activity, they can hydrolyze ATP -Upon ATP hydrolysis the ADP+Pi are produced resulting in conformational change in the myosin head, which causes the sliding of the actin filament over myosin filament. Muscle contraction Muscle fibre-approximately 50 m in diameter and up to several centimeters in length cytoplasm consists of myofibrils, which are cylindrical bundles of two types of filaments: -thick filaments of myosin (about 15 nm in diameter) - -thin filaments of actin (about 7 nm in diameter) Each myofibril is organized as a chain of contractile units called sarcomeres (2.3 µm long) which are responsible for the striated appearance of skeletal and cardiac muscle Actin -Myosin are motor proteins and contains a head like structure -Myosin head have ATPase activity, they can hydrolyze ATP -Upon ATP hydrolysis the ADP+Pi are produced resulting in conformational change in the myosin head, which causes the sliding of the actin filament over myosin filament. Myosin Contractile assemblies in nonmuscle cells Toward the end of mitosis in yeast and animal cells, a contractile ring consisting of actin filaments and myosin II assembles just underneath the plasma membrane, to facilitate cell division. Bipolar filaments of myosin II produce contraction by sliding actin filaments in opposite directions. Pseudopodia, lamellipodia- Actin filaments Filopodia or microspikes- Actin bundles Actin filaments supports a variety of cell structure Scientist working on Actin filaments  Microtubules Long, hollow cylinders, 25 nm in diameter, made of tubulin. The basic subunit is a heterodimer of α and β tubulin (55 kda) There are 13 protofilaments in a typical cylinder arranged parallelly in helical manner. There is a + end, fast growing and slow growing –ve end. The GTP’s are important in assembly They have MAPs, that help in linking them together, stabilizing them, or 25 nm 14 nm destabilizing them. They form a network, coming from the microtubule organizing centre (MTOC), known as centrosome, centrosomes determine the position of the nucleus Also form cilia and flagella, and spindle fibers in mitosis Functions: determine cell shape, variety of cell movements( cell locomotion and intracellular transport of organelles), separation of chromosomes during mitosis Assembly of Microtubules The role of GTP in microtubule polymerization. Tubulin dimers bound with GTP (β-tubulin) associate with the growing plus ends in a flat sheet manner first, which then zips up into the mature microtubule. Shortly after polymerization the GTP bound to β-tubulin is hydrolyzed to GDP. Since GDP-bound tubulin is less stable in the microtubule, the dimers at the minus end rapidly dissociate. Color coding-Green=alpha tubulin, blue=beta tubulin-GTP, purple=beta tubulin-GDP Microtubule Associated proteins Front. Pharmacol., 14 September 2022 Sec. Pharmacology of Anti-Cancer Drugs Volume 13 - 2022 | https://doi.org/10.3389/fphar.2022.935493  Microtubule motor proteins:Kinesin and Dynein Kinesin and Dynein move in opposite directions along microtubules, Kinesin moves toward the plus and Dynein moves toward minus ends of the microtubule. Kinesin Dynein Kinesin – 380kd, two heavy chains (120kd) and two light chains (64kd) The globular head domains of the heavy chains bind microtubules and are the motor domains of the molecule. Dynein – 2000kd, two or three heavy chains (500kd)in association with multiple light and intermediate chains(14- 120kd) The globular head domains of the heavy chains are the motor domains. Motor protein helps in Cargo transport and intercellular organization Transport macromolecules, membrane vesicles, and organelles through the cytoplasm of eukaryotic cells In Nerve cells- Kinesin transport the secretory vesicles containing neurotransmitters are carried from the Golgi apparatus In nerves cells dynein transports endocytic vesicles from the axon back to the cell body in reverse directions Kinesin I and other plus end-directed members of the kinesin family carry their cargo toward the cell periphery Cytoplasmic dyneins and minus end-directed members of the kinesin family transport materials toward the center of the cell. Microtubules and associated motor proteins position membrane- enclosed organelles (such as the ER, Golgi apparatus, lysosomes, and mitochondria) within the cell. Centrosomes Centrosomes are key organelles involved in cell division. Found near the nucleus in animal cells. Play a crucial role in organizing microtubules. Centrioles: Centrosomes contains a pair of cylindrical structures called centrioles, arranged 90 degree to each other. They are composed of microtubules arranged in a 9+0 pattern. Centrosome Function Microtubule Organization: Centrosomes act as the main microtubule organizing centers (MTOCs). Regulate the organization and stability of the microtubule network. Cell Division: Organize spindle fibers during mitosis and meiosis. Ensure accurate chromosome separation. Centriole collections of microtubules (9 triplets) found in pairs(1 pair = centrosome) Separate chromosomes Organization of Microtubule in Cilia and Flagella Microtubules are the central structural supports in cilia and flagella. Fundamental structure of both cilia and flagella is the axoneme Both can move unicellular and small multicellular organisms by propelling water past the organism. If these are anchored in a large structure, they move fluid over a surface. Cilia For example, cilia can sweep mucus trapped from the lungs. Cilia - occur in large numbers on the cell surface about 0.25 m in diameter and 2-20 m long Flagella- just one or a few flagella per cell Flagella are the same width as cilia, but 10-200 m long Flagella Structure of Cilia and Flagella Cilia and flagella are hair-like structures on the surface of many eukaryotic cells, and they are crucial for movement and fluid transport. Both cilia and flagella have a core structure known as the axoneme, which is composed of microtubules. The primary structural components include: Basal Body and Axoneme Basal Body also known as Kinetosome The base of the cilium or flagellum is similar to a centriole the basal body, which anchors the axoneme to the cell. It is structurally, consisting of a cylindrical arrangement of microtubules. Axoneme: The axoneme is organized into a "9+2" arrangement. This means there are nine pairs of microtubules (known as doublets) arranged in a circle around a central pair of single microtubules. Dynein Arms: Attached to the outer doublets are dynein arms, which are motor proteins that use ATP to produce movement. They create sliding forces between the microtubules. Structure of Cilia and Flagella Cilia and flagella are hair-like structures on the surface of many eukaryotic cells, and they are crucial for movement and fluid transport. Both cilia and flagella have a core structure known as the axoneme, which is composed of microtubules. The primary structural components include: Basal Body and Axoneme Basal Body also known as Kinetosome The base of the cilium or flagellum is similar to a centriole the basal body, which anchors the axoneme to the cell. It is structurally, consisting of a cylindrical arrangement of microtubules. Axoneme: The axoneme is organized into a "9+2" arrangement. This means there are nine pairs of microtubules (known as doublets) arranged in a circle around a central pair of single microtubules. Dynein Arms: Attached to the outer doublets are dynein arms, which are motor proteins that use ATP to produce movement. They create sliding forces between the microtubules.  The bending of cilia and flagella is driven by the arms of a motor protein, dynein. Addition phosphate group to dynein and its removal causes conformation changes in the protein. Dynein arms alternately grab, move, and release the outer microtubules. Formation of the mitotic spindle Interphase- centrioles and centrosomes duplicate Prophase- duplicated centrosomes separate and move to opposite sides of the nucleus Microtubules reorganize to form the mitotic spindle Metaphase- the condensed chromosomes aligned at the center of the spindle Activity 2: Study about the various drugs and chemicals that inhibit the mitotic spindle formation. Intermediate Filaments Intermediate filaments (IFs) are a crucial component of the cytoskeleton in eukaryotic cells, playing a key role in maintaining cell shape, providing mechanical support, and stabilizing cell structure. Size at 8 - 12 nm, specialized for bearing tension They are composed of various proteins, depending on the cell type and tissue mainly from a family of proteins called keratins These proteins form fibrous, rope-like structures that are more stable and less dynamic compared to microtubules and actin filaments. Shape of the microvilli in this Not directly involved in cell movement intestinal cell are supported by microfilaments, anchored to a network of intermediate filaments.  Assembly of intermediate filaments Central Rod Domain (α helix 310-350 amino acids) Monomers: Intermediate filament proteins are elongated, helical monomers that have a central alpha- helical rod domain. Dimer Formation: Two monomers form a coiled-coil dimer through interactions between their helical regions. Tetramer Formation: Two dimers associate antiparallel to form a tetramer, which is a basic building block of intermediate filaments. Assembly: Tetramers align and assemble into protofilaments, which then combine to form mature intermediate filaments. These filaments are more stable Protofilaments and less dynamic than microtubules or actin filaments. Intermediate filaments Examples of Intermediate filaments Keratin: Found in epithelial cells, including skin, hair, and nails. There are two main types: Type I (acidic keratins) and Type II (basic or neutral keratins). Vimentin: Found in mesenchymal cells, such as fibroblasts, and plays a role in supporting cell shape and integrity. Desmin: These are located in muscle cells, where it links myofibrils to the cell membrane and helps maintain muscle fiber integrity. Neurofilaments: These are found in neurons, where they support the structure of the axon and maintain its diameter. Lamins: Found in the nuclear lamina, a network of intermediate filaments located beneath the inner nuclear membrane, providing structural support to the nucleus. Intermediate filament associated proteins IFAPs: Examples Plectin: It is a linker protein protein that connects intermediate filaments to microtubules, actin filaments, and cell-matrix junctions. It plays a role in maintaining cell integrity and stability. Desmoplakin: It is a part of the desmosome complex, it connects intermediate filaments to cell- cell adhesion structures, crucial for tissue integrity, especially in epithelial cells. BPAG1 (Bullous Pemphigoid Antigen 1): Important in linking intermediate filaments to hemidesmosomes, contributing to the adhesion between the epidermis and dermis. Nestin: An IFAP associated with intermediate filaments in neural stem cells, playing a role in dynamic reorganization of the cytoskeleton during development.