The stability and arrangement of actin filaments as well as their properties and functions depend on which of the following? a. The structure of the actin filaments b. Microtubules c. Intermediate filament proteins d. Actin-binding proteins e. Motor molecules, such as kinesin

Microfilaments 7 nm filaments composed of actin Dense network of microfilaments beneath the plasma membrane are critical components of the cell cortex Involved with locomotion cytokinesis Cell5e-Fig-12-02-0.jpg

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. ADP-actin dissociates from filaments more readily than ATP-actin, so the critical concentration of actin monomers is higher for addition to the pointed end than to the barbed end of actin filaments. Treadmilling takes place at intermediate concentrations of monomers.

Treadmilling also occurs with microtubules Incorporation at plus end equals disassociation at minus end

Drugs that 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 polymerization and depolymerization.

Actin-Binding Proteins Assembly and disassembly of actin filaments within a cell is regulated by a diverse group of actin-binding proteins that act in diverse ways. Some actin-binding proteins bind along the length of actin filaments, stabilizing them or cross-linking them to one another. Others stablize by capping the ends and preventing dissociation. Others promote dissociation, while others regulate the exchange of ATP for ADP.

Actin bundles and networks Actin filaments are organized into: Actin bundles—actin filaments are cross-linked into closely packed parallel arrays. Actin networks—actin filaments are cross-linked in arrays that form 3-D meshworks with the properties of semisolid gels. Cross-linking proteins (actin-bundling proteins) are small rigid proteins with at least two domains that bind actin and align the filaments closely with one another. Proteins that organize actin filaments into networks tend to be large flexible proteins that can cross-link perpendicular filaments. Cell5e-Fig-12-09-0.jpg

Two types of Actin-bundling proteins 1. Parallel bundles—closely spaced actin filaments aligned in parallel, all with the same polarity, with barbed ends adjacent to the plasma membrane. Fimbrin is one protein in parallel bundles, and was first isolated from intestinal microvilli. 2. Contractile bundles—more widely-spaced filaments, cross-linked by α-actinin. Increased spacing between filaments allows the motor protein myosin to interact with the actin filaments. Cell5e-Fig-12-10-0.jpg Figure 16-51 Molecular Biology of the Cell (© Garland Science 2008)

Actin networks and filamin 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. 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. Cell5e-Fig-12-11-0.jpg Figure 16-49a Molecular Biology of the Cell (© Garland Science 2008)

Protein Functions Tropomyosin Stabilizes filaments Fimbrin Bundles filaments α-Actinin Filamin Cross-links filaments Spectrin I/II Cross-links filaments in membrane skeleton Gelsolin Fragments filament Myosin II Slides filaments in muscle Myosin I Moves vesicles on filaments Cap Z Caps plus ends of filaments Profilin Binds actin monomers

Spectrin The major structural protein is spectrin, a member of the calponin family of actin-binding proteins. It is a tetramer of two polypeptide chains, α and β. The ends of the spectrin tetramers associate with short actin filaments, resulting in the spectrin-actin network.

Other types of cells have similar linking proteins. Dystrophin, a calponin, links actin filaments to transmembrane proteins of muscle cell plasma membranes. The transmembrane proteins link to the extracellular matrix, which helps maintain cell stability during muscle contraction.

Duchenne and Becker Muscular Dystrophies Muscular dystrophy, an X-linked inherited disease, results in progressive degeneration of skeletal muscle. Dystrophin is absent in patients with Duchenne’s muscular dystrophy Dystrophin is abnormal (usually truncated) in patients with Becker’s muscular dystrophy.

Duchenne Muscular Dystrophy DMD: Clinical features Age of onset: male children at 3-5 years of age (I = 1/3500 ) Slowly progressive muscle weakness, resulting in awkward gait,inability to run quickly, and inability to climb stairs Pseudohypertrophy of calf increase in size of the calves calf muscles replaced by fat and fibrous connective tissue Pseudohypertrophy of Calf

DMD: Clinical features (Contd.) ● - Difficulty rising from the ground; use ‘Gower maneuver’ - rise by pushing on the ground with hands, and supporting the knee and the thigh - Affected boys wheel chair bound by age 11 - due to severe proximal muscle weakness - Subsequent deterioration lead to lumber lardosis, joint contracture - Death by age 18 - due to cardiorespiratory failure - Moderate intellectual compromise (IQ ~ 83) - Elevated serum creatine kinase (CK) level

Because of weakened leg muscles, boys with DMD have a distinctive way of rising from the floor, called the Gowers' maneuver. They first get on hands and knees, then elevate the posterior, then "walk" their hands up the legs to raise their upper body.

Dystrophin in Sarcolemmal complex Dystrophin is a membrane-associated intracellular protein (427 kDa), expressed predominantly in skeletal, smooth and cardiac muscles; also, in some brain neurons (hence, ↓ IQ)

Becker Muscular Dystrophy Clinical features very similar to DMD, but the disease process runs a much less aggressive course Mean age of onset 11 years Many patients remain ambulant until well into adult life Overall life expectancy slightly reduced Some form of dystrophin (usually truncated) is produced Truncated dystrophin-( shortened during protein synthesis)

Attachment of actin filaments to adherens junctions In sheets of epithelial cells, cell-cell contacts (adherens junctions) form a continuous beltlike structure (adhesion belt) around each cell. Contact is mediated by transmembrane proteins called cadherins. They bind to cytoplasmic catenins, which anchor actin filaments to the plasma membrane. Cell5e-Fig-12-17-0.jpg

a. Fibrous stromal connective tissue b. Parasympathetic ganglia A patient is diagnosed with a pleomorphic adenoma of the submandibular gland. The pathologist uses anti-vimentin antibodies with immunocytochemistry to stain the biopsy tissue. One would expect to find vimentin staining in which of the following structures? a. Fibrous stromal connective tissue b. Parasympathetic ganglia c. Serous acini d. Mucous acini e. Striated ducts A

Intermediate Filaments Intermediate filaments have diameters intermediate between actin filaments and microtubules. They are not directly involved in cell movements, but provide mechanical strength and a scaffold for localization of cellular processes. Intermediate filament proteins are tissue-specific!! Intermediate filament typing as a diagnostic tool for cancer Keratin filaments in epithelial cells

INTERMEDIATE FILAMENTS Tonofilaments Epithelium Keratinizing and (cytokeratins) non-keratinizing epithelia Vimentin Mesenchymal Fibroblasts, chondroblasts, cells osteoblasts, macrophages, endothelial cells, vascular smooth muscle cells Desmin Muscle Striated and smooth muscle Neurofilaments Neurons Most neurons Glial filaments Glial cells Astrocytes (GFAP)

Microtubules Two types of microtubules are responsible for many functions in the cell Axonemal microtubules (Found in cilia, flagella and basal bodies) Cytoplasmic microtubules (More loosely organized) Microtubules are composed of a-tubulin and b-tubulin heterodimers

The GTP cap and its role in the dynamic instability of microtubules

Microtubules in vivo MTs originate from microtubule-organizing centers (MTOC) within the cell Centrosomes: composed of 2 centrioles Pericentriolar material The role of g-tubulin in initiation Figure 16-31a Molecular Biology of the Cell (© Garland Science 2008) Figure 16-31b Molecular Biology of the Cell (© Garland Science 2008)

Centrosomes MTOCs ( microtubule organinizing centre) organize and polarize the microtubules within cells Minus ends are anchored in MTOCs Each MTOC has a limited # of nucleation and anchorage sites Fluctuation of pericentrin during cell cycle- highest at prophase and metaphase of mitosis Figure 16-30a Molecular Biology of the Cell (© Garland Sience 2008) Figure 16-30b Molecular Biology of the Cell (© Garland Science 2008) Figure 16-29 Molecular Biology of the Cell (© Garland Science 2008)

Structure of a centriole Centrioles have various satellites and appendages that are thought to interact with proteins in the centrosome matrix. Triplet microtubules contain modified α- and β-tubulins and the unique δ-tubulin. Centrin fibers extend out from the triplet microtubules and connect to the other centriole. Cell5e-Fig-12-48-0.jpg

Microtubules in vivo-continued Microtubule stability within cells is highly regulated Kinetochores stabilize MTs Microtubules are regulated by microtubule-associated proteins (MAPs) Motor MAPs (kinesin and dynein) Figure 16-85c Molecular Biology of the Cell (© Garland Science 2008) Microtubules in green Kinetochores in red Chromosomes in blue Figure 16-85a Molecular Biology of the Cell (© Garland Science 2008)

Drugs can affect the assembly of microtubules Colchicine, vinblastine, vincristine and nocodazol: inhibits MT growth Very important drugs For example: Taxol: stabilizes MTs used in breast cancer therapy (B) Microtubules Untreated (C) + Taxol Table 16-2 Molecular Biology of the Cell (© Garland Science 2008) Figure 16-23b,c Molecular Biology of the Cell (© Garland Science 2008)

Kinesin and Dynein Microtubule-associated motor proteins (motor MAPs) Two large families of motor proteins (kinesins and dyneins) are responsible for powering the movements in which microtubules participate. Most kinesins move along microtubules toward the plus end Dyneins move toward the minus end.

Kinesins Kinesins move along microtubules by hydrolyzing ATP Kinesins move to the “+” end of the microtubles Kinesins are a large family of proteins with varying structures and functions

Colchicine inhibits cell division by which of the following processes? Inhibiting DNA synthesis in mitochondria Preventing assembly of the mitotic apparatus Stabilizing microtubules into a rigid structure Preventing cytokinesis by inhibiting membrane flow Inhibiting the ATPase activity of G-actin

ANSWER-B The mitotic spindle does not assemble in the presence of colchicine. Colchicine inhibits microtubule assembly by binding to tubulin monomers and preventing polymerization and promoting disassembly of existing microtubule.

Spectrin filaments in erythrocytes Mitotic spindles of dividing cells Tubulin polymerizes into microtubules that constitute which of the following cellular elements? Cores of microvilli Spectrin filaments in erythrocytes Mitotic spindles of dividing cells Glial filaments in astrocytes Neurofilaments of axons and dendrites ANSWER= C

Attachment of actin microfilaments to the membrane of the cilium The beating of cilia and flagella is dependent upon which of the following processes? Polymerization and depolymerization of microtubules at the tip of the cilium Attachment of actin microfilaments to the membrane of the cilium Attachment and release of dynein side arms between adjacent microtubules of each doublets Attachment and release of doublet microtubules from the central pair of single microtubules Attachment and release of microtubule-associated proteins ( MAP) by doublet microtubules. C

ANSWER-C In cilia and flagella, the doublet microtubules in outer ring are associated with adjacent doublets by dynein side arms. The hydrolysis of adenosine triphosphate (APT) by the dyneins produce a sliding force between the doublets, producing motility in the form of beating.