Cell Biology: Cytoskeleton - Detailed Lecture Notes

INTERMEDIATE FILAMENTS

• Structure and Properties:
  • Strong and ropelike.
  • Provide mechanical strength to cells.
• Monomer Structure:
  • Central α-helical region with an NH2 terminus and a COOH terminus.
• Dimer Formation:
  • Two monomers form a coiled-coil dimer.
  • The dimer is approximately 48 nm in length.
• Tetramer Formation:
  • Two coiled-coil dimers associate in a staggered antiparallel manner to form a tetramer.
• Filament Assembly:
  • Tetramers associate laterally via noncovalent interactions.
  • Eight tetramers form a lateral array.
  • Tetramer bundles add to either end of the intermediate filament.
• Function:
  • Strengthen cells against mechanical stress.
• Types and Location:
  • Cytoplasmic:
    • Keratin filaments in epithelial cells.
    • Vimentin and vimentin-related filaments in connective-tissue cells, muscle cells, and glial cells.
    • Neurofilaments in nerve cells.
  • Nuclear:
    • Nuclear lamins in all animal cells.
• Nuclear Lamina Support:
  • The nuclear envelope is supported by a meshwork of intermediate filaments called nuclear lamins.
  • The nuclear lamina is located on the inner surface of the nuclear envelope.
• Linker Proteins:
  • Connect cytoskeletal filaments and bridge the nuclear envelope.
• Plectin:
  • Aids in the bundling of intermediate filaments.
  • Links intermediate filaments to other cytoskeletal protein networks.
  • Plectin is a key linker protein.
• Nuclear Envelope Structure:
  • The nuclear envelope consists of the outer and inner nuclear membranes, separated by the perinuclear space.
  • KASH-domain proteins and SUN-domain proteins are involved in linking the nuclear lamina to the cytoskeleton.

MICROTUBULES

• Structure:
  • Hollow cylinders made of tubulin protein.
  • Long and straight.
  • Typically have one end attached to a microtubule-organizing center called a centrosome.
  • Outer diameter of approximately 25 nm.
  • More rigid than actin filaments or intermediate filaments.
  • Rupture when stretched.
• Organizing Centers:
  • Centrosomes form poles of the mitotic spindle.
  • Basal bodies are associated with cilia.
• Structural Features:
  • Microtubules are hollow tubes with structurally distinct ends (plus and minus ends).
  • Tubulin dimer (=microtubule subunit).
  • Protofilament.
  • Lumen.
• Growth from Organizing Centers:
  • Microtubules grow from specialized microtubule-organizing centers.
  • Nucleating sites (γ-tubulin ring complexes) in the centrosome matrix.
  • Microtubules grow at their plus ends from γ-tubulin ring complexes of the centrosome.
• Dynamic Instability:
  • Microtubules display dynamic instability, growing and shrinking independently of their neighbors.
  • GTP hydrolysis controls the dynamic instability of microtubules.
• GTP Hydrolysis Mechanism:
  • Tubulin dimers with bound GTP (GTP-tubulin) add to the growing end of the microtubule.
  • If the addition of new GTP-tubulin dimers proceeds faster than GTP hydrolysis by the dimers, a GTP cap is formed.
  • If GTP hydrolysis is faster than the addition of new GTP-tubulin dimers, the GTP cap is lost, and protofilaments containing GDP-tubulin peel away from the microtubule wall.
  • GDP-tubulin is released to the cytosol.
• Organization of Cell Interior:
  • Microtubules organize the cell interior.
  • In nerve cells, microtubules transport materials to and from the cell body and axon terminal.
  • Outward transport is directed to the axon terminal, while backward transport goes to the cell body.
• Regulation by Binding Proteins:
  • Microtubule-binding proteins regulate microtubule dynamics and organization.
  • Examples include nucleating proteins (γ-tubulin ring complex), catastrophe-inducing motor proteins (e.g., kinesin-13), severing proteins (katanin), branching proteins (e.g., augmin), plus-end linking proteins, stabilizing proteins, and polymerizing proteins.
• Drugs Affecting Microtubules:
  • Taxol: Binds to filaments and prevents depolymerization.
  • Colchicine, colcemid: Forms a complex with tubulin dimers, preventing further polymerization.
  • Nocodazole: Binds tubulin dimers and prevents their polymerization.
• Motor Proteins:
  • Microtubule-associated motor proteins drive intracellular transport.
  • Examples include kinesin and cytoplasmic dynein.
  • Kinesins move towards the plus end of microtubules, while dyneins move towards the minus end.
• Motor Protein Mechanism:
  • ATP hydrolysis loosens the attachment of the head to the microtubule.
  • ADP release and ATP binding change the conformation of the head, which pulls the other head forward.
• Organelle Positioning:
  • Microtubules and motor proteins position organelles in the cytoplasm.
  • Examples include the endoplasmic reticulum and Golgi apparatus.
• Cilia and Flagella:
  • Cilia and flagella contain stable microtubules moved by dynein.
  • Microtubules in a cilium or flagellum are arranged in a "9 + 2" array.
  • Components include outer dynein arms, radial spokes, inner sheaths, central singlet microtubules, plasma membrane, A and B microtubules, outer doublet microtubules, inner dynein arms, and linking proteins.
• Dynein and Microtubule Movement:
  • In isolated doublet microtubules, dynein produces microtubule sliding.
  • In a normal flagellum, dynein causes microtubule bending.

ACTIN FILAMENTS

• Structure:
  • Thin and flexible helical polymers of the protein actin.
  • Diameter of about 7 nm.
  • Organized into linear bundles, two-dimensional networks, and three-dimensional gels.
  • Concentrated in the cortex, the layer of cytoplasm just beneath the plasma membrane.
• Polymerization:
  • Actin and tubulin polymerize by similar mechanisms.
  • Actin with bound ATP adds to the plus end of the filament, while actin with bound ADP is found at the minus end.
  • Treadmilling occurs as actin monomers are added to the plus end and removed from the minus end.
• Drugs Affecting Actin Filaments:
  • Phalloidin: Binds to filaments and prevents depolymerization.
  • Cytochalasin: Caps filament plus ends, preventing polymerization and leading to filament depolymerization at minus ends.
  • Latrunculin: Binds actin monomers and prevents their polymerization.
• Regulation by Binding Proteins:
  • Many proteins bind to actin and modify its properties.
  • Examples include severing proteins, cross-linking proteins, nucleating proteins (e.g., formin, ARP complex), monomer-sequestering proteins, bundling proteins (in filopodia), side-binding proteins (e.g., tropomyosin), capping (plus-end-blocking) proteins, and myosin motor proteins.
• Myosins:
  • Actin-binding motor proteins.
  • Myosin I has a head domain and a tail domain and can move vesicles along actin filaments.
• Cell Cortex and Crawling:
  • A cortex rich in actin filaments underlies the plasma membrane of most eukaryotic cells.
  • Cell crawling depends on cortical actin.
  • Actin polymerization at the plus end protrudes the lamellipodium.
  • Myosin motor proteins slide along actin filaments, causing contraction.
  • Focal contacts (containing integrins) attach the cell to the substratum.
• Protrusions:
  • Actin-binding proteins influence the type of protrusions formed at the leading edge.
  • Examples include filopodia and lamellipodia.
• Extracellular Signals:
  • Extracellular signals can alter the arrangement of actin filaments.
  • Rho, Rac, and Cdc42 activation can lead to different arrangements of actin filaments.

MUSCLE CONTRACTION

• Mechanism:
  • Muscle contraction depends on interacting filaments of actin and myosin.
  • Actin filaments slide against myosin filaments during muscle contraction.
• Myosin II:
  • Myosin II molecules have a head and tail.
  • Myosin II filaments have a bare region (myosin tails only) and myosin heads.
• Sarcomere Structure:
  • Each myofibril consists of a repeating chain of sarcomeres, the contractile units of the myofibrils.
  • The sarcomere is approximately 2.5 \mu m in length.
  • Sarcomeres contain thin filaments (actin) and thick filaments (myosin II).
  • The Z disc defines the boundaries of the sarcomere.
• Contraction Process:
  • During contraction, the actin filaments slide past the myosin filaments, shortening the sarcomere.
• Calcium Regulation:
  • Muscle contraction is triggered by a sudden rise in cytosolic Ca^{2+}.
• T-tubules and Sarcoplasmic Reticulum:
  • Action potentials trigger the release of Ca^{2+} from the sarcoplasmic reticulum via voltage-gated Ca^{2+} channels and Ca^{2+} release channels.
• Troponin and Tropomyosin:
  • In the absence of Ca^{2+}, tropomyosin blocks the myosin-binding site on actin.
  • In the presence of Ca^{2+}, the troponin complex moves tropomyosin, exposing the myosin-binding site, allowing contraction to occur.