B266 Chap. 17

• Cytoskeleton


  Intricate network of protein filaments that extend throughout cytoplasm

  
• Cytoskeleton


  • The primary support of large cytoplasm in animal cells (lack cell wall)
  • Highly dynamic: constantly rearranging itself

  
• Cytoskeleton is composed of three (3) main filaments

  • Intermediate Filaments
  • Microtubules
  • Actin Filaments

  
• Intermediate Filaments


  • Strongest type of filament found in the cell
  • Help cell withstand mechanical stress caused by cell becoming deformed or twisted, cell division, found in muscle and epithelial cells where they distribute effects of locally applied forces
  • Form a network extending through cytoplasm surrounding the nucleus and extending to periphery and anchored to plasma membrane
  • Indirectly connected to neighboring cells through a structure called desmosome
  • Compose the nuclear lamina network inside nucleus

  
• Intermediate filaments


  Diameter of about 10 nm, which is intermediate between the diameters of actin (about 8 nm) and microtubules (about 25 nm)

  
• Intermediate filament structure


  1. Monomer consists of α-helical monomer with terminal domains

  2. Stable dimers form as a result of intertwining of single monomers in coiled-coil interactions

  3. Two dimers will then associate (in opposite directions) to form a staggered tetramer

  4. Association of multiple tetramers via non-covalent interactions forms elongated intermediate filaments
• Both ends of intermediate filaments are the same (equal polarity)
• Intermediate filaments (IFs) are not directly involved in cell movement
• Main function of intermediate filaments

  Enable cells to withstand the mechanical stress that occurs when cells are stretched
• Four classes of IFs

  • 3 in the cytoplasm, 1 in the nucleus
• Between all four classes there are nearly 50 different types of IFs
• Nuclear lamina


  Intermediate filaments underlying the inner face of the nuclear envelope form the nuclear lamina, a fibrous network that supports the nuclear membrane and provides attachment sites for the chromatin

  
• The nuclear lamina differs from the cytoplasmic IFs in structure since it forms a meshwork as opposed to a rope-like structure
• Nuclear lamina disassembly and reassembly

  1. Disassembles with each cell division when the nuclear envelope breaks down

  2. Reassembly is regulated by phosphorylation of the lamina to cause disassembly, and dephosphorylation to allow for its reformation
• Mutations in a nuclear lamin protein are associated with progeria (premature aging in children) leading to death at a very young age
• Microtubules


  • Rigid, hollow rods approximately 25 nm in diameter
  • Dynamic structures that continually undergo assembly and disassembly
• Main functions of microtubules


  • Separation of chromosomes during mitosis
  • Intracellular transport of membrane-bound vesicles and organelles
  • Cell movement

  
• Tubulin


  • Microtubules are composed of a single type of globular protein, called tubulin
  • Tubulin is a heterodimer consisting of 2 closely related proteins, α-tubulin and β-tubulin
  • These dimers stack together via α-tubulin - β-tubulin interactions (non-covalent) to form a protofilament
  • This gives the filament polarity: α-tubulin is exposed at one end (the minus end) and β-tubulin is exposed at the opposite end (the plus end)
  • The plus end is considered the growing end
  • This polarity plays a role in determining the direction of movement along MTs
  • Each tube consists of 13 such protofilaments that arrange to give microtubule structure

  
• Microtubule-Organizing Centers (MTOC)


  Microtubules grow from specialized MTOC

  
• Centrosome


  • Microtubule organizing center of the cell
  • Composed of two centrioles and pericentriolar material
  • γ-tubulin rings serve as nucleation site
  • α-tubulin-β-tubulin dimers add to each γ-tubulin ring complex in specific orientation
  • Minus end is embedded in centrosome; growth takes place at plus end and extends into cytoplasm

  
• Dynamic instability of microtubules


  • Microtubules are highly dynamic; able to switch between polymerization and depolymerization
  • This dynamic instability is a result of tubulin dimers ability to hydrolyze GTP
  • During polymerization (growth), a tubulin dimer has a bound GTP molecule bound to the β-tubulin
  • GTP is hydrolyzed to GDP shortly after the dimer is added to the growing microtubule
  • Resulting GDP remains bound to the β-tubulin
  • During rapid polymerization, tubulin dimer addition proceeds faster than GTP hydrolysis, resulting in a GTP cap at the growing end
  • At times, tubulin dimers may hydrolyze their GTP before addition of new dimers, leading to loss of the GTP cap and depolymerization of the microtubule

  
• Microtubule motor proteins


  • Movement of membrane-bound vesicles and organelles along microtubules is based on the action of motor proteins
  • Motor proteins utilize energy derived from ATP hydrolysis to travel steadily along the microtubule in a single direction
  • Kinesins move toward the plus (+) end of a microtubule (away from the centrosome)
  • Dyneins move toward the minus (-) end of a microtubule (toward the centrosome)

  
• Structure of microtubule motor proteins

  • Each motor protein is composed of a dimer with two identical subunits
  • Each dimer contains two globular heads which bind and hydrolyze ATP and interact with MT
  • The tail end of the proteins binds specific cargo protein
  • The hydrolysis of ATP to ADP causes several conformational changes which allow the proteins to "walk" along MT

  
• Actin filaments are highly concentrated at the periphery of the cell, where they form a three-dimensional network underlying the plasma membrane

  
• Functions of actin filaments


  • Provide mechanical support
  • Determine cell shape
  • Enable cells to migrate

  
• Actin filaments


  • Made of the protein actin
  • Actin exists as a globular monomer called G-actin and as a filamentous polymer called F-actin
  • Actin monomers (G-actin) polymerize to form actin filaments (F-actin)
  • Actin filaments 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 the plus (+) and minus (-) ends) are distinguishable from one another
  • Like microtubules, new monomers are added to the plus (+) end of the filament

  
• Actin microfilament assembly


  1. Nucleation is not restricted to a particular region of the cell, but is stabilized by a protein complex called Arp2/3

  2. Microfilaments then grow by the reversible addition of monomers to the nucleated microfilaments

  3. Actin microfilament growth requires ATP

  4. ATP-bound G-actin polymerizes faster and dissociates slower than the ADP-bound form

  5. ATP-actin monomers associate with the fast-growing plus (+) end

  6. The ATP bound to actin is hydrolyzed shortly after polymerization

  7. Hydrolysis of bound ATP to ADP in actin filaments reduces the strength of binding between monomers and decreases the stability of the polymer, leading to dissociation of ADP-actin from the slow-growing minus (-) end

  8. Release of ADP-actin stimulates the exchange of bound ADP for ATP, resulting in the formation of ATP-actin monomers that can be re-polymerized into filaments

  
• Actin-binding proteins


  • Formins: proteins that promote actin polymerization
  • Thymosin: protein that prevents polymerization
  • Cofilin: binds to actin filaments and increases the rate of dissociation of actin monomers (bound to ADP) from the minus (-) end
• Myosin motor proteins


  • All actin-dependent motor proteins belong to the myosin family
  • Myosins bind and hydrolyze ATP, which provides the energy for their movement along actin filaments from the minus (-) end of the filament toward the plus (+) end of the filament
  • Two subfamilies: Myosin I (found in all types of cells) and Myosin II (most abundant in muscle)
• Myosin I


  • A single molecule with one globular head and a tail that attaches to another molecule or organelle in the cell
  • The head domain interacts with actin filaments and has ATP-hydrolyzing motor activity that enables it to move along the filament
  • The tail varies between the different types of myosin I and determines what cell components will be moved along by the motor
  • Can move vesicles along actin filaments towards the plus end, and can also attach the plasma membrane to the cortical actin filament network, pulling the plasma membrane into a different shape

  
• Myosin II


  • A dimer with two globular heads and a tail that forms a coiled-coil structure
  • Myosin II dimers associate through their coiled-coil tails forming a myosin II filament
  • The head domain interacts with actin filaments and has ATP-hydrolyzing motor activity that enables it to move along the filament, causing actin to contract, which happens in muscle cells

  
• Skeletal muscle fibers


  • Multinucleated individual cells formed by fusion of separate smaller cells
  • ~50 nm in diameter
  • Majority of the cytoplasm is composed of myofibrils
• Myofibrils


  • Contractile elements of the muscle cell
  • Made up of structures called sarcomeres
  • Each sarcomere is a highly-organized assembly of actin microfilaments and myosin II filaments
  • The plus (+) end of the actin filament is attached to a structure called the Z disc

  
• Muscle contraction


  1. During contraction, the myosin filaments start moving towards the plus end, pulling the actin filaments closer together

  2. Contraction is triggered by a rise in intracellular Ca2+ produced by stimulation from an adjacent neuron

  3. The calcium is released from a specialized region of the ER in skeletal muscle called the sarcoplasmic reticulum