Exam 3

• Three Types of Filaments in the Cytoskeleton

  • Ropelike fibers (flexible), made of different filament proteins
  • Thick and hollow cylinder (rigid), made of tubulin
  • Thin and flexible, made of actin

  
• Ropelike fibers


  • Provides mechanical strength to cells by interacting with many different proteins
  • Some filaments line inner face of nuclear membrane creating nuclear lamina
  • Used for intracellular transport and forms the mitotic spindle that segregates chromosomes

  
• Microtubules: Thick and hollow cylinder

  • They also form cilia and flagella (cell motility)
  • Determines cell shape; used for cell motility and muscle contraction
  • Highly concentrated just beneath plasma membrane

  
• Actin: Thin and flexible

  Made of actin

  
• Tubulin


  • Heterodimer made up of two different proteins
  • Tubulin dimer is stable; ⍺ and β subunits bind after translation and stay bound until degradation
  • β subunit can bind GTP and perform hydrolysis (GTP to GDP)
  • ⍺ subunit can bind GTP but cannot perform hydrolysis

  
• Growing microtubules


  1. Have GTP cap: addition of dimers with GTP occurs faster than GTP hydrolysis in β subunit

  2. Concentration of dimers with GTP in β subunit determines rate of synthesis

  3. Eventually GTP at plus end is hydrolyzed; dimers with GDP in β subunit dissociate (hydrolysis reduces binding affinity)

  
• GTP-containing tubulin subunits


  Create straight protofilament, very stable

  
• GTP hydrolysis


  Changes shape of filament making it less stable before GTP-tubulin is added to end

  
• GDP-containing tubulin


  Is curved at end and depolymerizes from protofilament; GDP-GTP exchange on free tubulin dimer happens spontaneously

  
• Centrosome


  Microtubule-organizing center (MTOC) because it has g-tubulin ring complexes: nucleation site for growth of one microtubule

  
• 1. tubulin ring complex


  Nucleates microtubule because it mimics a plus end and provides a template for 13 protofilaments

  
• Actin filaments


  • Helical polymers with a plus end (fast growing) and a minus end (slow growing); made of two protofilaments
  • Actin subunits are asymmetrical monomers with a plus and minus end
  • Actin filaments are composed of protein subunits called actin

  
• Actin polymerization

  1. Actin monomer hydrolyzes bound ATP to ADP after incorporation into actin filament

  2. Hydrolysis of ATP to ADP decreases stability of actin filament and promotes depolymerization

  3. Actin polymerization is dependent on amount of actin monomers available

  4. Treadmilling involves simultaneous gain of monomers at plus end and loss of monomers at minus end

  5. Dynamic instability involves rapid switch from growth to shrinkage; occurs only at plus end

  
• Arp2/3 complex


  • Nucleates actin filament growth by aggregating actin monomers
  • Nucleation-promoting factor binds to Arp2/3 complex; activated by shape change
  • Active Arp2/3 binds to minus end, promotes elongation of plus end
  • Arp2/3 complex nucleates most efficiently when it binds to a preexisting filament and grows branch

  
• Actin polymerization by Arp2/3 complex

  Causes membrane protrusion; membrane protrusion is used for cell motility

  
• Muscle contraction

  1. Myosin without ATP or ADP is tightly bound to actin filament

  2. ATP binds to myosin and causes myosin to detach from actin

  3. Myosin hydrolyzes ATP which changes position of myosin (ADP and phosphate are still attached)

  4. Myosin binds to weakly at new site on actin

  5. ADP and phosphate are released and myosin regains original shape (power stroke); myosin is tightly bound to actin filament

  
• Cell cohesion


  Critical for body architecture in multicellular organisms
• Epithelial tissues


  Cells are tightly bound by cell-cell junctions with very little extracellular matrix
• Connective tissues


  Formed from an extracellular matrix that is secreted by sparsely distributed cells (very little cell-cell junctions)
• Cadherins


  • Transmembrane adhesion proteins that mediate cell-cell junctions
  • Typically use homophilic binding for cell-cell junctions
  • Some cadherins linked to actin filaments (adherens junction), some linked to intermediate filaments (desmosome)
  • Cadherins on two different cells use calcium and lateral interactions for adhesion
  • Different cell types express different types of cadherins during development

  
• Cadherins


  • Allow cells of same tissue to preferentially adhere to one another
  • Homophilic binding allows similar cell types to bind to each other and segregate from other cell types during development
  • Two different cell types express two different types of cadherin: E-cadherin and N-cadherin
  • Mix cells together and homophilic binding allows different cell types to form aggregates (tissues)
• Extracellular matrix


  Composed of three major classes of macromolecules: glycosaminoglycans (GAGs), fibrous proteins, and proteoglycans
• Cadherins


  Transmembrane adhesion proteins that mediate cell-cell junctions
• Cadherins


  • Use homophilic binding for cell-cell junctions
  • Some linked to actin filaments (adherens junction), some linked to intermediate filaments (desmosome)
  • Use calcium and lateral interactions for adhesion
  • Different cell types express different types during development

  
• Cell adhesion


  • Cadherins allow cells of same tissue to preferentially adhere to one another
  • Homophilic binding allows similar cell types to bind to each other and segregate from other cell types during development
  • Two different cell types express two different types of cadherin: E-cadherin and N-cadherin
  • Mix cells together and homophilic binding allows different cell types to form aggregates (tissues)
• Extracellular matrix components


  • Glycosaminoglycans (GAGs)
  • Fibrous proteins
  • Glycoproteins

  
• Glycosaminoglycans (GAGs)


  Repeating disaccharides secreted by cells; occupy large amounts of space; enable matrix to withstand compression (e.g., cartilage); GAG attached to protein is a proteoglycan

  
• Collagen


  • Fibrous proteins secreted by cells that strengthen and help organize the matrix
  • Molecules typically form a triple-stranded helical structure; major component of skin and bone
  • Can aggregate to form bundles which strengthens connective tissue in skin

  
• Glycoproteins


  Secreted proteins that organize matrix and help cells attach; fibronectin has binding sites for adhesion and matrix proteins

  
• Cell signaling


  1. Reception

  2. Transduction

  3. Response
• Ion-channel-coupled receptors


  Alter permeability of plasma membrane to specific ions, which alters membrane potential
• 1. protein-coupled receptors (GPCRs)

  Activate membrane-bound GTP-binding proteins which activate enzyme or ion channel in plasma membrane
• Enzyme-coupled receptors


  Act as enzymes or associate with enzymes inside cell when stimulated by a signaling molecule
• GPCRs


  • Transmembrane receptor coupled to a heterotrimeric GTP-binding protein with three subunits (α, β, and ϒ)
  • No signal molecule = inactive GPCR, α unit bound to GDP and attached to β and ϒ subunits
  • Signal molecule = active GPCR, α unit bound to GTP dissociates from β and ϒ subunits
  • Both activated parts of G protein can stimulate different intracellular signaling molecules to trigger a response
  • Bind to a variety of extracellular signals (ligands) such as hormones, neurotransmitters, or lipids

  
• Cyclic AMP (cAMP)


  Second messenger made from ATP by adenylyl cyclase and degraded by phosphodiesterase
• Protein kinase A (PKA)


  Intracellular signaling molecule activated by cAMP
• Extracellular signal binds to GPCR

  Activates adenylyl cyclase; increase in cAMP activates PKA

  
• PKA phosphorylates and activates CREB transcription factor

  CREB binds to CRE element to alter gene expression (altered expression is response from signal)

  
• Enzyme-coupled receptors


  • Unlike GPCRs, they are single-pass transmembrane proteins so they cannot change shapes when bound by a ligand
  • Phosphorylation by protein kinase assembles signaling complex after binding of signal molecule
  • Often have a protein kinase in the cytoplasmic domain
• Receptor tyrosine kinases (RTKs)


  • Enzyme-coupled receptors that phosphorylate a tyrosine amino acid
  • Human cells use about 60 different RTKs that recognize different signaling molecules
  • Signal molecule causes two RTKs to dimerize
  • Dimerization activates kinase domains, each cytoplasmic domain phosphorylates the other at tyrosine residues
  • Phosphorylated tyrosines generate docking sites for intracellular signaling molecules (aka second messengers)
  • Protein kinases typically phosphorylate one amino acid in polypeptide sequence