Lecture 24-28: Cytoskeleton

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  • Cytoskeleton (Lec. 24)
    • 3 types of filaments
      • Actin filament (Lec. 25)
        • Actin filament structure
          • Functions
            • ex. cell migration, muscle contraction, cell division; etc
            • Depend on accessory proteins, ex. cross-linking, nucleating, bundling, motor proteins
          • Highly conserved in genome (humans have 6 actin genes)
        • Cell migration
          • Cross-linking actin cortex around cell fix it to substratum (plasma membrane)
            • Function as communication across the cell (malfunctioning --> melanoma: no cell migration
            • Jelly-like, elastic meshwork
          • Lamellipodium protrusion formed by actin polymerisation at (+) end
            • Protrusion 1: nucleation of actin filaments: activated ARP complex bind to actin at leading edge of cell
              • Destabilise: ATP actin hydrolysis recycles old filaments, freeing ARP to move forward and grow
            • Protrusion 2: add actin monomers to grow
              • Filopodia as pathfinders/ stabilisers in lamellipodia extension
              • Formin dimer protein promotes monomer addition
                • Similar to self-assembling crane or polymerase
          • Myosin contractile forces move the cell body forward
            • Use ATP hydrolysis energy to move forward/ put force on actin strands
              • 1) Tight bind between myosin-ATP actin
              • 2) Reduced affinity when myosin binds ATP
              • 3) myosin hydrolyzes to ADP/Pi, conformational change binds next actin
              • 4) Release of ADP/Pi returns myosin to tight bind w/ next actin
            • Myosin-I: 70nm: hydrolyses ATP in the (-)--> (+) direction (1-D movement)
            • Myosin-II forms dimers which squeeze the actin filament (2-D movement)
        • Actin in muscle
          • Muscle fiber made of myofibrils made of sarcomeres
            • Sarcomeres are myosin-II dimers assembled into bipolar thick filaments
              • Each (-) end of myosin thick filament attached to a (-) end of actin filament
                • (-) myosin contracts actin (+) towards center, then pushes outward (relax)
                  • Many sarcomeres contract in tandem to cause muscle movement
      • Microtubules (Lec. 26)
        • Microtubule structure
          • Polar polymers, ~25nm diameter
            • Protofilaments of a-b-a-b form tubes
              • Each subunit contacts 4 others for stability
                • Subunits: Dimer of alpha/beta tubulin; each binds 1 GTP
            • Polarity depends on which tubulin @ end; a is (-), b is (+)
            • Subunits more likely to add to (+) end
        • Dynamics
          • MTs organized by centrosomes
            • Nucleation facilitated by centrioles surrounded by y-tubulin ring complexes
              • Grows through addition of full rings of a- or b-tubulin
            • Forms branched network/ cloud of MTs
          • Dynamic instability of MT growth
            • Energy input for GTP hydrolysis (b-tubulin only) determines growing/shrinking of MTs
              • Allows MTs to search cellular space, be selectively stabilized to build ordered arrays
            • Growing MT: GTP added to GTP-unhydrolysed end (GTP cap), same as ATP/actin
              • Shrinking MT: If hydrolysis catches up (more energy added) then protofilaments will peel away
            • Drugs affecting MT stability
              • Colchicine: binds free tubulin dimers, preventing growth
              • Taxol: stabilises tubulin in MT lattice, preventing peeling --> cell death (cancer drug)
        • Functions in vivo
          • Internal organization of cell (moving vesicles)
            • Cell polarity
              • Neurons: proximal (-)--> distal (+) transport
              • Epithelial cells: apical (-)--> basolateral (+)
              • Pigment cells: center --> perimeter (cuttlefish)
            • Directed transport
              • Motor proteins use MTs as tracks
                • Kinesin moves toward (+)
                • Dynein moves (-)
              • Cargo can bind to motors to move in specific direction
            • Organize membranes/organelles, ex. ER, golgi, mitochondria
          • Mitotic spindle in cell division (chromosome segregation)
            • Centrosomes replicate during cell division
              • MTs randomly nucleate, are selectively stabilized towards center to engage chromosomes
            • Interpolar MTs stabilised by MT-associated proteins to push spindle apart
          • Cilia and flagella (moving cells in fluid)
            • Extend from centrioles, cylinder of 9+2 MT bound cylinder
            • Dynein motor on one strand causes bending/waving motion
      • Intermediate filaments (Lec. 27)
        • Structure
          • Assembly by coiled-coil formation
            • 7 AA's forming an amphipathic helix result in a twisting hydrophobic face
              • 2 helices wrap around each other to minimize hydrophobic face exposure
            • Can be homomeric or heteromeric
          • Ex. kinesin dimer, myosin-II
          • Strength derived from extensive contacts and hierarchical assembly
            • Forms staggered tetramers that wind around each other due to NH2-COOH interactions
            • Rope-like, flexible bc of breaks in the sequence
        • Types and functions
          • Cytoplasmic
            • Keratins (epithelia)
              • Allow sheets of cells to stretch w/o rupture in the skin, gut, etc.
                • 2 types- acidic and basic- form a heterodimer
            • Vimentin (muscle tissue)
            • Neurofilaments (nerve cells)
              • Filaments with branched proteins allowing neurofilaments to stabilize neuronal cells
          • Nuclear
            • Nuclear lamins (in animal cells)- network of filaments under nuclear envelope for structural integrity
              • Dissembles and reforms during cell division by de/phosphorylation
          • Human mutations of lamins show it is complex/ may be involved in gene expression
    • Prokaryotes vs. Eukaryotes
      • Diversity
        • Structural diversity in eukaryokes (change shape to adapt); metabolic diversity in prokaryokes (wide range of livable conditions)
      • Membranes
        • Eukaryokes have a membrane-bound nucleus and internal organelles
      • Genome
        • Eukaryotic genome is 3 to 1000x length of prokaryokes, to allow for structural evolution
    • Cytoskeleton assembly through protein-protein interactions
      • AA's/NTs covalently bond together into proteins (polymerase)
      • Macromolecular complexes
        • Self-assemble through random collisions
          • ex. ethanol diffuses into lipid bilayer of neurons in 5ns
          • Head-to-tail subunits build polymers to build a cytoskeleton
        • Binding affiliation depends on biological function
          • Most protein-protein Kd~10e-3M weak, 10e-9 tight bond
      • Proteins non-covalently bond into complexes, i.e. salt-bridge, van der Waals, ionic forces
    • Methods of studying
      • Immunofluorescence microscopy
        • primary antibody attaches to desired antigen; secondary fluorescent antibody seeks out and marks primary
      • GFP green fluorescent proteins fusion
        • GFP gene can be fused to any genome and expressed in vivo
      • Electron microscopy
        • Scale viewable down to 0.2nm
        • Thin section EM (view cross sections 100nm thick)
    • Integration of cytoskeletal systems (27)
      • Epithelia
        • Actin cortex for cell structure/ SA for nutrient uptake
        • MTs for directed transport apical--basolateral
        • Intermed filaments for tissue stability
      • Neurons
        • Actins extend cell
        • MTs grow in and stabilize the neuron/transport
        • Neurofilaments grow in behind
      • Cell division
        • Lamin phosphorylation before nuclear envelope breakdown
        • MTs form spindle for chromosome separation
        • Actin/myosin ring squeezes cell in half
      • Prokaryotic proteins similar to actin and tubulin form a "cytoskeleton"

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