Lecture 24-28: Cytoskeleton

Created by: annie.edge
Created on: 25-03-16 15:24

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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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Lecture 24-28: Cytoskeleton

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