module 3

• Cytoskeleton


  • Provides cells with structure so they can maintain their shape, internal organisation and polarity
  • Enables cells to change shape and move
  • Has to 'strike a balance' between stability to provide the mechanical support required for maintaining structure, and flexibility to allow cells to change shape, move and divide
  • Needs to be dynamic such that the cells can change their shape, internal organisation, or movement in response to changes in the cellular environment
  • Provides 'tracks' for motor proteins to transport macromolecules or entire organelles from one part to another part of the cells

  
• Main components of the cytoskeleton

  • Polymers of actin
  • Polymers of tubulin
  • Intermediate filaments
• Actin microfilaments

  • Extend throughout the cytoplasm
  • Have polarity
  • Polymerisation is ATP dependent
  • Dynamic ie polymerisation and depolymerisation constantly take place (treadmilling)
  • Tracks for myosin proteins (muscle contraction)
• Microtubules

  • Radiate out from the centrosome towards the cell periphery
  • Have polarity
  • Polymerisation is GTP dependent
  • Dynamic ie polymerisation and depolymerisation constantly take place (dynamic instabilities)
  • Tracks for Dynein and Kinesin (cargo transport, cilia/flagella)
• Intermediate filaments

  • Made from a myriad of units depending on the cell type
  • Extend through the cytoplasm or nucleus (depending on the type)
  • Do NOT have polarity
  • Polymerisation is independent of ATP or GTP
  • (mostly) static ie no treadmilling or dynamics instabilities
  • Not motor proteins
• G actin


  Globular actin, monomeric form, a 375-residue protein
• F actin


  Filamentous actin, polymeric form, made from two strands
• F actin has a (+) and a (-) end i.e. polarity
• Actin polymerisation

  1. Nucleation

  2. Elongation

  3. Steady-state
• Regulation of actin filament assembly

  • Formins - assemble unbranched filaments
  • Arp2/3 complex - nucleate branched filaments
• Regulation of actin filament assembly: formin


  1. Formin dimer binds to two G-actin subunits (early stage of nucleation)

  2. By rocking back and forth, additional subunits are added
• Regulation of actin filament assembly: Arp2/3


  1. Arp2/3 complex binds to the side of actin filaments (F actin) and generates a branch

  2. Arp2/3 needs an additional protein: WASp (verprolin in yeast) - results in nucleation of actin filaments
• Actin treadmilling at steady-state

  • ATP‐G actin subunits are preferentially added to the + end. After the G-actin is added to F-actin, ATP is hydrolysed.
  • G-monomers are preferentially lost at the ‐ end of the growing filaments.
  • On average, addition at the + end is faster than the – end, resulting in treadmilling.
• Controlling treadmilling by actin-binding proteins

  • Profilin - catalyses the exchange of ADP/ATP i.e. promotes the formation of ATP‐G‐Actin, providing a greater supply for binding to (+) end
  • Cofilin - binds along the filament, destabilises ADP‐actin in filaments, enhancing disassembly at (-) end
  • Thymosin B4 - sequesters away ATP‐G‐actin. Acts as a buffer for supply of ATP‐G‐Actin to (+) end
• Myosin

  • Myosin I found in the cell periphery
  • Myosin II, found in muscle and non‐muscle cells ‐ required for cytokinesis and focal adhesion (in non‐muscle cells)
  • Myosin V, required for organelle transport (e.g. melanosomes are transported from melanocytes to neighbouring keratinocytes)

  
• Myosin II motor protein


  1. Myosin head binds ATP, releases actin

  2. Head hydrolyses ATP to ADP + Pi, rotates to "cocked" state

  3. Cocked state binds actin

  4. Pi is released, myosin head moves along the filament: the "power" stroke

  5. Head remains bound to actin while ADP is present. When ADP is exchanged for ATP, the myosin head is released, and the cycle starts again
• The cytoskeleton is a dynamic structural network within cells comprising three filamentous components: Microfilaments, Microtubules and Intermediate filaments
• Actin can exist in G or F forms. G is the monomeric form, F is the filamentous form
• Actin polymerises in an ATP-dependent, 3-step process (nucleation, elongation, stead—state) with nucleation being the rate-determining step
• Unbranched and branched actin nucleation are regulated and assisted by the actin-binding proteins formin and Arp2/3, respectively
• Myosins are motor proteins of actin microfilaments
• Myosin walks "hand over hand" down actin filaments, driven by ATP hydrolysis
• The sarcomere is a unitary, contractile unit in muscle cells, and contains different zones where think or thin filaments are found in various arrangements
• Muscles contract due to the sliding of thick myosin filaments past thin actin filaments
• The filaments themselves do not contract
• Tropomyosin and troponin


  • Accessory proteins bound to actin thin filaments
  • In the absence of calcium, TM and TN molecules block the interaction of myosin with F actin
  • In the presence of calcium, TN induces TM to move to a new site, exposing the myosin-binding sites on actin
• Contraction and relaxation is ATP and Ca2+ dependent
• Ca2+ released from the sarcoplasmic reticulum binds to troponin, causing troponin and tropomyosin to move, which then exposes myosin-binding site on actin
• Cells use chemical gradients to direct their movements – chemotaxis
• Chemokines

  Secreted molecules that induce chemotaxis
• Chemokine receptors

  Receptors on the cell surface that 'sense' chemokines
• The binding of chemokines to chemokine receptors triggers cell-internal processes that control cytoskeleton components such as actin
• During chemotaxis, the cell re-organises the distribution of chemokine receptors such that more of them are located at the leading edge of the cell

  
• Cytoskeleton components

  • Processes that control cytoskeleton components such as actin
• Chemotaxis

  Cell re-organises the distribution of chemokine receptors such that more of them are located at the leading edge of the cell

  
• Microtubules are composed of 13 repeating units: proto-filaments (proto = protein)
• Tubulin

  Globular protein, 55 kDa, that microtubules are assembled from
• Microtubules

  • Assembled from hetero-dimers of α-tubulin and β-tubulin
  • Assembly happens at the (+) ends of microtubules
• GTP/GDP


  Regulates assembly and disassembly of microtubules
• γ-tubulin

  Nucleates and stabilises the (-) end of microtubules