Cytoskeleton Systems

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Chapter 15

• Cytoskeleton Systems

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Cytoskeleton

• Highly structured portion of the cytosol
• Network of interconnected filaments and tubules
  from the nucleus to the plasma membrane
• Functions as
• Architectural framework
• Internal organization moves organelles and
  cellular components
• Maintain complex shapes
• Very dynamic and changeable
• Important for cell movement and division

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Major Structural Elements

• Microtubules
• Microfilaments
• Intermediate filaments
• See similar things in bacteria assembly of
  fibers but not AA sequence
• All are linked structurally and functionally
• Each has its major structural component and other
  associated proteins hat all for diversity of
  function

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Study using EM and fluorescent antibody staining
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Know everything but size
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Drugs Used to Study

• Colchicine prevents tubulin polymerization
• Taxol forces microtubule formation
• Cytochalasin D and latrunculin prevents actin
  polymerization
• Phalloidin prevents the depolymerization of
  actin

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Microtubules (MT)

• Largest fiber
• 2 groups
• Axonemal MT specific cellular structures such
  as cilia, flagella and basal bodies
• Axoneme is the central shaft and has axonemal MT
  and associated proteins
• Cytoplasmic MT loosely organized and dynamic,
  variety of functions
• Maintain axons polarized shapes spatial
  disposition and directed movement of vesicles and
  organelles and mitotic and meiotic spindles

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Microtubules

• Tubulin heterodimers building blocks
• Straight hollow tubes of varying length
• Longitudinal array of linear polymers made up of
  protofilaments usually 13 around a lumen

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Tubulin

• ?-tubulin (dark orange) and ?-tubulin (yellow)
  linked non-covalently into a heterodimer that
  doesnt dissociate
• Share 40 AA homology but very similar shape
• 3 domains
• GTP-binding domain at N terminus
• Middle domain that can bind colchicine
• MT-associated protein (MAP) interacting domain at
  the C terminus
• Both ends of the MT are chemically and
  structurally distinct polarity
• Most organisms have related but not identical
  genes isoforms
• Differs mainly at the C end where they bind MAPs

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MT Formation
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MT Formation

• Reversible polymerization of dimers
• Clusters of dimers called oligomers and act as
  the nuclei of MT formation nucleation process
• See a lag phase as the start is very slow
• MT grows by addition of subunits elongation
  phase
• This phase is very fast
• Eventually the free tubulin becomes limiting
  factor so see a plateau phase

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Stages of MT Formation
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Tubulin Addition

• Faster growth at the plus end - ? tubulin end
• Concentration of free tubulin at either end will
  determine the rate of addition or subtraction
• Higher critical free tubulin at the plus end
  rather than the minus end will lead to growth at
  the plus and disassembly at the minus end
• Process called treadmilling

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MT Growth
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MT Formation Requires GTP

• Dimer bind 2 GTP molecules one on each subunit
• GTP on the ? subunit is hydrolyzed after addition
  to the MT
• GTP is essential but the hydrolysis to GDP is not

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Dynamic Instability Model

• 2 populations 1 growing and 1 shrinking
• Growing end has a stable tip that has a GTP-cap,
  adds new dimers
• If high tubulin then it is added quickly,
  otherwise it slows down
• Shrinking end had GDP and tip is unstable
  disassembles
• At some tubulin the hydrolysis of GTP on the ?
  subunit exceeds the addition of new dimers

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Dynamic Instability (cont)

• Alternate between growing/shrinking, usually at
  the plus end
• Growth then shrink MT catastrophe
• Shrink then growth MT rescue

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MT Organizing Centers (MTOC)

• Site of MT growth initiation and acts as anchor
• In mitotic cell it is the centrosome and is near
  the nucleus
• Animal cells also have 2 centrioles around the
  centrosome in a diffuse granular material called
  the pericentriolar material

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Centrioles

• 9 pairs of triplet MT at 90
• important in basal body formation for flagella
  and cilia
• No centriole then the spindles are poorly
  organized during cell division
• Not part of plants

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Centrosomes and MOTC

• Centrosomes use ?-tubulin
• Ring structure seen at the base of MT
• Acts as a nucleation site for new MT, anchored by
  the minus end, plus end moves to the cell
  membrane
• Fixed polarity
• MOTC influences the number of MT can change it
  during special cell functions
• Also helps stabilize the MT such as with the
  kinetochore on mitotic chromosomes or cell cotex
  and plus end tracking proteins

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Functions of MT
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Drug Effects on MT Assembly

• All are anti-mitotic drugs
• Colchicine binds to tubulin dimer and then
  binds to growing MT but no additional dimers can
  be added resulting in disassembly
• Vinblastine/vincristine acts as anti-cancer
  drug rapid cell growth makes them
  preferentially susceptible for drug
• Taxol stabilizes MT and arrests cells in
  mitosis
• Used in treating breast cancer

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MT-Associated Proteins (MAPs)

• Level of regulation to organize and function of
  MT
• MAPs bind at intervals along MT wall
  projections that interact with other filaments
  and cellular structures
• Also involved in MT assembly regulations,
  increases stability

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MAPs

• Motor MAPs
• Kinesin (moves to plus end) and dynein (moves to
  minus end)
• use ATP to drive transport of vesicles and
  organelles
• generate sliding forces
• Non-Motor MAPs
• control MT organization in cytoplasm specific
  for each type
• especially in neurons send out neurites which
  becomes axon carry electrical signals protein
  is MAP2, looser bundles
• MT bundles are denser in axons protein is Tau,
  tigher bundles

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Microfilaments (MF)

• Smallest filament contractile fibers
  interacting with myosin fibers
• In almost all cell types and have many functions
• amoeboid motion (along surface)
• cytoplasmic streaming (pattern of flow in cell)
• produce cleavage furrow
• attachment to adjacent cells
• cell shape cell cortex and in microvilli

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Actin

• Building block of MF have a binding site for
  ATP or ADP G-actin (globular)
• G-actin polymerizes to MF F-actin
• G/F-actin can bind many proteins actin binding
  proteins which regulates or modifies actin or are
  regulated/organized by association with actin
• Actin is highly conserved differs in many cells
  but can substitute in function

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Actin (cont)

• 2 major groups based on sequence
• muscle specific actins - ? actin
• non-muscle - ? and ? actins migrates to
  different areas of cell
• ? actin is predominately at the apical surface
• ? actin is concentrated at basal and sides of
  cells
• Actin-related proteins (Arps) less similar to
  actin

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Actin Polymerization

• G-actin monomers reversibly polymerize into
  filaments (lag-phase nucleation), more rapid
  polymerization elongation
• 2 linear strands of G-actin wind around each
  other to make F-actin, 13.5 actins per turn

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Actin and Myosin

• Inherent polarity structurally and chemically
  different ends
• Use myosin subfragment (S1) to determine
  direction
• Barbed end plus
• Pointed end minus

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Actin Polymerization

• Ends are important independently regulation of
  actin assembly/disassembly
• Add and lose faster at the plus end
• Plus end grows faster when conditions are
  favorable to adding monomers
• ATP is tightly bond to actin but its energy is
  not required for polymerization
• Plus end is ATP-actin
• Slowly, ATP is converted to ADP

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Actin Structures

• Cells regulate actin forms
• Lamellipodium and filopodia depends on actin
  filaments
• Stress fibers adhere to the surface
• Cortex under membrane gel or lattice of actin

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Lamellipodium and Filopodia

• Lamellipodia are less organized
• Filopodia are polarized cables with plus end
  pushing to the protrusion

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Proteins Affecting Actin Dynamics

• Controls nucleation and depolymerization
• Actin binding proteins, phosphotidylinositol and
  small regulatory G proteins (Rac, Rho and Cdc42)
• Amount of ATP-G-actin influences rate
• Cells regulate amount thymosin ?4 binds
  G-actin profilin transfers the G-actin from
  thymosin ?4 to filament
• ADF-Cofilin binds ADP-G-actin and F-actin
  causing turnover of ADP-G-actin at the minus end
• Capping protein prevents depolymerization
  stable actin filament

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Drugs Affecting Actin Dynamics

• Both drugs cause lose of MF from cell
• Cytochalasins prevents addition of monomers and
  results in depolymerization
• Latrunculin A sequesters actin monomer no
  growth

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Inositol Phospholipid Regulation

• IP3 is signaling molecule for G proteins
• Phosphotidyl inositol can be phosphorylated (by
  special kinase) to polyphosphoinositides that can
  bind actin-binding proteins
• Phosphotidyl inositol (4,5)-bisphosphate bind
  profilin and CAP Z to regulate their activity
  with actin
• PIP2 binds CAP Z and causes depolymerization

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Arp2/3 Complex

• Actin filaments can be branching dendritic
  network
• Plus or barbed end can branch via profilin and
  capped by capping proteins
• Actin-related protein (Arp) 2/3 complex helps
  with the branching, nucleation site for a branch
• Arp 2/3 activated by a family of proteins first
  found in Wiscott-Aldrich Syndrome, WASP
• Mutations result in platelet activation and clots
• Actin polymerization is independent of Arp 2/3
  but can form thru formins required for F-actin
  formation actin cables and contractile ring

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Rho, Rac and Cdc42

• Regulates actin polymerization cell must
  regulate movement and cytoskeletal changes
• Done by small monomeric G-proteins
• Essential for Growth Factors as PDGF and LPA
  (lysophosphatidic acid)
• Rac responds to PDGF to cause extension of
  lamellipodia
• LPA activates Rho to form the stress fibers
• Cdc42 activation forms filopodia

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Actin Binding Proteins

• Interactions between MF
• Involves cell cortex supports plasma membrane
  confers rigidity to cell surface, facilitates
  shape changes and cell movement
• Filamen cross-link actin-binding protein of
  cortex to MF 2 identical peptides joined
  head-to-head

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Other functions

• Other actin-binding proteins can break up MF
  network by either severing the MF or capping them
• Gelsolin can do both
• breaks actin filaments
• adds a cap to the plus end
• Regulated by polyphosphoinositides bind to
  gelsolin and can prevent capping and MF shortens

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Actin-Binding Protein Interactions
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Microvilli

• Ordered actin bundles
• Prominent in intestinal mucosal cells
• Helps to increase surface area of cell
• Tight bundle of MF plus end toward tip attach
  to an amorphous electron dense plaque also
  connected to plasma membrane by myosin I and
  calmodulin
• Bundle bound tightly together by cross-linking
  proteins fimbrin and villin

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Microvilli (cont)

• MF bundle extends into the terminal web, giving
  rigidity to microvilli
• Terminal web is made of myosin and spectrin
  connect the MF to each other, to proteins in the
  plasma membrane and to network of intermediate
  filaments

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Microvilli Structures
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Actin to Membrane

• Indirect linkage
• Requires 1 or more linker proteins, link MF to
  transmembrane proteins
• Cortex proteins
• spectrin
• ankyrin
• band 4.1
• ezrin
• radixin
• moesin (ferM) family

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Intermediate Filaments

• Sized between microtubules and microfilaments
• Play a structural or tension bearing role in
  cells
• Most stable, least soluble hard to remove from
  cytosol
• Acts as scaffold for cytoskeleton framework
• Not apparently polar

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IF
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Intermediate Filaments

• Differ in size and chemical properties
• Encoded by family of related genes
• Classified by AA sequences Class I VI
• Can distinguish cell type based on IF type
  present
• Aids in cancer metastasis diagnosis
• Cells retain their IF proteins even when they
  migrate somewhere else

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IF Assembly

• Made from fibrous subunits rather than globular
• Central rod like domain conserved in size and
  2 structure and to some extent in AA sequence
• 4 coiled helices with 3 linker segments
• N and C vary in size, sequence and function

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Assembly

• 2 IF polypeptides make coiled-coil structure
• N and C end are globular domains
• 2 coiled-coil structures pair to become a
  protofilament
• Associated in overlapping manner to build a
  filament
• 8 protofilaments thick and any given place
• End to end in staggered overlaps

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IF Bear Mechanical Stress

• Desmosomes tonofilaments of keratin loop thru a
  plaque that connects 2 adjacent cells
• Hemidesmosomes between basal surface of cell
  and extracellular matrix

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Dynamic Structures

• IF can be remodeled
• Scaffold of nuclear lamina has 3 IF fibers
  nuclear lamins A, B and C
• Phosphorylation of A, B and C cause them to
  dissociate to remove the nuclear membrane during
  mitosis
• Remove the phosphate and they reassemble

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Mechanically Integrated

• All 3 members of the cytoskeleton work together
• Special linker proteins combine the 3 (Chapter 17)