Molecular Motors

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Molecular Motors

• BL4010 12.07.05

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Outline

• Cytoskeletal components
• Vesicle movement
• dynein
• kinesin
• Cilia and flagella
• Muscle contraction
• tropomyosin
• regulation by calcium

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Actin filaments
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Swarming of Dictyostelium
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• http//www.biochemweb.org/fenteany/research/cell_m
  igration/movement_movies.html
• University of Illinois, Chicago

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Actin polymerization
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Tubulin and Microtubules

• Fundamental components of the eukaryotic
  cytoskeleton
• Microtubules are hollow, cylindrical polymers
  made from tubulin dimers
• 13 tubulin monomers per turn
• Dimers add to the "plus" end and dissociate from
  the "minus" end
• Microtubules are the basic components of the
  cytoskeleton and of cilia and flagella
• Cilia wave flagella rotate - ATP drives both!

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Tubulin is a anisotropic heterodimeric polymer
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• Tubulin polymerization is self-organizing but
  requires some help getting started
• Scaffolding proteins serve as microtubule
  organizing centers - centrioles are only one
  example

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Polymerization Inhibitors

• Vinblastine, vincristine inhibit MT
  polymerization
• anticancer agents
• Colchicine, from crocus, inhibits MT
  polymerization
• inhibits mitosis (larger plants)
• impairs white cell movement (gout)
• Taxol, from yew tree bark, stimulates
  polymerization but then stabilizes microtubules
• inhibits tumor growth (esp. breast and ovarian)

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MicrotubulesHighways for "molecular motors

• MTs also mediate motion of organelles and
  vesicles through the cell
• Typically dyneins move to -
• Kinesins move organelles - to

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Dynein

• Dynein proteins walk along MTs Dynein movement is
  ATP-driven

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Kinesin

• http//valelab.ucsf.edu/research/res_mec_dynein.ht
  ml

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Microtubules in Cilia Flagella

• MTs are the fundamental structural unit in cilia
  and flagella

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The dynein cargo in cilia movement is the
A-tubule, moves along the B-tubule
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Bending of cilia by MT sliding anchoring
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http//programs.northlandcollege.edu/biology/Biolo
gy1111/animations/flagellum.html
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Other uses for motorsDNA unwinding and packaging

• When stretched out to its full extent, the DNA is
  around 10µm long, 200 times the size of the
  capsid
• This motor can work against loads of up to 57pN
  on average, making it one of the strongest
  molecular motors reported to date. Movements of
  over 5µm are observed, indicating high
  processivity. Pauses and slips also occur,
  particularly at higher forces.

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Flagella
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Morphology of Muscle

• Four types skeletal, cardiac, smooth and
  myoepithelial cells

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Morphology of Muscle

• A fiber bundle contains hundreds of myofibrils
  that run the length of the fiber
• Each myofibril is a linear array of sarcomeres

• Surfaces of sarcomeres are covered by
  sacroplasmic reticulum
• Each sarcomere is capped by a transverse tubule
  (t-tubule), an extension of sarcolemmal membrane

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What are t-tubules and SR for? The morphology is
all geared to Ca release and uptake!

• Nerve impulses reaching the muscle produce an
  "action potential" that spreads over the
  sarcolemmal membrane and into the fiber along the
  t-tubule network
• The signal is passed across the triad junction
  and induces release of Ca2 ions from the SR
• Ca2 ions bind to sites on the fibers and induce
  contraction relaxation involves pumping the Ca2
  back into the SR

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Molecular Structure of Muscle

• Thin filaments are composed of actin polymers
• F-actin helix is composed of G-actin monomers
• F-actin helix has a pitch of 72 nm
• But repeat distance is 36 nm
• Actin filaments are decorated with tropomyosin
  heterodimers and troponin complexes
• Troponin complex consists of troponin T (TnT),
  troponin I (TnI), and troponin C (TnC)

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Muscle contraction
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Muscle fiber
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Titin

• Titin is a giant 3 MDalton muscle protein and a
  major constituent of the sarcomere in vertebrate
  striated muscle. It is a multidomain protein
  which forms filaments approximately 1 micrometre
  in length spanning half a sarcomere.
• At low force the whole I-band acts as an entropic
  spring. At higher forces elasticity is due to the
  reversible unfolding of individual immunoglobulin
  domains of the I-band.

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Thin filaments are actin tropomyosin
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Structure of Thick FilamentsMyosin - 2 heavy
chains, 4 light chains

• Heavy chains - 230 kD
• Light chains - 2 pairs of different 20 kD chains
• The "heads" of heavy chains have ATPase activity
  and hydrolysis here drives contraction
• Light chains are homologous to calmodulin

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Repeating Elements in Myosin

• 7-residue, 28-residue and 196-residue repeats are
  responsible for the organization of thick
  filaments
• Residues 1 and 4 (a and d) of the seven-residue
  repeat are hydrophobic residues 2,3 and 6 (b, c
  and f) are ionic
• This repeating pattern favors formation of coiled
  coil of tails. (with 3.6 - NOT 3.5 - residues per
  turn, ?-helices will coil!)

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Repeating elements in myosin

• 28-residue repeat (4 x 7) consists of distinct
  patterns of alternating side-chain charge ( vs
  -), and these regions pack with regions of
  opposite charge on adjacent myosins to stabilize
  the filament
• 196-residue repeat (7 x 28) contributes to
  packing and stability of filaments

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Associated proteins of Muscle

• ?-Actinin, a protein that contains several repeat
  units, forms dimers and contains actin-binding
  regions, and is analogous in some ways to
  dystrophin
• Dystrophin is the protein product of the first
  gene to be associated with muscular dystrophy -
  actually Duchennes MD

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Dystrophin

• Dystrophin is part of a large complex of
  glycoproteins that bridges the inner
  cytoskeleton (actin filaments) and the
  extracellular matrix (via a protein called
  laminin)
• Two subcomplexes dystroglycan and sarcoglycan
• Defects in these proteins have now been linked to
  other forms of muscular dystrophy

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Intermediate filaments
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The Dystrophin Complex

• Links to disease
• ?-Dystroglycan - extracellular, binds to merosin
  (a component of laminin) - mutation in merosin
  linked to severe congenital muscular dystrophy
• ?-Dystroglycan - transmembrane protein that binds
  dystrophin inside
• Sarcoglycan complex - ?, ?, ? - all transmembrane
  - defects linked to limb-girdle MD and autosomal
  recessive MD

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The Sliding Filament Model

• Many contributors!
• Hugh Huxley and Jean Hanson
• Andrew Huxley and Ralph Niedergerke
• Albert Szent-Gyorgyi showed that actin and myosin
  associate (actomyosin complex)
• Sarcomeres decrease length during contraction
• Szent-Gyorgyi also showed that ATP causes the
  actomyosin complex to dissociate

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The Contraction Cycle

• Cross-bridge formation is followed by power
  stroke with ADP and Pi release
• ATP binding causes dissociation of myosin heads
  and reorientation of myosin head

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Ca2 Controls Contraction

• Release of Ca2 from the SR triggers contraction
• Reuptake of Ca2 into SR relaxes muscle
• So how is calcium released in response to nerve
  impulses?
• Answer has come from studies of antagonist
  molecules that block Ca2 channel activity

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• http//www.blackwellpublishing.com/matthews/myosin
  .html

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Dihydropyridine Receptor

• In t-tubules of heart and skeletal muscle
• Nifedipine and other DHP-like molecules bind to
  the "DHP receptor" in t-tubules
• In heart, DHP receptor is a voltage-gated Ca2
  channel
• In skeletal muscle, DHP receptor is apparently a
  voltage-sensing protein and probably undergoes
  voltage-dependent conformational changes

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Ryanodine Receptor

• The "foot structure" in terminal cisternae of SR
• Foot structure is a Ca2 channel of unusual
  design
• Conformation change or Ca2 -channel activity of
  DHP receptor apparently gates the ryanodine
  receptor, opening and closing Ca2 channels

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The Ryanodine Receptor
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Ca 2 Regulates Contraction

• Tropomyosin and troponins mediate the effects of
  Ca2
• In absence of Ca2, TnI binds to actin to keep
  myosin off
• TnI and TnT interact with tropomyosin to keep
  tropomyosin away from the groove between adjacent
  actins
• But Ca2 binding changes all this!

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Ca 2 Turns on Contraction

• Binding of Ca2 to TnC increases binding of TnC
  to TnI, simultaneously decreasing the interaction
  of TnI with actin
• This allows tropomyosin to slide down into the
  actin groove, exposing myosin-binding sites on
  actin and initiating contraction
• Since troponin complex interacts only with every
  7th actin, the conformational changes must be
  cooperative

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Binding of Ca 2 to Troponin C

• Four sites for Ca2 on TnC - I, II, III and IV
• Sites I II are N-terminal III and IV on C term
  
• Sites III and IV usually have Ca2 bound
• Sites I and II are empty in resting state
• Rise of Ca2 levels fills sites I and II
• Conformation change facilitates binding of TnC to
  TnI

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Smooth Muscle Contraction

• No troponin complex in smooth muscle
• In smooth muscle, Ca2 activates myosin light
  chain kinase (MLCK) which phosphorylates LC2, the
  regulatory light chain of myosin
• Ca2 effect is via calmodulin - a cousin of TnC
• Hormones regulate contraction - epinephrine, a
  smooth muscle relaxer, activates adenylyl
  cyclase, making cAMP, which activates protein
  kinase, which phosphorylates MLCK, inactivating
  MLCK and relaxing muscle

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Smooth Muscle Effectors

• Useful drugs
• Epinephrine (as Primatene) is an over-the-counter
  asthma drug, but it acts on heart as well as on
  lungs - a possible problem!
• Albuterol is a more selective smooth muscle
  relaxer and acts more on lungs than heart
• Albuterol is used to prevent premature labor
• Oxytocin (pitocin) stimulates contraction of
  uterine smooth muscle, inducing labor

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