Transcript Molecular Motors

Molecular Motors
BL4010 12.07.05
Outline
• Cytoskeletal components
• Vesicle movement
– dynein
– kinesin
• Cilia and flagella
• Muscle contraction
– tropomyosin
– regulation by calcium
Actin filaments
Swarming of Dictyostelium
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• http://www.biochemweb.org/fenteany/researc
h/cell_migration/movement_movies.html
• University of Illinois, Chicago
Actin polymerization
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Tubulin and Microtubules
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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!
Tubulin is a anisotropic
heterodimeric polymer
Tubulin
polymerization is
self-organizing but
requires some help
getting started
•Scaffolding proteins
serve as microtubule
organizing centers centrioles are only
one example
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)
Microtubules
Highways for "molecular motors”
• MTs also mediate motion of organelles and
vesicles through the cell
• Typically dyneins move + to • Kinesins move organelles - to +
Dynein
• Dynein proteins walk along MTs Dynein
movement is ATP-driven
Kinesin
• http://valelab.ucsf.edu/research/res_mec_dynein.html
Microtubules
in Cilia &
Flagella
• MTs are the
fundamental
structural unit in
cilia and flagella
The dynein “cargo” in cilia movement is the Atubule, moves along the B-tubule
Bending of cilia by MT sliding +
anchoring
http://programs.northlandcollege.edu/biology/Biology1111
/animations/flagellum.html
Other uses for motors
DNA unwinding and packaging
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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.
Flagella
Morphology of Muscle
Four types: skeletal, cardiac, smooth and myoepithelial cells
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
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
Molecular Structure of Muscle
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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)
Muscle contraction
Muscle fiber
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.
Thin filaments are actin +
tropomyosin
Structure of Thick Filaments
Myosin - 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
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 sevenresidue 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!)
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
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
Intermediate filaments
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
The Sliding Filament Model
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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
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
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
• http://www.blackwellpublishing.com/matthews/myosin.
html
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
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
The Ryanodine Receptor
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!
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
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
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
Smooth Muscle Effectors
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Useful drugs
Epinephrine (as Primatene) is an over-thecounter 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