Cytoskeleton

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Cytoskeleton

• Werkzitting bij het college
• Celbiologie
• F. Wuytack

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Cytoskeleton cytobones and cytomuscles
monomer stability polarity diversity

structure Microfilaments (actin)
globular dynamic polar (/-) 18
genes instability Microtubuli globular dyn
amic polar (/-) ?, ?, ?, ?,
instability
?, ?, ? Intermediate filaments fibrous stabl
e non-polar many diff.
types
Only in eukaryotes?
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The missing links in Bacteria and Archaea
FtsZ a archaeal homologue of tubulin
MreB and Mbl two homologues of actin from
Bacillus subtilis form filamentous actin-like
structures in non-spherical bacteria that
determine cell shape
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(No Transcript)
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GTP hydrolysis controls the growth of microtubuli
? Tubulin ? Tubulin
GTP GDP
GTP
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Jan Löwe (MRC Cambridge UK)
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Dynamic instability of microtubules
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Taxus brevifolia is the source of taxol
Taxol binds to microtubuli and prevents them from
growing but not shrinking ? antimitotic drug ?
cancer therapy
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Vinca major source of Vinca alkaloids vincristin
and vinblastin
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Colchicine is derived from Colchicum autumnale It
binds to free tubulin and prevents polymerization
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Dinitroanilin herbicides bind selectively to
plant ?-tubulin (trifluralin, oryzalin)
Grow points of shoots and roots are disturbed In
the USA a form of goose-grass (Eleusine indica)
is found which is resistant against this
herbicide. The resistant form has one amino acid
difference with the wild type
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Actin isoforms
MW 42000 (374-376 aa)

• some protists (Amoeba proteus) 1 actin isoform
• most invertebrates and primitive chordates 2
  isoforms muscle actin
• non-muscle actin
• amniotic animals 6 actin isoforms
• ?sk skeletal muscle
• ?c cardiac muscle
• ?sm smooth muscle
• ? sm smooth muscle
• ?
  non muscle
• ? nm non muscle
• plants 6- 8 actin types or more
• highly conserved not present in prokaryotes

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ATP hydrolysis during actin polymerization
end
ATP
ATP
ADP
- end
Compare to tubulin which binds GTP/GDP Fungal
toxins cytochalasins prevent actin
polymerization
phalloidin (not permeable) prevents
depolymerization
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The polymerization of actin
Nucleation Elongation Steady state
Polymerization stimulated by increasing ionic
strength
nuclei
Mass of filaments
- nuclei
time
Filament Monomer
Balance point
Mass
Actin concentration
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Polymerization dynamics of actin
plus end
Cc- 0.8 ?M Cc 0.1 ?M
rate of polymerization
minus end
balance point
Cc
Cc-
actin concentration
Applies only to naked actin is modulated by
actin-binding proteins Relatively too much
monomeric actin (actin 0.5 mM 40
monomeric)
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Microfilament poisons

• Cytochalasins B D (cyto cell, chalasis
  relaxation)
• alkaloid produced by mold Helminthosporium
• membrane permeable
• caps end of microfilament
• also severs ? convert gel into sol
• fragmentation of microfilaments
• Phalloidin
• produced by mushroom Amanita phalloides (groene
  knolzwam)
• poorly membrane permeable
• binds at interface between actin subunits in
  filaments and locks them together
• stabilization of microfilaments

- end end
cyclic organic molecules reacting as bases
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Amanita phalloides (groene knolzwam)
3T3 fibroblasts stained with fluorescent
phalloidin
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Some actin-binding proteins (1)
Caps Severs Cross- Bundles
Attaches links
to PM









Sources blood plasma muscle cells macrophages,
platelets, brain brush border (intestin) blood
plasma Dictyostelium smooth muscle, macrophages
many mammal. cell types widely distrib. platelets
, muscle, fibroblasts Amoeba
Ca2 sens. - -
- - -
Protein
MW (kDa) 42 34-37 90-95 95 90 40 250-270
225-260 235-240 100-105 23-38
fragmin ?-actinin gelsolin villin brevin seve
rin filamin spectrin fodrin ?-actinin gelacti
n
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Some actin-binding proteins (2)
Caps Severs Cross- Bundles
Attaches links
to PM







(to microtubules)
(to
microtubules) binds actin monomers,
helps microfilament formation
keeps G-actin monomeric
Sources sea urchin oocytes muscle
cells, fibroblasts smooth muscle brush
border (intestine) brain brain widely
distributed widely distributed
Ca2 sens. - - - - - - -
-
Protein
MW (kDa) 58 130 215 68 280 55-62 12-15 5

fascin vinculin talin fimbrin MAP-2 tau prof
ilin thymosin
Note some actin-binding proteins (e.g. fragmin)
evolved from actin
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Growth of filopodia
Very long exploratory rapidly form and retract
Nerve cell axon
Compare to acrosomal reaction in sperm from sea
urchins
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GFP-tagged actin in fibroblasts and in
the neuronal growth cone
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Actin dynamics in melanoma cells
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Myosins are motor proteins 17 classes
calmod
IQ domain
calmod
Reverse motor
All motors except VI
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Unconventional myosins in inner-ear sensory
epithelia
Hasson T et al. (1997) J. Cell Biology 137,
1287-1307
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Gillespie PG Walker RG (2001) Nature 413,
194-202
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Gillespie PG Walker RG (2001) Nature 413,
194-202
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Myo1?
Gillespie PG Walker RG (2001) Nature 413,
194-202
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The essential light chain clips onto the myosin
heavy chain
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Ca2 Calmodulin binds to MLCK
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The cycle of myosin
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A more detailed model
The Myosin Home Page http//www.mrc-Imb.cam.ac.uk/
myosin/myosin.html
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Cross-bridge cycle
Muscle bearing weight
Muscle at resting length
Power stroke with Pi release
Almost irreversible
M.ADP.Pi complex has high affinity for actin
Rapid (10 ms)
reversible (Keq 10) Energy conserved
in M.ADP.Pi complex
Head released upon binding ATP
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The cycling cross bridge
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Vesicle transport and organelle movement

• Kinesin is a MT motor protein normally involved
  in
• anterograde ( to plus end of MT) transport of (1)
  ER,
• golgi-derived vesicles, pigment granules (2)
  actin - and
• intermediate filaments (vimentin)
• Evidence
• Movement of isolated MT over glass slide with
  fixed
• kinesins
• Transfection of cells with dominant-negative
  mutants
• of motors
• Inhibitory effect of anti-kinesin heavy chain
  (KHC) anti-
• bodies
• Inhibitory effect of anti-sense oligonucleotides
• complementary to KHC
• Kinesin KO mice

http//www.proweb.org/kinesin/index.html
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The Kinesin tree
(-) end directed motors
As of 4 March 2001, 268 sequences containing the
kinesin motor domain have been deposited in the
data bases. These proteins can be assigned to one
of ten subfamilies
Spindle kinesins
KIF1A transports specifically synaptic
vesicles KIF1B transports specifically
mitochondria
The Kinesin Home Page http//www.blocks.fhcrc.org/
kinesin/
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Organelle movement on microtubuli
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(No Transcript)
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The growth of ER along microtubules
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Cell stained with kinesin heavy chain antibodies
PtK1 cells were labeled for double
immunofluorescence microscopy with the H1
monoclonal antibody to kinesin heavy chain and a
polyclonal antibody to tubulin. Kinesin and
microtubules are shown in the rhodamine (red) and
fluorescein (green) channels, respectively. The
H1 antibody recognizes a subset of the total
cellular kinesin and stains a post-ER, pre-Golgi
system of membranes known as the intermediate
compartment. Kinesin is thought to be the motor
for moving these membranes from the Golgi to the
ER.
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Kinesin as a motor protein
? tubulin white ? tubulin green
Vale RD Milligan RA (2000) Science 288, 88-95
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Recessive lethal mutations in Drosophila kinesin
Heavy chain (Khc) gene http//www.proweb.org/kin
esin/WTKHClarvae.html
Wild type
Khc mutant
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laser tweezers
The strength of the force holding the bead is
adjusted by the laser intensity
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Axoneme-MTs moving on kinesin bound to a
coverslip
-------
fiber
Rate 1 micrometer/second Step-size 8 nm (tubulin
dimer) 100 ATPs split/s maximal (stalling)
force 5 pN ( force exerted by laser pointer on
screen or by gravity on a single bacterium) Work
force x distance 40 x 10-21 J
or efficiency 40
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MT are not the only tracks for organelle movements
Melanophores (fish and frogs) use MT and
heterotrimeric kinesin II or cytoplasmic dynein
for long-distance movements, actin and myosin
V for short distance cAMP induces anterograde
transport (neuronal or hormonal) Melanocytes
(mammals) secrete melanosomes which are taken up
by the keratinocytes Dilute mouse mutant is
defective in myosin V gene also MT are
involved Cross talk between both systems tails
of myosin V and conventional kinesin (?kinesin
II) interact (yeast two-hybrid system)
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Listeria monocytogenes, Shigella and viruses
(vaccinia) move through cytoplasm by recruiting
actin-filament nucleation factors They exploit a
mechanism employed by the cell to
move endocytotic vesicle away from the PM
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Listeria bacteria move in the cytosol of the host
cell
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Regulation of organelle transport
Loading movement
Conventional kinesin is heterotetrameric 2 heavy
chains- NH2 terminal head (catalytic motor
domain) - central stalk - coiled-coil
- 2
hinges -COOH terminal
globular tail 2 light chains Folded
(compact) ? extended form
inactive active
G proteins (?)
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Myosin and kinesin share Ras nucleotide-binding
fold
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Nothing in biology makes sense except in the
light of evolution
Th. Dobzhansky 1973