Cytoskeleton - Locomotion

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Cytoskeleton - Locomotion
Kohidai, Laszlo MD, PhD Med. habil., Assoc.
Professor Dept. Genetics, Cell Immunobiology,
Semmelweis University
www.dgci.sote.hu
Ecelctive Course / 2011
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Main functions of cytoskeleton

• Determines the shape of the cell
• Anchores organelles
• Movement of organelles
• Tensile strength
• Movement of chromosomes
• Polarity
• Motility

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Cytoskeleton

• Microfilaments (actin)
• Microtubuli (tubulin)
• Intermedier filaments
• Microtubule associated proteins (MAP-s)
• Motor proteins

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Microfilaments
Microtubuli
Intermedier filaments
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SLIDING
Globular proteins
Ca2
ATP
Motor proteins
Fibrillar proteins
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Microfilaments
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Polymerization of actin

ATP
ADP
Depolymerization
-
cytochalasin inh. phalloidin - stabilizer
ATP
ADP
Pi
Polymerization - slow
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Actin - still in Prokaryots !
((Ent et al. Nature 2001,413, 39)
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Cyclosis

• Transitional connections between actin and
  myosin
• Ca2, temperature- and pH-dependent

(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
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Fountain mechanism
Ca2-dep. requires ATP
Mono- Poly- Lobo- podial Filo- Reticulo-
Formation of pseudopodium
stress-fibrillums
integrins
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Cross-linking proteins of actin
contractile bundle a actinin in stress fibr.
gel-like network filamin - cortex
tight parallel bundle fimbrin in filopodium
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Migrating keratinocyte
15 mm/sec
Formation of lobopodium
microtubuli
actin-network
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Regulator proteins of actin polymerisation
gCAP39 Severin Gelsolin Villin CapZ
Tropomodulin
?
Cofilin Severin Gelsolin
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Actin polymerisation acrosomal-reaction
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
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• local actin polymerization
• speed 10 mm/min
• high ability to transmit
• in tissues

Listeria monocytogenes
actin
(Fred Soo Julie Theriot Laboratory)
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Model of actin nucleation

WASP Wiscott-Aldrich syndr. prot.
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Structure of cortical region
(Svitkina, TM, Borisy GG J. Cell Biol. 1999,
145, 1009)
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Actin membrane links
membrane
F-Actin
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Profilin-mechanism
Tb4 timozin b4
Proline-rich protein
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
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Filamin Membrane link
filamin
actin
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Structure of focal contact
actin filament
a actinin
vinculin

paxillin
talin
integrin
fibronectin
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A plasma membrane cortex links
((Lux SE, 1979 Nature 281426)
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E
Electromagnetic field induces the
transformation of cytoskeleton and formation of
pseudopodia
Adhesion plaque



-
-
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Myosin
head
Ca2-dependent phosphorylation and its effect on
the 3D strcture
light chain
heavy chain a helix
myosin I. 150 kD
monomer
myosin I I. 260 kD
Head - ATP-ase - motor
dimer
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Distribution of myosines in the migrating
Dyctiostelium and in dividing cell
myozin I. (green)
myozin II. (red)
(Fukui, Y. Mol. Cell Biol 2000, 785))
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Main types of interactions between the
globular and fibrillar components of
cytoskeleton
membrane
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MT-blocked
F-actin blocked
Non-treated
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Microtubules
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Tubulin still in Prokaryotes !
FtsZ
Tubulin
(Margolin Laboratory, University of Texas)
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Polymerization of tubulin
GTP
GTP
Polymerization - fast
GTP
GTP
Protofilament (strait)
GDP
GDP
GDP
GDP
Protofilament (curved)
Depolymerization
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Dynamics of microtubule-assembly
-

incorporation
balanced
release
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Role of g-tubulin in nucleation
(Wiease et al. Curr.Opin.Struct.Biol. 1999, 9,
250)
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Microtubular systems in the cells
- Centrosome - Cilia / flagellum - Mitotic
system - Vesicular transport
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MTOC Mikrotubul organizing center
g-tubulin
((Brinkley, B.R. Encyclop. Neurosci. 1987, 665)
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Network of microtubuli
Fibroblast
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Cilia
cilia
flagellum
Paramecium
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(No Transcript)
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Composition of dynein-arms
ATP-independent binding
ATP-dependent hydrolisis
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The role of dynein arms in beating of cilia
Bending
Telescoping
Proteolysis
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Molecules composing the cilia
more than 250 types of molecules

• 70 a and b tubulin
• dynein arms
• outer - 9 polypeptides - ATP-ase
• inner composition varies
• radial spokes - 17 polypeptides

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Microtubules of mitotic spindle and kinetochore
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Arrangement of actin during cell-division
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Intermedier filaments
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Mechanical characterization of cytoskeleton
components
deformation
force
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Role of intermedier filaments
Buffer against external mechanical stress
Tissue specificity
Nucleus lamines (lamina fibrosa)
Epithel keratin Connective tissue Muscles Neuro
glia Neurones - neurofilaments
vimentin
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Structure of intermedier filamentums
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
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Domain structures of intermedier filamentums
H2N-
a helical domain
-COOH
keratins
vimentin
neurofilam. prot.
nuclear prot
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Intermedier filaments
Keratin filaments
Vimentin-like filaments
! They DO NOT co-polymerise !
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Microvilli
myosin I.
actin
villin
terminal web

• a rigid bundle composed by 20-30 actin mol.s
• actin on the apical part
• villin is the linker molecule of actins
• terminal web intermed.fil. spectrin
• myosin I. and calmodulin anchore to the surface
  membrane

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SEM structure of microvilli
actin bundle
linker molecules
terminal web
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Intermedier filaments
Glial filaments few cross-linkers
Neuro-filaments many cross-linkers
The number of protein cross-links between the
intermedier filaments varies in different tissues
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Microtubuli associated proteins (MAP-s)
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Groups of MAP-s

• Structural MAP-s
• - MT-assembly
• - links to MF and to IF
• Motor proteins
• - sliding on MT
• Enzymes, signal molecules
• - glycolytic enzymek
• - kinases

Shape and polarity of the cell
Membrane transports
Assembly of molecules
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Motor-proteins
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Structure of motor-proteins
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Motor proteins
-

dynein
kinesin
microtubule
heavy chain
light chain
kinesin
dynein
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kinesin
-

dynein
cAMP
cAMP
pigment cells
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Kinesin
ADP
ATP
ADP
ATP
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MT-motor proteins and the transported elements
(Hirokawa, N. Science 1998, 279519
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Dynein membrane relations
(Hirokawa, N. Science 1998, 279519)
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There are other mechanisms over sliding
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Locomotion with spasmoneme ofVorticella
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Spasmoneme spring
Contracts 40 in few msecs Velocity 8 cm?/sec
Negative charges
Neutralization with Ca2
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Actin spring in sperm of horse-shoe crab Limulus
polyphemus
!
acrosome
actin bundle

• The extension does not involve a myosin
• motor or actin polymerization
• The bundle is crystalline in its coiled and
• uncoiled states

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Signalling mechanisms In a nutshell
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Polymerization of microtubules- Significance of
motor proteins
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Ameboid movement and microtubules
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Complex effects of focal adhesion kinase (FAK)
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LIM domains of proteins
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Central role of vimentin in signalling mechanisms
of cytoskeleton
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Phosphatases and cell migration