Transcript Citoszkeleton - Semmelweis University

Cytoskeleton - Locomotion
Kohidai, Laszlo MD, PhD
Med. habil., Assoc. Professor
Dept. Genetics, Cell & Immunobiology, Semmelweis University
Ecelctive Course / 2012
www.dgci.sote.hu
Main functions of cytoskeleton
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Determines the shape of the cell
Anchores organelles
Movement of organelles
Tensile strength
Movement of chromosomes
Polarity
Motility
Cytoskeleton
Microfilaments (actin)
 Microtubuli (tubulin)
 Intermedier filaments


Microtubule associated proteins (MAP-s)
Motor proteins
Microfilaments
Microtubuli
Intermedier
filaments
SLIDING
Globular proteins
Ca2+
ATP
Fibrillar proteins
Motor proteins
Microfilaments
Polymerization of actin
ADP
+
ATP
Depolymerization
ADP
ATP
cytochalasin – inh.
phalloidin - stabilizer
Pi
Polymerization - slow
Actin - still in Prokaryots !
((Ent et al. Nature 2001,413, 39)
Cyclosis
Moving
cytoplasm
Stationary
(cortical)
cytoplasm
Plasma membrane
Cell-wall
Actin filaments
Chloroplasts

Transitional connections between actin and myosin

Ca2+, temperature- and pH-dependent
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
„Fountain” mechanism
Ca2+-dep.
requires ATP
Formation of pseudopodium
stress-fibrillums
integrins
MonoPolyLobo- podial
FiloReticulo-
Cross-linking proteins of actin
contractile bundle
a actinin – in stress fibr.
gel-like network
filamin - cortex
„tight” parallel bundle
fimbrin – in filopodium
Migrating keratinocyte
15 mm/sec
Formation of lobopodium
actin-network
microtubuli
Regulator proteins of actin
polymerisation
gCAP39
Tropomodulin
Severin
+

-
Cofilin
Severin
Gelsolin
Gelsolin
Villin
CapZ
Actin polymerisation –
acrosomal-reaction
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
Listeria monocytogenes
• local actin polymerization
• speed: 10 mm/min
• high ability to transmit
in tissues
actin
(Fred Soo & Julie Theriot Laboratory)
Model of actin nucleation
WASP = Wiscott-Aldrich syndr. prot.
Structure of cortical region
(Svitkina, TM, Borisy GG J. Cell Biol. 1999, 145, 1009)
Actin – membrane links
membrane
Myozin I.
Arp2/3
F-Actin
Profilin
- G-aktin
Filamin
Integrin
Profilin-mechanism
Tb4 = timozin b4
Proline-rich
protein
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
Filamin – Membrane link
filamin
actin
Structure of focal contact
actin filament
a actinin
vinculin
+
paxillin
talin
integrin
fibronectin
A plasma membrane – cortex links
Thrombocyte
Glycophorin
Ankyrin
Spectrin
tetramer
Muscle
Epithel
((Lux SE, 1979 Nature 281:426)
E
Electromagnetic field
induces the transformation
of cytoskeleton and
formation of pseudopodia
-
Adhesion plaque
++
-
+
head
Myosin
Ca2+-dependent phosphorylation
and its effect on the 3D strcture
light chain
heavy chain
a helix
ATP - ADP
Pi
myosin I.
150 kD
monomer
myosin I I.
260 kD
dimer
Head: - ATP-ase
- motor
Distribution of myosines in the
migrating Dyctiostelium and in
dividing cell
myozin I.
(green)
myozin II.
(red)
(Fukui, Y. Mol. Cell Biol 2000, 785))
-
+
Main types of interactions
between the globular
and fibrillar components
of cytoskeleton
membrane
Non-treated
F-actin blocked
MT-blocked
Microtubules
Tubulin –
still in Prokaryotes !
FtsZ
Tubulin
(Margolin Laboratory, University of Texas)
Polymerization of tubulin
GTP
GTP
GTP
GTP
Polymerization - fast
Protofilament (strait)
GDP
GDP
GDP
Protofilament (curved)
GDP
Depolymerization
Dynamics of microtubule-assembly
Nucleation
Elongation
+
incorporation
balanced
release
-
Role of g-tubulin in nucleation
(Wiease et al. Curr.Opin.Struct.Biol. 1999, 9, 250)
Microtubular
systems
in the cells
Interphase cell
centrosome
Cilla
Basal body
- Centrosome
Dividing cell
spindle
- Cilia / flagellum
- Mitotic system
Neuron
centrosome
- Vesicular transport
axon
MTOC = Mikrotubul organizing center
g-tubulin
specialized
region of
the cortex
((Brinkley, B.R. Encyclop. Neurosci. 1987, 665)
Network of microtubuli
24 nm
ab dimer
Protofilaments
a tubulin
b tubulin
Fibroblast
Cilia
cilia
flagellum
Paramecium
tubulin
(13 ill. 11 protofilaments)
B
A
dynein-arms
nexin
Composition of dynein-arms
ATP-independent binding
ATP-dependent hydrolisis
The arm moves toward the
-
pole
The role of dynein arms
in beating of cilia
Bending
„Telescoping”
Proteolysis
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

Microtubules of mitotic spindle
and kinetochore
Arrangement of actin during cell-division
Intermedier filaments
Mechanical characterization
of cytoskeleton components
intermedier filament
i.e. vimentin
deformation
microtubule
= rupture
actin filament
force
Role of intermedier filaments
Buffer against external mechanical stress
Tissue specificity
Nucleus – lamines
(lamina fibrosa)
Epithel – keratin
Connective tissue
Muscles
Neuroglia
}
vimentin
Neurones - neurofilaments
Structure of intermedier filamentums
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)
Domain structures of intermedier filamentums
H2N-
a helical domain
keratins
vimentin
neurofilam. prot.
nuclear prot
COOH
Intermedier filaments
Keratin filaments
Vimentin-like filaments
! They DO NOT co-polymerise !
Microvilli
actin
myosin
I.
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
SEM structure of microvilli
actin bundle
linker molecules
„terminal web”
Intermedier filaments
Glial filaments
Neuro-filaments
– many cross-linkers – few cross-linkers
The number of protein cross-links between the
intermedier filaments varies in different tissues
Microtubuli associated proteins
(MAP-s)
Groups of MAP-s
•
Structural MAP-s
- MT-assembly
- links to MF and to IF
•
Motor proteins
- sliding on MT
•
Shape and polarity
of the cell
Membrane transports
Enzymes, signal molecules
- glycolytic enzymek
- kinases
Assembly of molecules
Motor-proteins
Structure of motor-proteins
asszoc.
motor domain
polypeptides
motor
domain
„stalk”
asszoc.
polypeptides
Kinesin
„stalk”
asszoc.
polypeptides
Myozin
Dynein
Motor proteins
-
+
microtubule
heavy
chain
light chain
kinesin dynein
kinesin
dynein
kinesin
-
+
dynein
cAMP
pigment cells
cAMP
Kinesin
ADP
ATP
ADP
ADP
ATP
ATP
ADP
ADP-Pi
MT-motor proteins and the transported elements
(Hirokawa, N. Science 1998, 279:519
Dynein – membrane relations
(Hirokawa, N. Science 1998, 279:519)
There are other mechanisms
over sliding …
Locomotion – with spasmoneme of
Vorticella
Spasmoneme spring
Contracts 40% in few msecs
Velocity: 8 cm˛/sec
Negative
charges
Neutralization
with Ca2+
Actin spring in sperm of horseshoe 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
!
Signalling mechanisms
– In a nutshell –
Polymerization of
microtubules
Significance of
motor proteins
Ameboid movement
and microtubules
Complex effects of focal adhesion
kinase (FAK)
LIM domains of proteins
Central role of vimentin in signalling
mechanisms of cytoskeleton
Phosphatases and cell
migration