Zelluläre Bewegungsmechanismen “Intro cytoskeleon and actin”

Zelluläre Bewegungsmechanismen “Intro cytoskeleon and actin”

Zelluläre Bewegungsmechanismen
Vorlesung im WS
Mo 16-17 Uhr, Mi 16-17 Uhr
“Intro cytoskeleon and actin”
Uwe Wolfrum
Institut für Zoologie
[email protected]
Text books:
Literature
Bruce Alberts et al.:
Molecular Biology of the Cell 4th Ed. 2002
Dennis Bray:
Cell Movements 2nd Ed. 2001
Harvey Lodish et al.: Molecular Cell Biology
4th Ed. 2001
Kleinig / Sitte Zellbiologie
Fischer 1999
Bershadsky / Vasiliev Cytoskeleton Plenum 1988
Web
- pages: see
http://www.uni-mainz.de/FB/Biologie/Zoologie/abt1/Wolfrum/
1
Function of the cytoskeleton
• stabilization
• intracellular transport
• signal transduction
Composition of the cytoskeleton
•
•
•
•
actin filaments (6-10 nm)
microtubules (25 nm)
& associated proteins
intermediate filaments (11 nm) & molecular motors
nano filaments (2-6 nm)
Cytoskeletal elements differ in diameter.
Motility and trafficking
microtubules
kinesins
dyneins
actin filaments
myosins
2
Molecular motors
nano filaments
?
?
microtubules
actin filaments
microtubules
intermediate
filaments
Nature Reviews
Cytoskeletal elements in eukaryotes
nano filaments
actin filaments
intermediate filaments
microtubules
6-10 nm
10-13 nm
25 nm
according to Alberts et al.
3
Distribution of different cytoskeletal elements
in the same cell
nano filaments
(e.g. centrins)
actin filaments (F-actin)
(rhodoamin-phaloidin)
intermediate filaments (IF)
(anti-vimentin)
microtubules MT)
(anti-tubulin)
Bundles of actin filaments in cells
ruffles
microvilli
contractile bundles
in the cytoplasm
(stress fibers linked
to focal contacts)
= muscle
lamellipodia
(sheetlike)
filopodia
contractile ring
during
cell division
(fingerlike)
4
Actin features?
• 6 actin genes in birds and mammals (only one in yeast)
• 2D-gel electrophoresis differentiates actin in: α-, β-, and γ-actin
- 3 α-actins: expressed in the 3 types of muscle cells
- β-actin:
cytoplasmic actin
2D-gel electrophoresis
(20 human pseudogenes)
- γ-actin:
1 cytoplasmic actin
2 smooth muscle actins
Actin is the most abundant protein in the cell:
- 2% of total protein in a regular cell
- 10-20% in muscle cells
• G-actin polymers to actin filaments (F-actin)
Actin filaments (F-actin)
actin molecule = G-actin (2.73 nm)
10 nm
{
plus end
38 nm
= 14 G-actins
minus end
• double helical structure
• polarity
5
Actin filament polarity
minus end
A
plus end
minus end
pointed end
plus end
minus end
100 nm
B
Myosin II digestion
plus end
barbed end
Actin polymerization II
minus end
ATP
ADP
plus end
6
Polymerization
Distrubution of globular G-actin and filamentous
F-actin in the same cell
F-actin: OregonGreen 488 phalloidin
G-actin: Texas Red deoxyribonuclease I
in vivo: G-actin : F-actin 1:1
in vivo: [G-actin] ~ 50-200µmol
(2-8 mg/ml)
in vitro polymerization of pure
G-actin at [G-actin] 1 µmol
- in vivo most actin should be
polymerized to F-actin.
in vivo regulation of actin polymerization
by actin-binding proteins
cultured bovine pulmonary artery endothelial cell
7
actin monomers
monomer
-sequestering
protein
nucleation protein
thymosin, profilin
profilin
severing protein
gelsolin
bundling protein
(in filopodia)
actin filaments
motor protein
cross-linking protein
(in cell cortex)
myosins
filamin
side binding protein
tropomysoin
caping (end-blocking) protein
gelsolin
Classes of actin-binding proteins
Bundles of actin filaments in cells
ruffle
tail
tail
filopodia
ruffle
tail
lamellipodia
filopodia
lamellopodia
cortex
8
Actin-binding proteins in vertebrate cells
FUNCTION
filaments
strengthen
filaments
bundle
filaments
cross links
filament into gel
E.G.
FEATURES
370 x 42 kDa/µm
actin
tropomyosin
fimbrin
α-actinin
- end
+ end
2 x 35 kDa
14 nm
68 kDa
40 nm
2 x 100 kDa
filamin
2 x 270 kDa
2 x 265 plus 2 x 260 kDa
attach sides of
filaments to
plasma membrane
spectrin
contractile units
myosins
β
myosin I
α
150 kDa
myosin II
monomer
binding proteins
ACTIN-INTERACTION
2 x 260 kDa
thymosin
myosin II
5 kDa
Actin filaments are polermerized at tip of
leading edge
Entire F-actin:
FITC-phalloidin
Newly formed F-actin:
Rhodamin-actin
Experiment:
Fibroblasts - permeabilization with detergent - rhodamin-actin incubation
- 5 min actin polmerization - FITC-phalloidin staining
9
Monomer actin-binding proteins
protein binds and
keeps actin in
ADP-bound form
thymosin
- 4β: • size 5 kDa
• binds to G-actin 1:1
• sequestering activity
inhibits actin polymerization
and nucleotide exchange
site that binds
to F-actin
protein bound over
actin-binding site
profilins: profilin:actin complex = 1:1
• complicated role in actin polymerization!
• enhances exchanges of nucleotides of G-actin
• bind to G-actin, to Arp2/3 complex, to VASP, ...
and is associated with the plasma membrane.
Actin polymerization in vivo
10
Actin polymerization cycle in vivo
fast growing end
(+ end; barbed)
k+ 5 µM-1s-1
k- 1 s-1
slow growing end
( - end; pointed)
k+ 0.1 µM-1s-1
k- 0.2 s-1
UW Bewegungsmechanismen 2002
Filipodium formation
unactivated
PIP2 = Phosphatidyl inositol 4-diphosphat
activated
• profilin-actin elongates fast growing end
• profilin binding increases exchange nucleotides
11
Acrosomal process in sea urchin sperms
profilin-actin complex
pH increase
profilin
actin
Tilney et al. 1982
Acrosomal process in Limulus sperm
Sucrin:
102 kDa
Ca2+-binding protein
EF-hand protein
sucrin-sucrin bridges
between parallel
actin filaments
Actin filament bundle induced by [Ca2+] concentration increase
12
Microvilli
myosin I + calmodulin
villin, fimbrin
actin filaments
plasma membrane
glycocalyx
microvilli with core filaments
Terminal web of microvilli
spectrin
myosin II
tropomyosin
13
Actin-binding proteins in vertebrate cells
FUNCTION
filaments
strengthen
filaments
bundle
filaments
cross links
filament into gel
E.G.
FEATURES
370 x 42 kDa/µm
actin
tropomyosin
fimbrin
α-actinin
ACTIN-INTERACTION
2 x 35 kDa
14 nm
68 kDa
40 nm
2 x 100 kDa
filamin
2 x 270 kDa
2 x 265 plus 2 x 260 kDa
attach sides of
filaments to
plasma membrane
spectrin
contractile units
myosins
β
myosin I
α
150 kDa
myosin II
monomer
binding proteins
+ end
- end
2 x 260 kDa
thymosin
myosin II
5 kDa
Actin related proteins Arp´s
• Arp´s are closely related to actin.
• Arp-classes:
Arp 1 (~ 50% homology to actin),
Arp 2 (40-50%), and Arp 3 (30-50%)
Arp 1 (centractin):
• component of the
dynactin complex of
cytoplasmic dynein
(MT minus end motor)
• connects motor complex
to cargo vesicle
14
Actin nucleation: ARP2/3 complex
Actin nucleation: ARP2/3 complex
15
Actin filament branching at the Arp2/3 complex
4 µM G-actin (unlabeled)
&
Red rhodamin-phalloidin
(mother filament)
&
Green Alexa 488-phalloidin
& Arp2/3 complex
(a,b: bovine; c-e: amoebae)
5 µm
Wiskott-Aldrich Syndrome protein (WASP)
• Human Wiskott-Aldrich Syndrome is a rare recessive X-linked
immunodeficiency syndrome effecting young boys.
• WASP has been shown to be defective in patients.
• WASP is an actin nucleation factor involved in phagocytosis.
WASP activation
WASP domain structure
actin Cdc42 profilin
actin
Arp2/3
inactive
activated
N-WASP: brain isoform
Cdc42 binds to
activation domain
binding sites
accessible
16
Arp 2/3 mediated actin nucleation at the plasma
membrane
• WASP and VASP (vasodilator stimmulation phospho-protein)
act as actin nucleation factors.
• Zyxin anchors VASP at the integrin complex of the membrane.
zyxin
17
Actin filament polymerization by Listeria
monocytogenes: intracellular motility of bacteria
Actin filament
(FITC-phalloidin)
ActivinA (ActA)
(rhodamin)
Listeria infection of a fibroblast
Listeria monocytogenes
• Das Bakterium Listeria monocytogenes tritt in verdorbenen
Lebensmittel auf und eine Listeria-Infektion führt beim
Menschen zu einer Lebensmittelvergiftung.
• Bei der Infektion einer Wirtszelle nutzt das Bakterium das
“System” der Zelle aus:
1. Aufnahme von Listeria durch Phagocytose durch einen
“zipper mechanism”: Internalin 1 A (In1A) ein Oberflächenfaktor des Bakteriums bindet über sein LRR-Motiv an CellCell Adhersionsproteine, E-Cadherine der Wirtszelle.
Ausbildung eines Phagocytose Vesikels incl. Bakterium.
2. Verlassen des Phagocytosevesikels und Übertritt in das
Cytoplasma der Wirtzelle.
3. Fortbewegung des Bakteriums im das Cytoplasma der
Wirtzelle (11 µm/sec) durch die Polymerization von
Actinfilament.
4. Ausbildung von “microspikes” zur Infektion von
Nachbarzellen.
free Listeria
host cell
phagocytosis
escape
actin polymerization
motility
microspikes
infection of neighboring cell
18
TEM of actin filament polymerization by Listeria
actin tail
infection of neighboring cell via micrspikes
Mechanism of Listeria actin tail formation
F-actin nucleation is madiated by
Arp2/3 in eukaryotic cells
F-actin nucleation at the Listeria tail
is also mediated by Arp2/3
Listeria protein ActivinA (ActA)
= Zyxin + VASP = WASP
19
Drugs that affect actin filaments and
microtubules
ACTIN-SPECIFIC DRUGS
Phalloidin
Cytochalasin
Swinholide
Latrunculin
binds and stabilizes filaments
caps filament plus ends
severs filaments
binds subunits and prevents their
polymerization
MICROTUBULE-SPECIFIC DRUGS
Taxol
Colchicine, colcemid
Vinblastine, vincristine
Nocodazole
binds and stabilizes microtubules
bind subunits and prevents their
polymerization
20