Biophysics of Molecules
Cytoskeletal filaments, Actin
polymerization and Actin Treadmilling
Part 1 (26.11.2012)
Dr. Carsten Grashoff
MPI of Biochemistry
E-mail: [email protected]
Lecture Outline – The Cytoskeleton
1. Actin dynamics and the f-actin network
2. Tubulin dynamics, the tubulin network
and intermediate filaments
2
What is the cytoskeleton ?
-
highly organized
system of filamentous
networks
-
consists of highly
dynamic networks with
specific functions
-
essential for life !
3
The three cytoskeletal networks
f-actin
tubulin
intermediate
filaments
4
What does the cytoskeleton do ?
- the cytoskeleton
has many and very
diverse functions
cell shape
cell division
- the cytoskeleton
is involved in the
most fundamental
processes such as
cell division, cell
migration and cell
differentiation
- all organism have
a cytoskeleton
cell migration
mechanical integrity
5
Why do you need to know this ?
The cytoskeleton is fundamental to most if not all
cell biological processes
Development and homeostasis depends on the
cytoskeleton
Many disorders are caused by cytoskeletal defects
Many therapies interfere with the cytoskeleton
6
The actin cytoskeleton
7
The actin monomer is the smallest subunit
- the smallest subunit of the
actin network is the actin
monomer
- actin is a very abundant
protein; in the muscle 1020 % of all protein is actin
- the actin molecule has a
plus (+) end and a minus (-)
end
- the actin molecule has a
nucleotide binding site (for
ADP or ATP)
8
Fibers are formed by polymerization
- the actin network
consist of f-actin cables
that are polymerized actin
The actin monomer is
called g-actin
The polymerized actin is
called f-actin
(for globular actin)
(for filamentous actin)
9
Three steps of actin polymerization
Actin polymerization
involved three steps:
-
nucleation
-
elongation
-
steady-state
(equilibrium)
the resulting actin
filament has a polarity:
-
fast-growing (+) end
(barbed end)
-
slow-growing (-) end
(pointed end)
10
Actin polymerization: nucleation
-
actin nucleation is the rate-limiting step and takes the longest
- three actin monomers form a nucleus
- the actin monomer is bound to ATP; upon polymerization ATP is
quickly hydrolyzed to ADP (not instantaneous though)
11
Actin polymerization: filament elongation
-
elongation of an existing actin filament is fast
- the rate of polymerization (kon) depends on the concentration of
free actin monomers
- the depolymerization rate (koff) does not depend on the
concentration of the free subunit
12
Actin polymerization: the steady state
- as the filament grows a critical concentration (CC) will be reached
at which subunits addition equals subunit dissociation
13
Actin treadmilling
-
the critical concentration CC at the (+) end is different from the
CC at (-) end (because on-rates are different)
- if the concentration is above CC for the (+) end but below CC of the
(-) end, filaments will undergo an assembly at the (+) end and
disassembly at the (-) end
- despite the constant assembly and disassembly the length of
the filament remains constant – this is called treadmilling
14
The actin network is highly dynamic
15
Inhibition of actin dynamics are toxic
phalloidin
Amanita phalloides
(Knollenblätter-Pilz)
-
phalloidin is a toxin from Amanita phalloides that binds f-actin
and strongly reduces rate constant for actin depolymerization
- labelled phalloidin is used to visualize the f-actin cytoskeleton
- do not eat that mushroom !
16
Other toxins that affect actin dynamics
Latrunculina magnifia
latrunculin
latrunculin binds to
actin monomers and
prevents polymerization
Helminthosporium
cytochalasin
cytochalasin binds to
actin monomers and
prevents polymerization
17
Effects of latrunculin on the actin network
18
Regulation the actin network
The problem
The critical concentration for actin polymerization is ~ 1 μM.
The concentration of g-actin in the cytoplam is ~ 100 μM.
How does the cell maintain a pool of
unpolymerized actin ?
19
Regulating monomeric actin concentration
-
profilin binds g-actin and
alters its conformation
-
profilin binding to actin
stabilizes ATP-actin
-
profilin inhibits actin
nucleation but promotes
polymerization at the (+)
end
-
thymosin keeps a pool of
free actin monomers
-
thymosin prevents actin
polymerization
20
Regulating actin nucleation
The problem
Actin nucleation is the rate-limiting step. It takes 3 actin subunit to
form a nucleus.
Often the cell needs to induce actin polymerization very quickly
(for example during cell migration).
How does the cell control actin polymerization ?
21
Actin nucleation by Arp2 and Arp3
-
Actin-related proteins (Arp) are 45 % identical to actin
-
Arp2 and Arp3 are at the core of the Arp2/3 complex
22
Actin nucleation by the Arp2/3 complex
-
the Arp2/3 complex
consists of 7 proteins
-
the complex needs to be
activated (i.e. by WAVE
and WASP proteins)
-
the activated Arp2/3
complex bypasses the
rate-limiting step of actin
nucleation
- Apr2/3 nucleation is
especially important
during cell migration
23
Arp2/3 actin networks are branched
-
the Arp2/3 complex nucleates actin most efficiently when
bound to an existing filament
- Apr2/3 dependent actin nucleation leads to branched, gel-like
actin networks
24
Actin elongation by formins
-
formins are a family of
dimeric proteins
-
each formin subunit
binds an actin monomer
-
as the filament grows
formins remain
associated with the (+)
end of the actin polymer
- filopodia are
characterized by parallel
actin bundles
25
Controlling actin polymerization
The problem
How to stabilize actin networks?
26
Controlling actin dynamics by capping
-
capping proteins can interact with the (+) end (CapZ) or the
(-) end (tropomodulin) to stabilize the actin filament
27
Stabilizing f-actin fibers by crosslinking
-
crosslinking proteins contain actin-binding domains
-
by binding two actin filaments at the same time the filaments
are bundled and stabilized
28
Crosslinking f-actin fibers
-
actin-crosslinking
proteins are
characterized by actinbinding domains
- usually cells expressing
many actin-binding
proteins at the same time
(i.e. a-actinin)
29
Generating forces across f-actin fibers
-
myosins are an important family of motor proteins that can
crosslinks (bundle) f-actin
- the actomyosin network can generate mechanical forces
30
The actomyosin network in muscle cells
-
muscle cells have a very
extensive actin-myosin
network to generate
mechanical forces
31
Disassembling the cytoskeleton
The problem
If a cell divides or migrates very fast, it has to quickly
disassemble the f-actin structures.
How can f-actin fibers quickly be removed?
32
Actin-depolymerizing factor (ADF) Cofilin
-
the ADF / cofilin family of
proteins bind to actin
monomers and filaments
- cofilins have a higher
affinity for ADP-actin
- severing of filaments created more
(+) ends and thereby lead to
increase actin dynamics
- at very high concentrations, cofilin
can nucleate new filaments
- newly generated fibers,
which still contain ATPactin, are not affected
- binding to f-actin induces
a twist in the filament
which increases the
chance of severing
33
Fine tuning the actin cytoskeleton
The problem
Cells have to be able to respond to external cues
such as growth factors or mechanical forces.
How can the actin cytoskeleton be modulated
from the outside ?
34
Fine tuning the actin cytoskeleton
small Rho-GTPases have
specific effects on the actin
cytoskeleton:
-
activation of Rho
induces myosin II and
leads to strong actin
fibers (stress-fibers)
-
activation of Rac
induces Arp2/3 and
lamellipodia formation
-
activation of Cdc42 leads
to filopodia formation
35
Rho-GTPases induce complex cell signaling
36
The regulation of actin networks is complex
-
many proteins can
directly or indirectly
interact with actin
- many proteins have more
than 1 isoform (there are 4
profilins, 3 cofilins, many
myosins, etc.)
- spatiotemporal regulation
within the same cell
In humans there are 6 actin genes !
37
Let’s summarize…
- the smallest subunit of the actin network is the actin monomer
- the actin monomer can be in the ADP or ATP bound form and
undergo polymerization
- we distinguish: nucleation, elongation and the steady state
- actin networks are highly dynamic structures (see treadmilling)
- cells express proteins which modulate the actin network:
- thymosin, profilin (monomer regulation)
- Arp2/3 (actin nucleation)
- formins (actin elongation)
- crosslinking proteins (network formation, stabilization)
- cofilin (actin severing, filament breakdown)
- these processes are regulated by signaling networks (i.e. Rho GTPases)
38
Why is this important ?
Biological functions of actin networks
39
Keeping cells in shape
40
Cell migration critically depends on actin
lamellipodium:
Arp2/3-dependent network
retraction area:
high myosin II activity
41
Cell migration and the immune response
42
Generating and sensing mechanical forces
-
through the actin myosin
network and cell matrix
adhesion cells generate
mechanical forces
-
examples: heart and
skeletal muscle
-
cells can also sense
mechanical forces with
the actin network
43
Actin fibers connect to focal adhesions
focal adhesions
F-actin fiber
44
Actin signaling is altered in cancer
-
the cell signaling cascades
that regulates actin
networks are altered in
many cancers
-
often Ras- and RhoGTPases
are activated
45
Actin dynamics during pathogen infections
Listeria monocytogenes:
Bacterium
(causes listeriosis)
Listeria motility
offers a biochemically
tractable system
for reconstituting actin polymerization dependent motility
46
Actin dynamics and Listeria infections
47
Summary of biological importance
cells critically depend on actin networks, because:
- actin networks maintain cell shape and stability
- actin polymerization and contraction is essential for cell migration
- actin-myosin networks are critical for stable cell adhesion to the ECM
- actin-myosin networks generate and sense mechanical forces
many diseases affect the actin cytoskeleton:
- signaling networks are affected in cancer
- actin integrity is compromised in muscular dystrophies and myopathies
- some pathogens use actin dynamics
48
The three cytoskeletal networks
f-actin
tubulin
intermediate
filaments
49