Biophysics of Molecules
Cytoskeletal filaments, Actin
polymerization and Actin Treadmilling
Part 2 (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
Why do you need to know this ?
Microtubules are essential for cell division
(and therefore essential for life)
Important diseases affect the tubulin network
Most chemotherapies interfere with the
tubulin network
3
The tubulin network
f-actin
tubulin
4
The tubulin subunits
- the smallest subunits of the
tubulin network are a-tubulin and
b-tubulin
- a-tubulin and b-tubulin form a
heterodimer
- both subunits have a GTPbinding site
- the GTP on a-tubulin is trapped
in the heterodimer and can never
be hydrolyzed; b-tubulin can be
in a GDP-or GTP-bound form
5
Microtubule formation
- tubulin heterodimers
form protofilaments,
which have a polarity
- 13 protofilaments form
a microtubule, which
resembles a hollow
cylinder
- due to multiple
molecular interactions
between subunits,
microtubules are very stiff
6
Microtubules are very stiff
- individual protofilaments
are thermally unstable
- assembled microtubule
filaments are thermally
stable
7
Assembly of microtubules
Microtubule assembly
involves three steps:
-
protofilament assembly
-
sheet assembly
-
elongation
-
shortly after subunit
incorporation, GTP is
hydrolyzed
-
if the rate of subunit
addition is faster than
hydrolysis a GTP-cap is
formed
8
Tubulin polymerization
-
each subunit carries a GTP (T)
before incorporation
-
hydrolysis reduces the
binding affinity and GDPtubulin (D) is more likely to
dissociate
-
on-rates are depending on the
concentration of the free
subunit
-
addition of GTP-tubulin to the
(+) end is fast but slow for the
(-) end
9
Tubulin treadmilling
-
the critical concentration of
GTP-rich (+) end and GDPrich (-) end are different
-
at the appropriate subunit
concentrations, when
CC(T)<C<CC(D), tubulin
filaments will undergo
treadmilling
10
Dynamic instability of tubulin networks
-
if the GTP-cap is lost, a
rapidly growing filament
may quickly start
shrinking, a process
called catastrophe
-
if a GTP-cap is regained,
a disassembling
filament may start
rapidly growing, a
process called rescue
-
this inherently
metastable behavior is
known as dynamic
instability
11
Dynamic instability of tubulin networks
dynamic instability can be frequently observed in living cells
12
Dynamic instability in living cells
13
GTP-hydrolysis affects tubulin structure
-
GTP stabilizes a straight
conformation
-
GTP hydrolysis leads to
a structural change and
protofilaments are
bending
-
the GTP-cap structurally
stabilizes the tip of
growing microtubules
14
Growing and shrinking tubulin filaments
15
The tubulin network is highly dynamic
16
Regulation the tubulin network
The problem
Cells make a lot of a- and b-tubulin.
How do cells prevent tubulin polymerization ?
17
Stathmin-tubulin interactions
- stathmin can interact with
tubulin heterodimers
- stathmin-bound tubulin
heterodimers can not
polymerize
- stathmin is regulated during
mitosis by phosphorylation
- mutations in stathmin cause
cancer (stathmin is also known
as “oncoprotein 18”)
18
Regulation the tubulin network
How is microtubule nucleation regulated ?
19
g-tubulin-dependent tubulin nucleation
- tubulin nucleation is
facilitated by a third tubulin
molecule: g-tubulin
centrosome
a-tubulin
g-tubulin
- g-tubulin concentrates in
the microtubule organizing
center (MTOC) called
centrosome
20
The centrosome
- microtubules are nucleated at the centrosome
- microtubules (+) ends point outward towards the cell periphery
21
Centrosome architecture
- each centrosome contains a pair of centrioles (a mother and a
daugther centriole)
- centrioles consist of a short cylinder of microtubules and are
connected by accessory proteins
22
Centrosome replication during the cell cycle
- centrosomes replicate during the cell cycle to generate two
centrosome at the beginning of mitosis
- centrosome replication is not fully understood, but many
cancer cells have too many centrosomes
23
Regulation the tubulin network
The problem
Microtubules are very instable.
(dynamic instability)
How do cells stabilize microtubules ?
24
Stabilizing microtubules at the (+) end
-
XMAP215-like proteins can
be found in every
eukaryotic organism
studied so far
- XMAP215 stabilizes the
weakly bound tubulin
dimer thereby decreasing
dissociating rate
25
Stabilizing microtubules by MAPs
-
microtubule-binding proteins (MAPs) stabilize microtubules
by direct binding
-
MAPs are highly expressed in the brain (i.e. in neurons)
26
High MAP expression in the brain
Distribution of two
MAPs in a neuron
(green: tau; red: MAP2)
27
The special case of the Tau protein
-
the tau protein binds to
microtubules and
stabilizes them
-
expression of tau in cells
induces formation of
microtubules
-
tau gets modified (phosphorylation,
glycosylation, ubiquitinylation,
cleavage, crosslinking, etc.)
-
tau also binds other proteins
-
the physiological role of tau is not
totally clear
28
Tau aggregates cause Alzheimer’s disease
29
Regulation the tubulin network
The problem
In preparation for cell division cells have to disassemble
interphase microtubules quickly.
How do cells break or disassemble
microtubules ?
30
Katanin, the cellular samurai
hexameric AAAATPase complex
-
katanin is an AAA family
ATPase
-
katanin forms a hexameric
complex which uses ATP
hydrolysis to break bonds
between tubulin heterodimers
-
katanin is regulated by
phosphorylation
31
Catastrophe factors
-
some kinesins such as
Kinesin-8 and Kinesin-13
use ATP-hydrolysis to
facilitate depolymerization
-
kinesins can walk on
microtubules and induce
depolymerization at the
ends
32
Regulating tubulin dynamics
33
Fine tuning the tubulin network
-
many proteins are
involved in the
regulation of the tubulin
network
-
spatiotemporal
regulation (regulation in
time space and time)
within the same cell
-
there are many things
we still need to learn
34
Let’s summarize…
- the smallest subunits of the tubulin network is the tubulin
heterodimer consisting of an a-tubulin and a b-tubulin
- the b-tubulin subunit can be in the GDP or GTP-bound form
- heterodimers form protofilaments; 13 protofilaments form a microtubule
- tubulin networks are highly dynamic; they undergo treadmilling, rapid
shrinking (catastrophe) and rescue; this phenomenon is called dynamic
instability
- nucleation of tubulin networks occurs at the centrosome using g-tubulin
- cells express proteins which modulate the tubulin network:
- stathmin to sequester heterodimers
- microtubule-associated proteins (MAPs) to stabilize
- katanin and catastrophe factors to break and depolymerize
- these processes are regulated by complex signaling networks
35
Why is this important ?
Biological functions of tubulin networks
36
Microtubules essential role for cell division
37
Microtubules capture chromosomes during mitosis
Growing microtubules
Shrinking microtubules
38
39
Figure 16-85c Molecular Biology of the Cell (© Garland Science 2008)
Why this knowledge might save your life
The problem with cancer is that cells
division is not controlled.
We need cancer cells to stop
proliferating.
40
Tubulin drugs to fight cancer
paclitaxel
Taxus brevifolia (Eibe)
-
paclitaxel is a toxin of the taxane family
-
paclitaxel binds to b-tubulin and prevents depolymerization of
microtubules
- paclitaxel is used in chemotherapies against many cancers such
as breast cancer, prostate cancer or lung cancer
41
Other toxins that affect tubulin
Catharanthus roseus
Vincristine
Vincristine binds to
tubulin heterodimers and
inhibits filament
assembly (for example
used against lymphoma)
Colchicum autumnale
Cholchicine
Colchicine inhibits
microtubule filament
assembly
42
Microtubules are also important for transport
-
microtubules are used for transport
-
in very long axons many microtubules are aligned
-
we distinguish: anterograde and retrograde axonal transport
43
Vesicle transport in cells
video: Daniel v. Wangenheim
44
Microtubules build cilia and flagella
- many bacteria but also
mammalian cells such as sperm
cells use flagella to move forward
45
Most of our cells have a primary cilium
primary cilia in
epithelial cells
- most of our cells have a primary
cilium ( i.e. fibroblast, epithelial
cells, neurons, chondrocytes, etc.)
- primary cilia sense mechanical
flow and are connected to
mechanosensitive calcium channels
- loss of the primary cilium on
kidney cells leads to polycystic
kidney disease
46
Summary of biological importance
cells critically depend on the tubulin network, because:
- microtubuli are essential for cell division
- microtubuli are the intracellular highways
- the microtubular network supports cell shape and mechanical stability
important diseases are connected to the tubulin network:
- cancer
- Alzheimer disease
47
The intermediate filament network
48
The intermediate filament network
- not all organisms have
intermediate filaments
- the smallest subunit are
elongated coiled-coil
proteins (i.e. keratin)
- parallel dimers
assemble into tetramers
- intermediate filaments
do not have a polarity and
do not bind nucleotides
49
Keratin networks in skin cells
- the integrity of
keratinocytes (skin cells)
depends on keratin-based
intermediate filaments
- intermediate filaments fulfill
important mechanical
properties and influence the
elasticity of cells
50
Keratin networks in skin cells
- loss of the intermediate filament system in the skin leads to
severe skin disorders such as epidermolysis bullosa
51
The three filament networks
f-actin
tubulin
intermediate
filaments
thin (7 nm)
thick (25 nm)
intermediate (10 nm)
ADP/ATP-binding
GDP/GTP-binding
no nucleotide binding
polarity
polarity
no polarity
decentralized
centralized
decentralized
52