Biophysics of Molecules
Concepts of cell adhesion and cellular
mechanostransduction – part I
Dr. Carsten Grashoff
MPI of Biochemistry
E-mail: [email protected]
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
2
Mechanosensitivity
- the ability to sense
and respond to
mechanical forces
is called
mechanosensitivity
osteocytes
myocytes
endothelial cells
- all prokaryotic and
eukaryotic cells are
mechanosensitive
epithelial cells
cardiomyocytes
keratinocytes
3
Many disorders are ‘mechanical diseases’ !
- endothelial cells line the blood
vessels and are constantly
exposed to the shear flow of
the blood
- endothelial cells sense and
respond to the shear flow
DeBakey et al.
1985
atherosclerosis
- the first atherosclerotic
plaques form in areas of
disturbed blood flow
4
Cancer cells are mechanosensitive
- primary tumors are always
more rigid than their
environment (tumor cells are
more soft though)
- increased tissue rigidity
promotes tumor growth
Levental et al., Cell, 2009
5
Many biological processes are mechanosensitive
push
- cells generate mechanical
forces to migrate
- but they also sense their
mechanical environment
pull
- cells will usually move
towards stiffer substrates; this
process is called durotaxis
6
What we would like to understand
1. How does the cell recognize its
mechanical environment ?
2. How does the cell transduce mechanical
information into a biological response ?
3. How can we measure that ?
7
What we would like to understand
1. How does the cell recognize its
mechanical environment ?
What is the mechanical environment ?
8
What characterizes the mechanical environment ?
-
the mechanical
properties of the
cellular environment
are characterized by
the extracellular
matrix (ECM)
-
the ECM dictates
the biochemical
and biophysical
properties of the
environment
9
The extracellular matrix (ECM)
The ECM consists of many
different macromolecules.
We distinguish:
-
filamentous proteins
-
glykosaminoglycanes
(GAGs)
-
proteoglycanes
-
adhesion proteins
10
Collagen: the most abundant EMC protein
- collagen is the most
abundant protein in the
human body
- we know 28 different
collagen subtypes
consisting of 46 distinct
polypeptides
- collagens are
evolutionary conserved
11
Collagen and the collagen triple helix
- collagen is characterized
by a typical amino acid
repeat:
(G-X-Y)n
G: glycine
X: proline
Y: hydroxy-proline
12
Collagen and the collagen triple helix
- collagen is characterized
by a typical amino acid
repeat:
(G-X-Y)n
G: glycine
X: proline
Y: hydroxy-proline
- the collagen repeat leads to
the formation of a helix with
one repeat per turn
- three helices assemble to
form the collagen triple-helix
13
The role of hydroxyproline
(G-X-Y)n
prolin
X: often Prolin
Y: often Hydroxy-Prolin
prolylhydroxylase
- hydroxyprolin stabilizes
the triple-helix through
stereo-electronic effects
+ Vitamin C
hydroxyprolin
- hydroxyproline is not a
natural amino acid and has
to be synthesized by the cell
- lack of hydroxyproline
leads to scurvy
14
Collagen fibers are very long
- an average collagen triple
helix is 300 nm long and
displays a diameter of less
than 2 nm
- collagen fibers can get 1
cm long and reach a
thickness of 500 nm
- this is achieved through
collagen fibrillogenesis
15
Collagen fibrillogenesis is a multi-step process
- collagen synthesis occurs within the cell
- collagen fibrillogenesis occurs extracellulary
16
Lysyloxidase (LOX) catalyzes collagen crosslinking
- collagen fibers are
crosslinked during
collagen fibrillogenesis
- lysyloxidase (LOX)
catalyzes the oxidation of
lysine groups
- LOX-expression correlates
with malignancy of breast
cancer
- most primary tumors
are more rigid because of
increased collagen
crosslinking
17
Collagens can form many distinct networks
- collagens can form
sheets, fibrils, anchors,
membranes, etc.
- these networks have
distinct biochemical and
mechanical properties
18
Defects in the collagen protein cause diseases
Scurvy
(general)
Osteogenesis
Imperfecta (Col1A)
Morbus Ehlers-Danlos
(Col1A, Col3A, Col5A)
Stickler-Syndrom (Col2A,
Col2A, Col11A)
19
Mechanical properties of the ECM
The problem
Collagens mediate tensile strength.
But the ECM has to be elastic as well.
What mediates matrix elasticity ?
20
Our tissue needs to be elastic !
skin
blood vessels
21
Elastin gives tissues their elasticity
- many tissues need to be
elastic such as the skin, lung or
blood vessels
- elastin content increases by
500 % in the uterus during
pregnancy
- the typical elastin repeat is
very similar to the collagen
repeat:
(P-G-V-G-V-A)n
- but elastin is essentially
unstructured and behaves as
an entropic elastomer
22
Elastin fibers behave as entropic springs
- individual elastin fibers are
cross-linked through covalent
bonds
- ΔS
+ ΔS
- each elastin molecule in the
network can extend and
contract in a manner
resembling an entropic spring
so that the elastin fiber will
recoil after transient stretch
23
Elastomeres and the elastic module
- ideal elastomeres show a
linear-elastic behaviour
linear-elastic
non-linear elastic
elastic modulus:
stress σ [ N / m2 ]
non-elastic
break
E=σ/ε
[ N / m2 ] = Pa
[ N / mm2 ] = MPa
[ kN / mm2 ] = GPa
Erubber ~ 0.01-0.1 GPa
strain ε [ Δl / l ]
Ecollagen ~ 1.2 GPa
24
Examples of naturally occurring elastomers
elastin
(P-G-V-G-V-A)n
flagelliform
(G-P-G-G-A)n
resilin
(G-G-R-P)n
E ~ 0.0011 GPa
E ~ 0.003 GPa
E ~ 0.002 GPa
Erubber ~ 0.01-0.1 GPa
Ecollagen ~ 1.2 GPa
25
Elastin concentration in the aging skin
- the elastin concentration gradually decreases in aging skin and
tissue elasticity is reduced
26
Mechanical properties of the ECM
The problem
Collagens mediate tensile strength and elastin confers elasticity.
How about compressibility ?
27
What mediates compressibility ?
28
GAGs mediate compressibility
- glycosaminoglykane
(GAGs) consist of
repeating sugar subunits
- depending on the sugar
side-chain we distinguish:
hyaluronan
chrondoitin sulfate
dermatan sulfate
keratan sulfate
heparan sulfate
29
GAGs occupy a large volume
- polysaccharide chains are too stiff
to adopt a compacted structure and
are very hydrophilic
- even at low concentration GAGs
form gels, which attract cations
(such as Na+) causing water
incorporation and swelling
- GAGs fill most of the space in the
cartilage
30
GAG example - Hyaluronan
- the concentration of hyaluronan is reduced in osteoarthritic joints
- injection of hyaluronan into the joint (vascosupplementation) is
used to reduce symptoms
31
Proteoglycanes
- proteoglycanes are
composed of GAGs covalently
linked to a core protein
- proteoglycanes are a very
heterogenous group of
molecules and can be complex
- they have many functions
and may serve as adhesion
proteins (syndecan), linker
between other matrix proteins
(decorin) or as a sponge
(aggrecan)
32
Proteoglycane - Aggrecan
33
Adhesive glycoproteins
- glycoproteins are smaller and more flexible than proteoglycanes
- glycoproteins only bind small saccharide side chains
- they often connect to other matrix molecules (such as collagen)
- they often self-assemble (i.e. laminin, fibronectin)
34
The adhesive glycoprotein laminin
- laminin can self-assemble (trimer formation)
- laminin interacts with proteoglycanes (perlecan)
- laminin is recognized by cell surface receptors called integrins
35
Summary - ECM
- the ECM composition determines the biochemical and
biophysical properties of the cellular environment
- the ECM contains a large variety of molecules that can
be classified into proteins, glycosaminoglycanes (sugars),
proteoglycanes and glycoproteins
- collagens mediate tensile strength
- elastins mediate elasticity
- GAGs mediate compressibility
36
Summary - ECM
sugars
elastomers
triple helix
37
What we would like to understand
1. How does the cell recognize its
mechanical environment ?
How does the cell sense the ECM ?
38
Direct and indirect sensing mechanisms
- ion-channels can rather
unspecifically sense
mechanical forces
(stretch-dependent ionchannels)
- cells express surface
receptors which bind
distinct ECM molecules:
- we distinguish:
cell-cell adhesions
cell-ECM adhesions
39
Overview of cell adhesion structures
40
Integrins mediate cell-matrix adhesions
α
β
- integrins are heterodimeric
transmembrane proteins and
consists of an a and a b-subunit
extracellular
- integrins bind to various ECM
proteins such as collagens,
laminins or fibronectin
intracellular
- integrins are the main ligands for
the ECM
41
Human cells express many integrins
a1
a2
a3
a4
a5
a6
a7
a8
a9
a10
a11
aV
aIIb
aD
aX
aL
aM
aE
α
β
extracellular
b1
b2
b3
b4
b5
b6
b7
b8
intracellular
42
Integrin receptor classes
43
Integrins are very important
- without integrins
embryos die even
before implantation
into the ueterus !
44
Integrins are important for platelet activation
- platelets need to be activated
for aggregation
- b1 and b3 integrins are
essential for platelet
activation
inactive
active
- defective integrins on platelets
leads to thrombastenia
(bleeding disorder)
45
Integrins can be active or inactive
EZM
inactive
conformation
active
conformation
Integrin activation is regulated !
46
Intracellular activation by talin and kindlin
Kindlin
Talin
no (αIIbβ3): no platelet aggregation
no talin-1: no platelet aggregation
no kindlin-3: no platelet aggregation
47
Integrin-connection to the cytoskeleton
The problem
Integrins can not bind to the cytoskeleton directly.
How do integrins connect the ECM with the
cytoskeleton?
48
Focal adhesions form at integrin tails
- upon integrin activation and
ECM binding cells form
intracellular complexes called
focal adhesions (FAs)
- FAs consist of 100‘s of proteins,
which link integrins to the
cytoskeleton
>100 Proteine
- FAs are very dynamic and
undergo a constant turnover
- FAs are the cells’ feet !
49
FAs affect many cellular processes
- FAs contain many different
proteins (structural proteins,
enzymes, etc.)
adapter proteins
kinases, phosphatases,
proteases, etc.
cell division
cell migration
mechanical integration
50
Summary – cell adhesion and integrins
- cells adhere to each other using cell-cell contacts
- cells adhere to the ECM using a family of cell surface
receptors called integrins
- integrins have to be activated by intracellular proteins
- integrins connect to the cytoskeleton in subcellular
structures called focal adhesions
51
What we would like to understand
2. How does the cell transduce
mechanical stimulation into a biological
information ?
How do integrins (FAs) sense
mechanical forces ?
52
Integrin-dependent force transduction
- cells pull on the ECM at
FAs !
- the ECM pulls at cells in
FAs!
What happens on the
molecular level ?
53
Talin can interact with integrins and actin
FERM domain
(integrin binding)
vinculin binding domain
actin binding domain
dimerization domain
vinculin binding
domains
54
A model of force transduction in FAs
f-actin-talin
binding
55
A model of force transduction in FAs
forces of the actin
cytoskeleton
56
A model of force transduction in FAs
vinculin
recruitment
forces of the actin
cytoskeleton
57
A model of force transduction in FAs
vinculin
recruitment
to talin
58
A model of force transduction in FAs
higher forces at the
integrins
59
A model of force transduction in FAs
integrin clustering
60
Measuring force transduction
The problem
We need to know which molecules are exposed to mechanical
forces within the cells.
How can we measure mechanical forces in cells ?
61