Molecular mechanisms of
mechanotransduction
Summer Topic 2011
Tom Seegar
Implication of mechanotransduction in
disease
And you thought reading and watching TV
at the same time is hard
Several different cellular components have been proposed to act as mechanosensors enabling
cells to respond to the extracellular environment.
-Ion Stretch Channels
-Cell-ECM and Cell-Cell contacts
-Cytoskeleton Contacts at the cell surface and nuclear membrane
-Cell Surface Glycocalix layer (endothelial cells)
Current tools utilized to study
mechanotransduction
Mechanisms in which cells convert force
into a chemical signal
• Mechanosensitive Ion Channels
• Cryptic Binding Sites
– Catabolism
– Phosphorylation
– Protein-Protein interaction
• Catch Bonds
Mechanosensitive Ion Channels
Mechanosensitive Ion Channels can be sub-divided into
two categories in respect to the force vector the channels
respond:
-force perpendicular to the lipid bilayer
ex: Ca2+ ear hair cell channels
-force parallel/in plan with the lipid bilayer
ex: bacteria mecanhosensitive K+ channel, MscL
Stretch of the Lipid Bilayer control MscL
opening
MscL and MscS channels have been shown to be responsible for recovery of bacteria from
hypo-osmotic shock by opening in response to membrane swelling
Patch-Clamp studies showed that upon
changing the pressure across the lipid
bilayer caused an opening of an ion
channel.
In the resting closed confirmation the
channel remains closed with a primarily
desolvated core. Upon membrane
stretch the force of separation from the
lipid polar heads and the outer ring
cause the channel to open like an iris in
a camera.
Cryptic Binding Sites
• Talin
– Cryptic protein-protein interaction domain
• Von Willebrand Factor
– Cryptic cleavage site
• P130cas
– Cryptic phosphorylation domain
Von Willebrand Factor
Von Willebrand Factor(vWF) is a multidomain protein secreted from Weibel-Palade
bodies into the blood in response to thrombogenic stimuli.
In its ultra large form (typically containing >200 monomers) vWF aids in the formation of
a hemostatic platelet plug through interactions of the A3 Domain binding collagen and A1
Domain binding platelet glycoprotein Ib molecule. The hemostatic potential of vWF is
directly correlated with its length, therefore cleavage on the A2 Domain by ADAMTS13 is
an important mechanism in vWF catabolism.
After secretion, VWF is digested in ~2hr by ADAMTS13 to yield a pool of circulating small
vWF.
Hypothesis: Large vWF under shear stress exposes a cryptic site with in A2 domain for
ADAMTS13 permitting cleavage into the inactive small fragments
Is Force a Cofactor for ADAMTS13 cleavage
of vWF?
Through the use of Laser trap tweezers, the A2 domain was cycled through a series of force
pulling to observe a transition from folded partially unfolded fully extended
conformation of the the A2 domain.
Unfolding and force clamping the A2 domain at
5pN(140sec refold rate) ADAMTS13 was added and the
cleavage of the A2 domain was observed by a complete
loss in force across the protein tether.
Cells Respond to Stretch and Contraction
via MAPK pathway
Using a flexible polymer based membrane, stretch/contractile force was applied to cells.
The biochemical response was an increase in the MAPK pathway leading to an increase in
p38 phosphorylation mediated by Rap1.
How does stretch activate Rap1?
Stretch-dependent tyrosine phosphorylation of Rap1 was further shown to be performed
in detergent-insoluble cytoskeletal complexes involving an increase in the phosphorylation
of p130Cas by Src family kinases.
Stretch dependent phosphorylation of
p130cas
P130cas has been reported to be involved in various different cellular event such as
migration, survival, transformation, and invasion. P130cas is localized to focal
adhesions through the amino-terminal SH3 domain and carboxyl-terminal Src-Binding
domain. Between the two domains is the cas substrate domain, containing fifteen
tyrosine residues (YxxP).
Stretching the cells by 10% revealed an increase in cas phosphorylation mediated by
Src family kinases(CGP77675 SFK inhibitor)
In vitro Protein Extension Assay
Using an in vitro protein extension assay,
upon stretch of the polymer membrane
bound protein, the CasSD domain become
partially unfolded. This unfolding allows
recombinant SFKs to phosphorylate the
CasSD in a stretch dependent mechanism.
P130cas extension is localized to the
periphery of the cell
The anti-cas1 was developed to
recognize the extended CasSD.
Using Trition X-100 treated
cytoskeletons, anti-Cas1 is capable
of interacting p130cas in a stretch
dependent mechanism. The
localization of the stretched
p130cas was found on the
periphery of the cells during
spreading where traction force is
thought to be elevated.
Talin: Vinculin recruitment to focal complex
upon force resistance
Using a optical trap, FN7-10 fragment
coated beads were trapped at focal
adhesions and the amount of vinculin-GFP
accumulation was monitored in the
presence of external force.
Without force the small surface area
beads (1um) were insufficient in recruiting
vinculin. Although once an external force
was applied, vinculin recruitment was
greatly enhanced to the focal complex.
How does force alter protein localization?
Talin contains cryptic binding sites for
Vinculin
Talin is a large protein capable of creating a physical link of the cellular cytoskeleton to the ECM
by interacting via its N-terminal FERM domain with the tail of integrin Beta1 and attachment to
actin with its C-terminal 5helical bundle.
Between the binding domains is a flexible talin rod domain consisting of 11 vinculin binding
sites, majority of which are buried within the amphipathic helical bundle.
Hypothesis: Physiological force generated across a focal adhesion results in the unfolding of the
talin rod domain exposing cryptic sites for the vinculin head domain to bind.
TR unfolding Force Assay Reveals a cryptic
protein-protein interaction domain
-A portion of the TR Domain was immobilized via a
6xHis Tag and C-terminal biotin group.
-The TR domain was mixed with the Vh domain
labeled with alexa488 under different force pulling
conditions.
-The bound Vh-alexa488 was measured by single
molecule TIRF photobleaching.
TR Domain
Alpha-actinin, served as
a negative control since
it has only one surface
exposed VBS
Vh-alexa488 increases the number of bound molecules to the TR Domain upon addition
of force. Suggesting that the number of vinculin molecules bound to talin is directly
dependent on the applied force.
Bond stability under force
Applying force to protein-protein interactions has the ability to change the k
off rate, favoring an unbound state vs the bound state. This is measured
experimentally by observing a faster disassociation as force increases. This is
typical for most protein-protein interactions. These bond types are called
“Slip Bonds”
Although this is not the case for all protein-protein interactions. In which
applied force does not decrease bond strength but has the ability to increase
bond strength. These types of bonds are called “Catch Bonds”
Catch Bond: Integrin Alpha5 Beta1
Using atomic force microscopy, the binding lifetimes
of FN7-10 to various forms of alpha5-beta1 were
measured under different forces in various different
divalent cation conditions.
Generating force-scan traces:
-The plate is brought into contact with the Tip (Blue)
-Immediately retracted to reduce nonspecific
adhesion (Green)
-Bond formation was allowed to form for
0.5sec(Brown)
-The dish was slowly retracted (Red)
Binding of FN7-10 to Alpha5-Beta1 became
progressively more frequent when the cation
condition was changing from Ca2+/Mg2+ to
Mg2+/EGTA and to Mn2+.
The binding was specific, as it was abolished
by the addition of HFN7.1 (anti-FN) antibody
as well addition of free peptide.
Catch Bond in Integrin Alpha5 Beta1
Bond lifetime measurements under different
forces revealed the existence of a catch
bond:
As force increased, bond lifetimes first
decreased to a minimum, then increased to a
maximum and decreased again, exhibiting a
triphasic transition from a slip bond to a
catch bond back to a slip bond.
In the case of CA2+/Mg2+ the second slip
bond appears between the FN-alpha5beta1
protein-protein interaction. Although the
second slip bond in the Mg2+/EGTA and
Mn2+ appears to possibly occur between the
alpha5beta1FC-GG7.
The catch bond between Fn and alpha5beta1 appears to exist in a range of 10-30pN.
Black line
Grey line
Mechanisms in which cells convert force
into a chemical signal
• Mechanosensitive Ion Channels
• Cryptic Binding Sites
– Catabolism
– Phosphorylation
– Protein-Protein interaction
• Catch Bonds