Molecular mechanisms of mechanotransduction

1. Molecular mechanisms of mechanotransduction Summer Topic 2011 Tom Seegar

2. Implication of mechanotransduction in disease

3. 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)

4. Current tools utilized to study mechanotransduction

5. Mechanisms in which cells convert force into a chemical signal • Mechanosensitive Ion Channels • Cryptic Binding Sites • Catabolism • Phosphorylation • Protein-Protein interaction • Catch Bonds

6. 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

7. 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.

8. Cryptic Binding Sites • Talin • Cryptic protein-protein interaction domain • Von Willebrand Factor • Cryptic cleavage site • P130cas • Cryptic phosphorylation domain

9. 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

10. 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.

11. 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.

12. 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)

13. 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.

14. 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.

15. 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?

16. 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.

17. 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.

18. 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”

19. 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.

20. 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 alpha5-beta1 appears to exist in a range of 10-30pN. Grey line Black line

22. Mechanisms in which cells convert force into a chemical signal • Mechanosensitive Ion Channels • Cryptic Binding Sites • Catabolism • Phosphorylation • Protein-Protein interaction • Catch Bonds