Project Description
This study shows the dynamic and cooperative binding of multiple vinculins to a stretched antiparallel dimer of talin using super resolution microscopy.
These findings were published in Nano Letters in 2016.
Hu X, Jing C, Xu X, Nakazawa N, Cornish VW, Margadant FM, Sheetz MP. 2016. Cooperative Vinculin Binding to Talin Mapped by Time-Resolved Super Resolution Microscopy. Nano Lett. Jul 13; 16(7):4062-8. doi: 10.1021/acs.nanolett.6b00650.
More information on the Sheetz lab.
Figure: 1. Talin monomer is tagged at the N-terminal with GFP and the C-terminal with mCherry 2. In a talin dimer, the separation between the N-termini is 162 ± 44 nm, whereas the C-terminal dimerization domains colocalize 3. Upto 10 vinculins binds the talin dimer when talin is stretched to 180 nm. (* Note that the 3 part of the gigure can have 180nm separation between the two N-termini.
This study shows the dynamics of binding of vinculin to talin, both focal adhesion (FA) proteins, using time –resolved super resolution microscopy. FAs are multi-protein structures that connect cells to their extracellular matrix. FA proteins play roles in sensing the mechanical micro environment of the cells and in transducing forces that influence cellular processes such as cell migration.
Here the researchers used two fluorophores to tag the N-terminal (GFP) and C-terminal of talin (mCherry) to visualize the antiparallel talin dimer and measure changes in talin length as talin stretches in response to force. They further used a third fluorophore (Atto 655) to tag vinculin and monitor the dynamics of vinculin binding to stretched talin.
This work reveals the cooperative binding of multiple vinculins to a stretched anti parallel dimer of talin.
From the paper – This video shows the co-operative binding of multiple vinculin molecules (pink) to a stretched antiparallel talin dimer that has its N-termini (green) separated and the C-termini (red) co-localized and mobile
Understanding the basics
Talin is a cytoskeletal protein that is localized at cell- ECM junctions and links the cytoskeleton to the cell membrane by binding directly to both the actin filaments and the integrin cytoplasmic tails at the focal adhesions (FA). Talin plays a key role in mechanotransduction and is required for the initiation of FAs. Talin contains a 47-kDa N-terminal head, a neck and a 220kDa rod domain. The head domain comprises four subdomains termed F0, F1, F2 and F3, with the latter three forming a three-lobed FERM domain. Integrin tail binding occurs via the F3 phosphotyrosine binding (PTB) domain via a unique interaction with the integrin membrane proximal region, which is sufficient for integrin activation. The rod contains an additional integrin-binding site (IBS2), two actin-binding sites (ABD) and several vinculin-binding sites that are shown to be exposed by stretch in response to force. Vinculin binding reinforces and increases the stability of adhesion sites.
Vinculin frequently links adhesion receptors (e.g. integrins) to the contractile actin-myosin cytoskeleton by binding either talin through its amino-terminal globular head domain, or paxillin through its rod-like tail domain. Vinculin generally forms two structural states, an open (active) and closed (inactive) state, which are controlled by interactions between the head and tail domains. Whether vinculin can bind to other factors depends both allosterically and sterically on the formation of the complete open state. This in turn is favored by combinatorial binding of ligands namely talin, phosphatidylinositol 4,5-bis-phosphate [PIP2] and actin.
Super-resolution microscopy overcomes the diffraction limit of conventional light microscopy by at least a factor of two, providing a resolution of 100 nm or smaller. Diffraction is a manifestation of the wave properties of light. In a typical light microscope operating in the visible spectral range (400-700 nm) (see Figure below), diffraction determines the smallest focal volume that light can be focused into. The diffraction limit of light microscopes, known as the point spread function (PSF), limits resolution to ~250 nm in the X-Y image plane and ~500 nm along the Z optical axis . In conventional light microscopes such as confocal or TIRF (Total Internal Reflection Fluorescence), structural features lying closer to each other than the PSF length scale cannot be resolved. This includes cellular structures that contribute to cell motility, force generation and mechanosensing such as actin filaments, microtubules and focal adhesion complexes. This limitation has been overcome with the development of various super resolution microscopy methods. In Superresolution microscopy, the subcellular structures are tagged with fluorescent molecules. These molecules are then excited, causing them to emit light. This fluorescence is then captured by sensors and cameras attached to the microscopes.
The Study in Detail
Key Findings
- In a talin dimer, the separation between the N-termini are 162 ± 44 nm, whereas the C-terminal dimerization domains colocalize and are mobile
- Optimal vinculin binding occurred when talin was stretched to 180 nm
- When talin is stretched between 50-180 nm, the vinculins bound to the N-termini of talin whereas at high stretches >210 nm, the binding occurred at the C termini of talin
- Upto 10 vinculins bound to an antiparallel, stretched talin dimer in 1 s in a highly cooperative manner
- (Note: The number of vinculins observed in this study provides a quantitative lower bound since Atto655 does not bind to every functional recombinant vinculin used in the study. Also there will be endogenous non fluorescent vinculins that bind talin.)
Methods and Controls used in the study
- The separation between the two N-termini or C-termini in a talin dimer was measured by extending a previously described method for measuring talin length in vivo by tagging its N-terminal with GFP and C-terminal with mCherry .
- A third fluorophore Atto655, which was spectrally discernible ( different wavelengths and different excitation dynamics) from GFP and mCherry, was used to tag vinculin and visualize its binding to talin while tracking talin length
Applications and Future Directions
- This method can be applied to study the dynamics of binding of other proteins that undergo stretch-relaxation cycles within the cell such as focal adhesion kinase, α-actinin, filaminA and p130Cas
- This method can be used to study the order of binding of proteins in the focal adhesions in response to force
- Allows for quantification of stretch and binding dynamics
