Cells in tissues exert forces as they squeeze, stretch, flex and pull on each other. These forces are incredibly small, on the scale of piconewtons, but they are essential for cell survival and growth. Key proteins involved in sensing mechanical forces, are the cadherin family of cell-cell adhesion proteins. Cadherins mechanically couple neighboring cells, transmit extracellular forces to the cytosol and transduce mechanical forces into biochemical signals. However, the biophysical mechanisms by which cadherins sense and interpret mechanical forces and regulate adhesion are unknown. Our research targets this critical gap.


How do cells sense and respond to mechanical signals

How do classical cadherins respond to mechanical tension and regulate adhesion? Classical cadherins are essential in the formation and maintenance of tissues. Disruption in adhesion result in diseases like cancer. Our lab is a leader in resolving the biophysical mechanisms by which cadherins tune adhesion in response to mechanical force. Using single molecule Atomic Force Microscope (AFM) force measurements and predictive computer simulations, we showed that cadherins bind in three distinct conformations that form distinctly different types of adhesive interactions (PNAS 2016, Nature Commun 2014; PNAS 2012). We also showed that cadherins tune adhesion by switching between these conformations and identified key amino acids involved in adhesion. We also used single molecule Fluorescence Resonance Energy Transfer (FRET) to resolve the mechanism by which cadherins mediate cell adhesion (Structure 2009, PNAS 2009). Our data showed that cadherins initially interact with each other in a transient fashion and subsequently undergo a structural change that strengthens adhesion.

Cadherins bind in multiple conformations with different biomechanical properties
Cadherins bind in three conformations with different biomechanical properties. X-dimers form catch bonds which counter-intuitively lock in the presence of tensile force. Strand-swap dimers form slip bonds which become shorter lived when pulled. Finally, the intermediate conformation forms ideal bonds that are insensitive to mechanical stress.

While our previous work systematically characterized cadherin conformational changes in cell-free systems, a key question in the field is how cells and tissues regulate adhesion in vivo. Our ongoing work addresses this critical question. We have developed technology to measure the interactions between cadherins on live cells with single molecule resolution. We have also developed a high-resolution single molecule force microscope-fluorescence microscope to measure the effects of force on cadherin organization on the cell surface. By integrating these bioengineering tools with cell biological manipulations of cadherin-cytoplasmic protein interactions, we have obtained the first direct evidence that cadherin conformational changes and organization on the cell surface are regulated using an ‘inside-out’ signaling mechanism. Our data indicates that cytoskeletal tension allosterically regulates the conformation adopted by the cadherin extracellular region and drives cadherin clustering in cells to form robust adhesions. In the future, we are interested in how cadherins transduce extracellular mechanical stimuli into intracellular biochemical signals. Finally, we also plan to resolve the biophysical links between cadherin adhesion and cancer metastasis.

How do desmosomes assemble and adhere? Desmosomes are an essential intercellular adhesive organelle that mediates the integrity of the epidermis and heart which are exposed to significant levels of mechanical stress. Desmosomes are composed of two types of desmosomal cadherins. Currently, little is known of the biophysical mechanisms that drive the assembly of desmosomes. Using cell-based and cell-free single molecule binding assays, we previously assigned unique roles to different desmosomal cadherins in adhesion (J. Cell Sci. 2014). Using single molecule AFM force measurements and super resolution imaging of desmosomes in keratinocytes, we have recently resolved the key molecular steps in desmosome assembly (eLife 2018). In the future, we plan to characterize how desmosomes are compromised in skin and heart disease.


Bioengineering tool development

Microscope for ultrasensitive-measurement of single-molecule interaction and conformation: Transformative advances in mechanobiology have been constrained by the lack of automated, high throughput, high resolution, techniques that can quantify the effect of mechanical force on protein structure-function. We are developing the world’s first fully-automated, microscope that can simultaneously measure interaction forces between single molecules and their corresponding force-induced conformational changes (Nano Letters 2009, Scientific Reports 2018).

Ultrastable force-fluorescence microscope
Left: Images of an AFM tip  in the ultra-stable force-fluorescence microscope. Center and Right: Drift of the AFM tip measured without/with feedback. Feedback stabilizes the AFM tip at the sub-nm level.

Our instrument integrates two distinct components: (i) an ultra-stable AFM which eliminates thermal drift using fast feedback technologies, and enables mechanical interrogation of single molecules with ultra-high accuracy, precision and throughput, and (ii) single molecule FRET, which can measure force-induced conformational transitions in mechanosensitive proteins, with single molecule resolution. We anticipate that this instrument will enable mechanical processes in biology to be investigated in previously unparalleled ways by enabling a user to directly apply force on a single molecule while simultaneously imaging force-induced conformational changes, binding events, or catalytic reactions. It can also be used to track downstream cellular response following mechanical signals that are involved in cancer metastasis and stem cell differentiation.

Simultaneous 3D super-resolution fluorescence imaging and single-molecule mechanical manipulation: While conventional super-resolution fluorescence microscopes can image single molecules with nm lateral resolution (in the x- and y- direction), these technologies have poor axial resolution (along the z-axis). To overcome this limitation, we recently invented a fluorescence technique, called Standing Wave Axial Nanometry (SWAN), to localize single molecules along the z-axis of an optical microscope, with previously unachievable sub-nm resolution (Nano Letters 2012, US Patent 9,103,784).

Standing Wave Axial Nanometry (SWAN)
A standing wave, generated by positioning an AFM tip over a focused laser beam, is used to excite fluorescence. Axial position is determined from the phase of the emission intensity.

We are currently integrating SWAN with 2D super-resolution fluorescence microscopy to image biological structures in 3D, with nm resolution in all spatial dimensions. In the future, we plan to integrate this 3D super-resolution technology with an ultra-stable AFM to develop the world’s first microscope capable of building biological structures by precisely positioning individual fluorescent biomolecules, one molecule at a time, and imaging them in 3D. Such a technology has numerous transformative applications, such as building miniaturized biomedical devices by placing biomolecules with exquisite control and for assembling biomolecular networks which develop new functionalities depending on the arrangement of their constituents. With this technique, functional aspects of complex protein machines - such as different combinations of enzymes, and how close together they must be located in order to perform coupled reactions - can be tested directly.