We are developing real-time microfluidics magnetic tweezers to effectively monitor conformational change under physiological force range in subnanometer resolution, which can detect small changes in elasticity. This Bio-CS collaboration aims to build software and hardware to support and speed up magnetic tweezer experiments. We are currently designing and building a new CMT (covalent magnetic tweezer) model where the magnetic forces are controlled using electromagnets instead of physical movement: this presents new issues with heating and control, which will require complex real-time error correction systems.
Cellular behavior such as development, migration and structure maintenance are evidently dependent on mechanical crosstalk between extracellular matrix (ECM) and focal adhesion (FA), the supramolecular complex at the interface of cell and ECM. Notably, perturbation in elasticity of ECM causes change in cell shape and behaviour. For instance, cell produces short actin filaments to migrate slowly on soft matrix while they migrate fast on stiff matrix by producing long stress fibers. Notably, the proteins associated in FA are highly elastic in nature, for instance Talin can stretch up to 400nM and quench to 60nM. Thus, it is obvious that the elasticity of proteins must impact on FA by tuning mechano-transduction. However, the role of elasticity on focal adhesion, cell development and migration is missing. This data is also very hard to collect due to noise.
The cellular interactome network, created in the FA junction, is controlled by mechanical forces (1.5-40 pN). When stretched, they expose new recognition sites and interact with various partner proteins, creating a mechanical-tension regulated interactome. Notably, most of these interactions have been studied biochemically, in the absence of force. We aim to bridge this knowledge-gap by adding the force-dimension in adhesion dynamics by developing a state-of-the-art single molecule technology, and directly show how force controlling the mechano-interactome network or how the force transduce from ECM to cell.
We plan to combine fluorescence with covalent-magnetic-tweezers to monitor binding-unbinding dynamics of the interactome under force. We hope to determine elasticity of seven wild type focal proteins (talin, vinculin, syndecan-4, kindlin, α-actinin, paxillin, zyxin) individually and collectively in single molecule resolution. These multidomain proteins, involved in force axes, behave as molecular spring while transmitting the bidirectional mechanical force. After this, we hope to examine the force-dependent binding interactions between the focal adhesion proteins.
(Collaboration with Prof. Subhasis Haldar.)