Research

Mechanotransduction, the process by which cells transduce mechanical stimuli into biochemical signals, plays a central role in many physiological and pathophysiological processes. A better understanding of mechanotransduction pathways would, therefore, allow for greater control, manipulation, and treatment of mechanosensitive processes and diseases.

A key step in mechanotransduction is tension-induced conformational changes across a mechanosensitive protein. These conformational changes can lead to subsequent recruitment, or disassociation, of the protein’s interacting partners. Changes in protein interaction can, then, facilitate downstream mechanoresponse. Elucidation of these tension-sensitive interactions would, therefore, increase our understanding of mechanotransduction pathways. However, previous work in this regard has either been performed outside of the cellular context, lacked molecular specificity, or biasedly selected candidate proteins to investigate. Therefore, the goal of my thesis work is to develop a technique to unbiasedly detect molecular-tension-sensitive interactions and understand these interactions’ role in cellular processes.

Conceptualizing Mechanotransduction

Mechanotransduction can be conceptualized as four steps: (1) mechanotransmission - force across a mechanosensitive subcellular structure, (2) mechanosensing - a conformational change across a mechanosensitive protein due to said force, (3) mechanotransduction - altered protein-protein interactions due to said conformational change, and (4) mechanoresponse - downstream changes in signaling pathways and cellular function due to the altered protein-protein interactions.

We are interested in the third step, as it is the hinge point leading to mechanoresponse.

Invetigating Tension Sensitive Proximal Interactions via Mass Spec

In order to unbiasedly detect tension-sensitive protein proximal interactions, we turn to proximity dependent biotinylation. Specifically, we tag the mechanosensitive protein vinculin and a unloaded vinculin mutant, I997A, with the engineered biotin ligase. This allows for biotinylation of proteins within a 10 nm radius of either loaded or unloaded vinculin. These labeled proteins are then analyzed by mass spectrometry.

Identification of Vinculin-Tension-Sensitive Proximal Interactions

Based on the mass spectromety results we found several vinculin-specific proximal interaction partners that were either enhanced or repressed by vinculin tension. To our knowledge this is the first use of mass spectrometry for the identification of tension-sensitive interactions.

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