Investigating vinculin-tension-sensitive protein-protein interactions by mass spectrometry

Introduction:: The cells’ extracellular microenvironment is composed of hundreds of external cues that the cell must sense, integrate, and respond to. Many of these cues come in the form of physical stimuli, which are detected by the cell and transduced into biochemical signals in a process termed mechanotransduction. These mechanotransduction pathways have increasingly been shown to play a critical role in physiological and pathophysiological processes, such as differentiation, migration, and tumorigenesis [1-3]. A key step in mechanotransduction is the change in load-bearing proteins’ conformation, therefore changing their function, which can lead to subsequent recruitment of structural and signaling proteins. The formation of these force-mediated protein-protein interactions is a critical step in facilitating cellular mechanoresponse, and elucidating the molecular mechanisms responsible for the proteins’ recruitment will lead to a better understanding of mechanosensitive processes. However, most work conducted in this regard either lacks the proper cellular context, lacks molecular specificity, or biasedly selects candidate proteins to investigate. Therefore, the goal of this work is to develop a method to unbiasedly detect molecular-tension sensitive protein-protein interactions.


Materials and Methods:: In order to do this, we adapted proximity-based biotin labeling for our application. Specifically, the engineered biotin ligase, TurboID, was attached to a load-bearing protein of interest [4]. In this study, the mechanical linker protein vinculin was selected. Vinculin is a key component of focal adhesions and was selected due to its ability to bear physiological load, its multitude of interactions within the integrin adhesome, and the presence of a vinculin mutant, I997A, known to bear no significant loads in cellulo [5,6,7]. Therefore, vinculin-TurboID and vinculinI997A-TurboID constructs were made and validated, along with a cytosolic TurboID control [8]. These constructs were separately transduced into vinculin -/- mouse embryonic fibroblasts (MEFs) and the cells were then treated with biotin. After a 30-minute incubation the cells were lysed, and the lysates were subjected to a biotin-streptavidin pulldown and prepared for analysis by mass spectrometry. Mass spectrometry results were used to identify proteins enriched in the wildtype vinculin and I997A vinculin populations, which can be classified as protein-protein interactions enhanced or repressed by vinculin tension, respectively.


Results, Conclusions, and Discussions:: Results: An initial pilot experiment, using qualitative protein analysis by mass spectrometry, identified 1795 unique proteins, with 941 of those proteins enriched in the vinculin and vinculinI997A populations compared to the cytosolic control. As an initial validation of our experimental technique, we checked whether our results contained proteins from the consensus integrin adhesome, a set of proteins that comprise the cell’s core adhesion machinery [6]. We found that forty-four out of the sixty consensus adhesome proteins were identified within our sample population, verifying that this method probes protein-protein interactions at focal adhesions (Figure 1). In addition to identifying known components of focal adhesions, this initial pilot experiment was successful in unbiasedly detecting molecular-tension sensitive protein-protein interactions. Using the log2 fold change of spectral counts from the vinculin-TurboID and vinculinI997A TurboID populations, we were able to identify proteins enriched in either population (Figure 2). Proteins enriched in the wildtype vinculin population can be classified as vinculin-tension enhanced interactions and include key proteins such as migfilin, zyxin, and vinexin. Likewise, proteins enriched in the vinculinI997A population can be classified as vinculin-tension repressed interactions and include such proteins as Beta-PIX and BCAR1. These proteins, among others, represent interesting candidate proteins for future studies investigating mechanosensitive processes.

Discussion: Overall, this initial pilot study supports the continued use, development, and expansion of our technique. Future experiments will use label-free quantitative mass spectrometry for protein quantitation. Subsequent work will also study the role of the mechanical microenvironment, such as substrate stiffness, on force-sensitive protein-protein interactions. Additionally, this technique is generalizable. While, to our knowledge, this was the first use of proteomics to identify molecular-tension sensitive protein-protein interactions, the technique can easily be adapted to investigate protein recruitment due to load across other mechanosensitive proteins.

Conclusion: This work, in conjunction with similar studies, will greatly enhance our mechanistic understanding of mechanotransduction pathways. This will lead to better control of mechanosensitive cellular processes and potential improvements in therapies for mechanosensitive diseases.


Acknowledgements (Optional): :

References (Optional): : [1] Engler et al., Cell, 2006; [2] Shellard et al., Dev. Cell, 2021; [3] Butcher et al., Nat. Rev. Cancer, 2009; [4] Cho et al., Nat. Protocols, 2020; [5] Grashoff et al., Nature, 2010; [6] Horton et al., Nat. Cell Bio., 2015; [7] Rothenberg et al., Biophys. J., 2018; [8] Tao et al., Dev. Cell, 2023