Actin Binding Site Mutations in Talin Regulate Cell Spreading and Force, but do Not Prevent YAP Activation

Introduction: Cells sense mechanical signals from their surrounding extracellular matrix (ECM) using integrin-based adhesions. These cues drive growth, migration, and matrix remodeling [1]. The focal adhesion protein Talin has long been established as essential for matrix mechanosensing through integrins [2,3]. Recent work has also identified the third actin-binding site (ABS3) in Talin as being directionally mechanosensitive, with asymmetric catch bond behavior resulting in a drastically increased bond lifetime when F-Actin is pulled toward its pointed (-) end [4]. Previously, we have shown that complete mutation of ABS3 by replacement of the R13 domain of Talin (actin binding) with the R6 domain (non-actin binding) results in abnormal focal adhesion growth, dynamics, sliding, vinculin recruitment, and molecular forces when measured using a FRET-based molecular tension sensor [5]. Additionally, there is a separate actin-binding site in Talin (ABS2) that is involved in additional actin binding in mature and stable adhesions. However, it is unclear how these impact downstream cellular pathways that are involved in matrix mechanosensing. The YAP/TAZ mechanosensing pathway, which plays a prominent role in fibrosis is regulated by integrin-based cell spreading, is of particular interest. Identifying the significance of ABSs in upstream mechanosensing could allow for the regulation of aberrant ECM growth that exists in fibrotic remodeling. The primary goal of this work is to identify the influence of ABS3 and ABS3/ABS2 double mutants on cell spreading, Talin force, and YAP activation.

Materials and

Methods: 3T3 cells were cultured in DMEM media with 10% FBS and 1% pen-strep. Talin tension sensor constructs with WT (TS) or mutant actin binding sites (R6 and ABS2-R6) were compared to a no-force WT C-terminal control sensor (CTS). Mutant or WT Talin constructs were transfected into 3T3 cells (Polyplus jetPrime®), seeded on fibronectin-coated coverslips for 24 hours, and then fixed with 4% PFA for FRET and YAP localization analysis. FRET samples were mounted with Fluormount-G® (SouthernBiotech) and imaged with 63x oil objective using a Zeiss AxioObserver microscope for ratiometric analysis of GFP-RFP FRET (488/507 nm eGFP; 488/607nm FRET; 584/607 nm mRFP). FRET index was calculated from shade, bleed, and cross-excitation corrected images using MATLAB as previously described [5]. YAP samples were fixed, permeabilized, blocked, and stained with Alexa-488 Phalloidin, YAP primary antibody w/ goat anti-mouse Alexa-647 secondary, then mounted w/ DAPI Fluoromount-G ®. YAP localization was quantified from 20x images for RFP-positive cells using MATLAB. Cell area was quantified in ImageJ.

Results, Conclusions, and Discussions: R6 and ABS2-R6 show significantly reduced average cell areas compared to WT control (Fig. 1A, B). Interestingly, the ABS3 mutant (R6) resulted in relatively little change in YAP nuclear localization, while the ABS2-R6 double mutant increased nuclear localization (Fig 1C). The Wild-type Talin tension sensor (TS) displayed a low FRET index (high force) and the no-force control sensor (CTS) showed a high FRET as expected. Talin ABS mutants showed significant increases in the FRET index in R6 and ABS2-R6 indicating reduced force acting on Talin for both mutants (Fig. 1D, E).

Current results indicate that loss of function of the Talin ABS3 and ABS2 sites significantly alters force on Talin, adhesion size, and cell spreading. However, these have relatively little influence on the mechanosensitive YAP signaling pathway. Previous work has shown an important role of nuclear deformation in regulating YAP [6,7,8]. The reduction of cell area due to decreased F-actin-Talin binding and reduced Talin forces may be insufficient to prevent nuclear deformation-based activation of YAP. Future work will test the influence of these mutations on directional mechanosensing processes such as contact guidance and durotaxis.

[1] Kechagia+ Nat Rev MCB (2019), [2] Dunn+ PNAS (2021), [3] Brown+ Dev Cell (2002), [4] Zhang+ Nat Cell Bio (2008), [5] Driscoll+ PNAS (2020), [6] Driscoll+ Biophysical Journal (2015) [7] Elosegui-Artola+ Cell (2017). [8] Kirby & Lammerding+ Nature Cell Biology (2018),

Acknowledgements (Optional): Claudia Villalobos, Garrett McDaniel, Ananya Naha, and Dr. Christina Holmes