Actin Branching Regulates Cell Spreading and Force on Talin, but not YAP Activation

Introduction: Extracellular matrix (ECM) forces regulate the YAP/TAZ pathway, which is critical in development, homeostasis, and disease [1]. ECM force transmission occurs through both integrin-based adhesions [2] and the linker of the nucleoskeleton and cytoskeleton (LINC) complex [3], allowing for nuclear envelope deformations [4]. Cell spreading uses polymerization of branched actin at the leading edge which engages integrin receptors, stretches the nuclear envelope, and regulates YAP activation [5]. The Arp2/3 complex and Rac GTPase both regulate the polymerization of actin and the balance of protrusion speed versus stability [6]. Cell spreading is well established as an important regulator of YAP activation [3], but the importance of actin branching in this process is unclear. Here, we reduce cell spreading through actin branching perturbations to assess their influence on YAP activation, force on the integrin adapter molecule talin, and traction stress on the ECM.

Materials and

Methods: NIH 3T3 cells were cultured in DMEM supplemented with 10% FBS and 1% pen-strep. For YAP quantification, cells were seeded on fibronectin coated glass coverslips in the presence of Arp2/3 inhibitor (CK666, 10μM), Rac inhibitor (NSC23766, 10μM), or control (DMSO), for 2 hours and then fixed with 4% PFA, and stained with YAP, Alexa-488 phalloidin, and DAPI then imaged at 20x. Arp2/3 was also perturbed by the overexpression of actin debranching protein GMFβ [7]. MATLAB was used to quantify cell area and the nuclear to cytoplasmic ratio of YAP. A fluorescence resonance energy transfer (FRET) based molecular tension sensor was used to measure forces on the focal adhesion adapter protein talin. Cells were transiently transfected with talin tension and control sensor using jetPrime transfection reagent and cells were seeded on fibronectin coated coverslips 36-72 hours post transfection. Cells were imaged at 63x and MATLAB was used to calculate FRET index values. For traction force microscopy (TFM), PDMS substrates (Sylgard 184, B/C ratio of 55/1 for 10kPa) [8] were coated with fluorescent beads, and cells were seeded in the presence of DMSO, CK666 (10μM), or Blebbistatin (10μM) for imaging at 63x under phase contrast (for cell area) and epifluorescence (for beads) before and after cell lysis with SDS. TFM images were processed with the FTTC method using a previously developed MATLAB code [9], assuming a Poisson’s ratio of 0.5 and a regularization of 0.0001.

Results, Conclusions, and Discussions: Results and

Discussion: The CK666 and NSC23 treatment resulted in significantly reduced cell spreading (Fig 1 A,B). However, there was no change in the nuclear localization of YAP (Fig 1 A,C). These results reinforce the important role of Arp2/3 and Rac signaling in cell spreading and actin branching, while indicating that perturbations to cell spreading do not regulate YAP directly. Similarly, debranching of acting via GMFβ overexpression reduced cell spreading and did not prevent YAP activation (Fig 1 D-F), instead increasing YAP activation. Possibly due to a change in actin structure that alters force transmission to the nucleus. Talin tension sensor experiments resulted in a slight increase in average FRET with inhibition of branching, corresponding to a decrease in talin force (Fig 1 G, H). Next, traction forces were measured in cells treated with DMSO, CK666, or the myosin inhibitor Blebbistatin (as a low force control). There was no significant difference in the average stress (Fig 1I, J) for the Arp2/3 inhibited cells, indicating that this perturbation to cell spreading does not affect average traction force per cell area but does reduce the average force per talin adapter. Additional experiments with simultaneous measurement of traction stress and force on talin showed similar results, indicating a distinct difference in the influence of branching on molecular scale vs. cellular scale forces.

Conclusions: These results demonstrate that YAP mechanosensing is maintained with the inhibition of actin branching-based cell spreading. This is shown through the nuclear localization of YAP, despite reduced cell spreading and reduced force on Talin. This indicates that while cell spread area can correlate with YAP activation, this regulation is indirect and independent of branched actin and Rac. This is consistent with previous work that has identified nuclear envelope forces as the primary driver of YAP mechanosensing [4, 5].

References: [1] Cai+ Front in Cell and Dev Bio (2021); [2] Sun+ JCB (2016); [3] Dupont+, Nature (2010); [4] Driscoll+, Biophysical Journal (2015); [5] Elosegui-Artola+ Cell (2017); [6] Papalazarou+ Cur Op Cell Bio (2021); [7] Haynes+ JCB (2015); [8] Gutierrez+ PLoS one (2011); [9] Han+ Nat Methods (2015).

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