Blebbistatin paradoxically induces ROCK-dependent junctional enrichment of p-MLC and adherens junction components in chick embryo epidermis.

The phosphorylation level of myosin light chain (p-MLC) is widely employed as a biochemical surrogate for cellular contractility in tissue mechanics studies, while small-molecule inhibitors such as Blebbistatin are routinely used to suppress non-muscle myosin II (NMII) contractile output without perturbing upstream kinases. Contrary to this expectation, we observed in E6 chick embryo dorsal skin explants that Blebbistatin treatment did not attenuate junctional p-MLC signals; instead, it induced pronounced, dose-dependent spatial enrichment of p-MLC at epidermal cell boundaries, accompanied by parallel co-enrichment of the adherens junction (AJ) core components E-cadherin and β-catenin. The selective ROCK inhibitor Y-27632 produced opposing effects on all three markers. To dissect the role of ROCK in this paradoxical enrichment, we performed both forward-rescue experiments (Y-27632 added after Blebbistatin-induced enrichment was established) and reverse-blockade experiments (Y-27632 pre-treatment before Blebbistatin), demonstrating that ROCK kinase activity is required for both the initiation and the maintenance of the compensatory junctional enrichment of p-MLC and AJ components. Integrating these molecular observations, we propose a conceptual framework in which active contractile tension and passive junctional scaffolding are independently regulated and jointly contribute to epidermal morphogenesis. These findings indicate that, when myosin ATPase inhibitors are used to perturb tissue mechanics, in situ p-MLC fluorescence intensity may become decoupled from actual physical tension and should not be interpreted as a sole readout of contractility.