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Protein complexes, assembled by scaffold proteins, act as molecular machines driving development. The mechanosensitive adapter protein Zyxin is a key example, integrating actin cytoskeleton dynamics with gene expression. However, the developmental regulation of its interactions and post-translational modifications remains poorly understood. Here, we characterize the dynamic Zyxin interactome across three early developmental stages of Xenopus laevis (from gastrulation to neurulation) using co-immunoprecipitation coupled with quantitative mass spectrometry (DDA and DIA). We identify stage-specific changes in Zyxin's association with core focal adhesion components, transcriptional regulators and kinases. Furthermore, we uncover developmentally regulated phosphorylation events on isoforms, suggesting dynamic post-translational control of its interactions. Our work provides a comprehensive resource that positions Zyxin as a central orchestrator of cell adhesion, survival, and gene regulatory programs during morphogenesis. These findings underscore the role of Zyxin as a multifaceted regulatory hub, with important implications for understanding tissue homeostasis and related pathologies.
Figure 1. Stage-resolved analysis of the Zyxin interactome in Xenopus laevis embryos. (a) Schematic of the three analyzed developmental stages: early gastrula (stage 10), early neurula (stage 14), and late neural fold (stage 16). (b) Experimental workflow. Endogenous Zyxin complexes were isolated by co-immunoprecipitation (co-IP) from embryo lysates using a validated antibody cocktail and analyzed by quantitative mass spectrometry. (c) Western blot validation of co-IP specificity. Zyxin was efficiently and specifically immunoprecipitated across all stages. Non-specific rabbit IgG served as a negative control. Lysate inputs are shown as a reference. (d) Complementary mass spectrometry strategies: Data-Dependent Acquisition (DDA) for deep interactome discovery and Data-Independent Acquisition (DIA) for accurate, label-free quantification across stages.
Figure 2. Relative stage-specific dynamics of Zyxin-associated structural and adhesion proteins. Heatmap depicts the stage-resolved association patterns of core cytoskeletal and focal adhesion proteins with the Zyxin complex. The color scale (red/blue) represents row-wise Z-scores, indicating whether the Z/C ratio for a given protein at a specific stage is above (red) or below (blue) its own mean value across all three stages. This normalization highlights the temporal dynamics of each protein relative to its own baseline. Notable patterns include the stable, constitutive association of the adhesome core components Talin-1 and Vinculin, the sharp, transient recruitment peak of α-actinin-3 and α-actinin-4 at stage 14, the progressive engagement of Myosin-9, and the absence of specific enrichment for Integrin subunits.
Figure 3. Relative stage-specific dynamics of Zyxin-associated regulators. Heatmap depicts the stage-resolved association patterns of regulatory proteins with the Zyxin complex. The color scale (red/blue) represents row-wise Z-scores, indicating whether the Z/C ratio for a given protein at a specific stage is above (red) or below (blue) its own mean value across all three stages. This normalization highlights the temporal dynamics of each protein relative to its own baseline. Notable patterns include the constitutive, high-level signal of Caspase-3 (casp3.2), the late-stage-specific emergence of YAP1-A, and the transient peak of CDK4 at stage 16.
