The Impact of Zyxin Modifications on Its Intracellular Distribution in the Xenopus Laevis Embryo Model

Abstract

Objective: The aim of this study is to investigate the mechanisms underlying the intracellular transport of the mechanosensitive protein zyxin. Zyxin, a conserved LIM domain protein, plays a critical role in regulating the assembly of F-actin filaments at cellular junctions. In response to mechanical stimuli such as cell stretch, zyxin has the capacity to translocate into the nucleus to influence gene expression, or it may exit the nucleus. Importantly, zyxin is recognized as a tumor marker, making the exploration of its post-translational modifications and the dynamics of its nuclear-cytoplasmic shuttling significant for advancements in molecular diagnostics related to disease. Methods: To elucidate the nuclear entry dynamics of zyxin, we employed site-directed mutagenesis to selectively target palmitylation sites, O-GlcNAcylation sites, and specific amino acids at both the N- and C-termini. This investigation was conducted using Xenopus laevis gastrula embryos, which serve as an effective model for studying protein trafficking. Results and Discussion: Our findings reveal that the introduction of a Flag epitope at the C-terminus of the zyxin molecule results in a loss of its nuclear translocation capability. Conversely, mutating the palmitylation sites leads to a noticeable decrease in the nuclear presence of zyxin, while modifications to amino acids responsible for O-GlcNAcylation result in an increased accumulation of zyxin in the nucleus. Conclusions: The data obtained from this study highlight the significant impact of these specific post-translational modifications on the subcellular localization of zyxin. These findings contribute to the broader research knowledge regarding the mechanistic aspects of the localization dynamics of zyxin and related mechanosensitive proteins. Given that the disruption of intracellular localization can have profound implications in the development of cancer and cardiovascular diseases, this research bears both fundamental and medical importance.