Isoforms of the cytoskeletal lim-domain protein zyxin in the early embryogenesis of Xenopus laevis

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Abstract

Zyxin is a conserved mechanosensitive LIM-domain protein that regulates the assembly of F-actin filaments at cell junctions. At the same time, under mechanical stress, it can move from focal adhesions to stress fibrils and into the nucleus and affect gene expression. In Xenopus laevis embryonic cells, western blots with antibodies against Zyxin’s N-terminal and C-terminal LIM-domain regions revealed two full-length and two short isoforms. The intracellular localization of these isoforms and the number depending on the stage of embryo development were determined. According to our study, full forms with different electrophoretic mobility are localized differently in the cell, and the shortest isoform containing LIM-domains is stable during development, mainly located in the nucleus, and participates in gene expression regulation. This study may be of great value to understanding how the LIM-domain mechanotransducer proteins jointly influence morphogenesis and differentiation in vertebrates at early stages of development.

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About the authors

E. D. Ivanova

Pirogov Russian National Research Medical University

Email: martnat61@gmail.com
Russian Federation, ul. Ostrovitianova 1, Moscow, 117997

E. A. Parshina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: martnat61@gmail.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

A. G. Zaraisky

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Email: martnat61@gmail.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

N. Y. Martynova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry RAS

Author for correspondence.
Email: martnat61@gmail.com
Russian Federation, ul. Miklukho-Maklaya 16/10, Moscow, 117997

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2. Fig. 1. Schemes of the domain and spatial organization of the zyxin molecule and deletion constructs used in the work: (a) – scheme of the domain structure of the full-length zyxin molecule, the numbers of amino acid residues for all zyxin domains shown in the figure are indicated at the top: P-domain 120–147 aa (proline-rich domain), proteolysis 334–340 aa (site of possible proteolytic cleavage), NES 438–458 aa (nuclear export signal) and LIM-domain region 469–659 aa; (b) – scheme of the “closed” conformation of the zyxin molecule; (c) – spatial three-dimensional structure of zyxin, obtained according to the PhosphoSitePlus® database (https://www.phosphosite.org). (g) – diagram of the C-terminal deletion mutant used to obtain antibodies to C-zyxin; (d) – diagram of the N-terminal deletion mutant used to obtain antibodies to N-zyxin; (e) – diagram of the deletion mutant of zyxin (∆zyxin) truncated at the proteolysis site.

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3. Fig. 2. Zyxin isoforms, their stability and distribution between the nucleus and cytoplasm: (a) – zyxin isoforms detected by antibodies to C-zyxin and antibodies to N-zyxin in the nuclear and cytoplasmic fractions of embryonic cells at the 11th stage; (b) – changes in the number of truncated forms of zyxin during development, detection by antibodies to C-zyxin and antibodies to N-zyxin, α-tubulin was used as a reference band; (c) – electrophoretic mobility of the truncated mutant ∆zyxin (334–664 aa) coincides with the endogenous isoform of C-zyxin 37 kDa, detection by antibodies to C-zyxin.

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