INTRODUCTION

The functioning of highly conserved mechanosensitive proteins, including zyxin, is of great interest to study, because the shape of embryos of all animals during embryogenesis is formed by the concerted action of morphogenetic processes, including stretching, bending, or folding of embryonic cell sheets, as well as by a strictly controlled transition of cells to the differentiated status. The conservatism of the early stages of embryogenesis in vertebrates allows the use of animals whose development occurs in the external environment (Xenopus laevis (X. laevis), Danio rerio) as model organisms, and, therefore, studies on these models are not only of fundamental value, but can also be useful in medical research.

The zyxin molecule from X. laevis consists of 664 aa and contains three regions conserved in all vertebrate genomes: a proline-rich N-terminal region, a leucinerich nuclear export sequence (NES), and a C-terminal fragment with three LIM domains (Fig. 1a) [1].

Fig. 1.
figure 1

Schemes of the domain and spatial organization of the zyxin molecule and deletion constructs used in the work: (a) diagram of the domain structure of the full-size zyxin molecule (the numbers of amino acid residues for the zyxin domains shown in the figure are given 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 sequence), and LIM domain region 469–659 aa; (b) diagram of the closed conformation of the zyxin molecule; (c) 3D structure of zyxin, retrieved from the PhosphoSitePlus database (https://www.phosphosite.org); (d) diagram of the C-terminal deletion mutant used to obtain antibodies to C-zyxin; (e) diagram of the N-terminal deletion mutant used to obtain antibodies to N-zyxin; and (f) diagram of the deletion mutant of zyxin truncated at the site of proteolytic cleavage (∆zyxin).

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The N-terminus of zyxin is required for interaction with cytoskeletal proteins, primarily with the actin filament cross-linking protein α-actinin [2, 3], the actin assembly modulator Ena/VASP [4], and the cytoskeletal proteins LASP-1 and LASP-2 [2]. The proline-rich repeats in zyxin are similar to the proline-rich sequences in the ActA protein of the intracellular bacterium Listeria monocytogenes [5], whose pathogenicity is associated with its ability to assemble actin filaments on the cell surface. The ActA protein attracts Ena/VASP cell contact proteins to the site of actin assembly [6]. Due to the presence of proline-rich repeats, zyxin, like ActA, mediates the assembly of members of the Ena/VASP family with actin and is involved in transformations of the actin cytoskeleton in eukaryotic cells [7].

NES—leucine-rich region is involved in the binding of zyxin to the CRM1 protein for exit from the cell nucleus into the cytoplasm [5, 8].

The C-terminus of zyxin contains three LIM domains. The LIM domain is a Cys- and His-rich sequence 60 aa long. Each LIM domain has a two-zinc-finger structure. LIM domains mediate specific protein–protein and protein–DNA interactions [9]. A study on the participation of LIM-domain proteins in mechanotransduction showed that zyxin binds by its LIM domain region to F-actin under stress conditions and is distributed along stress fibrils [10]. Interestingly, the free zyxin molecule in the cytoplasm has a closed head-to-tail conformation, which must be phosphorylated at Ser142 under AKt2 kinase catalysis [11, 12], as well as acetylated [13, 14] or palmitylated [15] to allow zyxin to form complexes with other proteins (Fig. 1b). The LIM domains of an open zyxin provide a platform for the assembly of various protein complexes, primarily an ensemble of transcription regulators [16].

Recently, Sabino et al. [17] reported a study of peptide repertoire in wound exudates in mammals. Among the identified protein sequences, two truncated zyxin fragments were found. It was shown that zyxin is subject to proteolysis at the serine peptidase HtrA1 site to form a light form of 254–572 aa, which is able to move into the nucleus and take part in the activation of a number of transcriptional regulators involved in enhancing the adaptive properties in the wound surface. Evidence was obtained showing that this truncated zyxin is produced at high cell densities and may regulate the number of wound-associated proteins during cutaneous wound healing [17]. It is also known that zyxin produced by cell-free synthesis can be cleaved by caspases in vitro when incubated in a cell-free apoptotic lysate S100 and in vitro in a lysate of the HEK293 cell line with activated apoptotic activity [18].

It is noteworthy that, against the background of quite active research into the functions of zyxin in cell cultures, only few studies have been focused on the functions of zyxin in embryogenesis. Among the most interesting works we can mention the research of Lecroisey et al. [19], who explored the role of zyxin in stabilizing synaptic contacts during the development of synapses in mechanosensory neurons of the roundworm Caenorhaditis elegans. Zyxin from C. elegans, like its vertebrate homolog, contains an N-terminal proline-rich region and three C-terminal LIM domains arranged in tandem. An unexpected finding of the cited work was that the neuronal function of zyxin is mediated by a short C-terminal isoform containing only LIM domains, i.e. this isoform acts as a fully functional protein capable of transducing mechanical reactions absolutely independently of the N-terminal domain [19].

The primary objective of our present study was to identify endogenous zyxin isoforms from X. laevis and to obtain information about changes in their abundance and intracellular localization that may occur with these isoforms during embryogenesis.

RESULTS AND DISCUSSION

Endogenous proteins are mostly detected by means of specific antibodies. In our study we made use of polyclonal antibodies specific to the C-terminal region of zyxin, containing the NES and three LIM domains (438–663 aa), which we obtained previously [1] (Fig. 1d). These antibodies have a disadvantage that they detect only the LIM domain region of zyxin and, accordingly, cannot detect its N-terminal truncated isoforms. Therefore, we obtained polyclonal antibodies to the N-terminal proline-rich region of zyxin (1–373 aa), which is involved in the interaction with actinin and other cytoskeletal proteins (Fig. 1a, diagram of the full-length zyxin protein).

Thus, we had in our hands antibodies capable of specifically detecting the C-terminal and N-terminal fragments of zyxin.

Xenopus laevis embryos were obtained according to a standard established procedure [20]. Embryos were collected at the stages from the beginning of cleavage (32 blastomeres), the gastrula stage (stage 11) to the motile tadpole stage (stage 26), 20 embryos per stage, and separated into the nuclear and cytoplasmic fractions for each stage, following the procedure described [21]. The nuclear and cytoplasmic fractions were used to prepare samples for Western blotting after separation on 10% PAGE by the Laemmli protocol. Zyxin isoforms were detected using polyclonal antibodies to N- and C-zyxin and alkaline phosphatase-conjugated anti-rabbit secondary antibodies.

The use of antibodies specific to various domains of the zyxin protein allowed us to detect its isoforms truncated at the N- and C-termini, trace their nucleus– cytoplasm distribution, and identify transformations of these isoforms during embryonic development. First of all, we identified the full-length forms of zyxin with the molecular weights 105 and 70 kDa, which were detected by antibodies to both the N- and C-domains (Fig. 2a). Since the estimated molecular weight of the unmodified protein without post-translational modifications is ~70 kDa, the full-length zyxin with the molecular weight 105 kDa is a modified isoform.

Fig. 2.
figure 2

Zyxin isoforms and 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, reference antibodies to α-tubulin; and (c) electrophoretic mobility the truncated mutant ∆zyxin (334–664 aa) coincides with that of the endogenous 37 kDa C-zyxin isoform, detection by antibodies to C-zyxin.

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The most studied modification of mammalian zyxin is phosphorylation at the Ser 142 residue [11, 12]. Along with this residue, zyxin contains 35 more potential phosphorylation sites (according to the PhosphoSitePlus database, https://www.phosphosite.org). Since phosphorylation is known to greatly slow down the electrophoretic mobility of proteins [22], phosphorylation is the most likely modification of the full-length form. Some evidence has been reported for the possibility of reversible modifications of the full-length zyxin, such as palmitoylation and acetylation [13–15]. There a few modifications, which are likely to slow down the electrophoretic mobility of the full-length zyxin concurrently.

The separation of embryonic cell lysates into the nuclear and cytoplasmic fractions allowed us to show that the modified and unmodified forms of the full-length in X. laevis zyxin have different intracellular localizations: the 105 kDa form predominates in the cytoplasmic fraction, and the 70 kDa form predominates in the nuclear fraction (Fig. 2a); and in the process of embryogenesis, the level of these forms does not change significantly. When the level of zyxin expression was reduced by suppressing its mRNA translation by morpholine oligonucleotides, we observed a decrease in the intensity of both bands in the Western blot analysis of the lysates from stage 13 embryos, using antibodies to C-zyxin (data not shown).

In the Western blot analysis of the lysates from embryos at early stages of development (starting from the 32-cell embryo), we noticed that antibodies to different zyxin domains detected lighter bands with different electrophoretic mobilities. Thus, with antibodies to C-zyxin, a band with a mobility in the region of 37 kDa was detected, and with antibodies to N-zyxin, a band in the region of 45 kDa was detected (Fig. 2b).

Therewith, it is worth noting that cross-staining is completely excluded; antibodies to different domains detect strictly specific bands, i.e. it is safe to state that we were able to “catch sight of” the two halves of the zyxin molecule formed most likely by proteolytic cleavage. The strongest band associated with the truncated zyxin was a band of 37 kDa, which was detected by antibodies to C-zyxin in both the nuclear and cytoplasmic fractions at early stages, but starting from the gastrula stage, its intensity in the cytoplasm dropped sharply until it disappeared with further development, whereas in the nucleus it remained well-defined until the stage of the mobile tadpole (stage 26) (Fig. 2b). Since this band is stained only by antibodies to C-zyxin, we can suggest that this is the C-terminal, LIM-domain part of the zyxin molecule truncated at the N-terminus (C-zyxin).

In the case of antibodies to N-zyxin, in the samples from the lysates of early stage embryos (32 blastomeres) we observed a 45 kDa band in the nuclear and cytoplasmic fractions. This band is much weaker compared to the 37 kDa band and tended to attenuate with further development until it almost completely disappeared by the 26th stage, which was especially characteristic of the nuclear fraction (Fig. 2b). At the same time, the 105 and 70 kDa bands did not change their intensity depending on the stage. The 45 kDa band was stained only by antibodies to N-zyxin and not detected by antibodies to C-zyxin, implying that it belongs to an isoform truncated at the C-terminus (N-zyxin). In all experiments, monoclonal anti-tubulin antibodies (Sigma, USA) were used as loading controls.

Thus, we were able to detect two stable nuclear isoforms of zyxin: the full-length unmodified isoform (70 kDa) and the LIM-domain C-terminal truncated isoform (37 kDa). The 105 kDa isoform is also detected to a minor extent by antibodies to N-zyxin, thereby providing evidence showing that it is present in a small amount in the nucleus. This finding may be explained by some modifications of the N-region of this isoform. The 105 kDa isoform is the major and most stable in the cytoplasmic fraction and is detected by two types of antibodies. The isoform with an electrophoretic mobility of ~45 kDa, which is detected by antibodies to N-zyxin, was unstable and clearly detected only up to the gastrula stage.

The evidence for the presence of truncated forms of zyxin in X. laevis embryos, obtained in the present work, correlate with the discovery of Sabino et al. [17] of truncated C-terminal forms of zyxin in their study of the peptide repertoire in mammalian wound exudates. The site of proteolytic cleavage by HtrA serine peptidase 1, indicated in the cited work, coincides with the 332–338 aa range for X. laevis, has a highly conserved amino acid sequence APGF/GSF/G in the 332–338 aa range. An analysis of the spatial structure of zyxin from X. laevis revealed a structural loop in the outer part of the molecule, which is accessible for proteolysis, while the LIM domain region is located in the center of the molecule and has a compact tertiary structure comprising alternating helices and zinc finger structures (Fig. 1c ).

In confirm that proteolysis occurs precisely at this site, we created a deletion mutant Δzyxin (334–664 aa) (Fig. 1f) and found that its electrophoretic mobility completely coincided with the mobility of the endogenous 37 kDa fragment, whereas the exogenous fragment is localized predominantly in the cytoplasm (Fig. 2c). At the same time, abundant experimental evidence is available showing that most of the endogenous 37 kDa zyxin is present in the nuclear fraction.

Furthermore, using specific antibodies, we showed that the N-terminal fragment of zyxin is also present in the nucleus. This is an interesting and new result, since the N-terminal region is known to interact with cytoskeletal proteins: the 16–36 aa sequence is a binding site for α-actinin. Therefore, one might hypothesize that the biological function of zyxin proteolysis is the dissociation of its LIM domain region from the region associated with cytoskeletal proteins and its translocation into the nucleus, where it becomes able to interact with regulatory transcription factors.

However, according to the results of our studies, the N-terminal fragment of zyxin with a molecular weight of 45 kDa is present in the nucleus at the early stages of development. At the same time, no information has yet been reported in the literature on the function of the N-terminal fragment in the nucleus. It is believed that this region is responsible for the interaction with cytoskeletal proteins, and, therefore, the phenomenon of the appearance of the N-terminal fragment in the nucleus at the early cleavage stages deserves further study.

No less interesting and important for further research is the discovery of the endogenous C-terminal zyxin fragment containing the LIM domain with a mass of 37 kDa and its nuclear localization. Previously, using a model of X. laevis embryos, we showed that in embryogenesis, precisely due to its LIM domain region, zyxin performs important functions for development: 1) modulates the activity of the regulator of the forebrain, the transcription factor Xanf1 [23, 24]; 2) regulates the activity of SHH (sonic hedgehog) signaling pathways [25]; 3) affects the stability of the mRNA of pluripotency markers of the Pou 5F3 family, homologs of the known stem cell factor Oct 4 and the retinoid receptor Rxrg [26, 27].

In all studies using the yeast two-hybrid system and coimmunoprecipitation methods, we proved that transcription factors such as Xanf1, Gli1, Zic1 and Ybx1 interact specifically with the LIM domain region, and for all these proteins it was shown that the interaction with full-length zyxin is significant weaker than the interaction with its LIM domain fragment. For coimmunoprecipitation, we used an exogenous C-terminal fragment carrying peptide tags to allow coprecipitation of protein complexes using resins with immobilized commercial antibodies. Expression of peptide-labeled proteins in embryonic cells is achieved through microinjections of synthetic mRNA into embryos at the stage of the first division, and the amount of such exogenous proteins exceeds the level of endogenous ones.

Therefore, the weakening of the binding of the studied factors to endogenous full-length zyxin did not raise any doubts in our minds. The identification of a truncated endogenous C-terminal fragment containing LIM domains raises the question of which (full-length or truncated) zyxin isoform is involved in the interaction with previously found partners—transcription factors, and whether the truncated isoform can modulate the activity of the corresponding signaling cascades.

Since in this work we obtained unique data on the intracellular distribution of modified and unmodified full-length forms of zyxin, as well as truncated isoforms of zyxin in X. laevis, further study of the occurrence and possible influence of these isoforms on gene expression during embryogenesis is necessary.

EXPERIMENTAL

Preparation of monospecific antibodies to the N- and C-fragments of zyxin. Antibodies to the C-terminal fragment of zyxin we obtained previously by the procedure described in [1].

Antiserum against the N-terminal fragment of zyxin (UniProt: A5H447) was prepared by immunizing a rabbit with a truncated protein (1–373 aa). The corresponding cDNA insert was cloned into the pQE80 vector (Qiagen, USA) and the fusion protein with six His residues and a Myc peptide at the N-terminus was expressed in E. coli DH-5α. For immunization, the protein expressed in the bacterial system was purified by Ni–NTA chromatography, and the amount and degree of purification were detected by immunoblotting with alkaline phosphatase-conjugated antibodies to 6His and Myc peptide.

Immunization of a rabbit (healthy female, 1 year old, weight 1.8 kg, Breeding Facility for Laboratory Animals, Stolbovaya Branch, Russia) was performed by the procedure in [1] using complete and incomplete Freund’s adjuvant (Sigma, USA).

Monospecific antibodies were purified by affinity chromatography on CNBr-Sepharose (Sigma, USA) with an immobilized N-fragment of zyxin hybrid with GST (glutathione-S-transferase, glutathione-binding protein). To prepare the column, the sequence encoding the zyxin N-domain (1–373 aa) was cloned into the pGEX-4T-1 vector (Pharmacia, Sweden); and the GST-N-zyxin protein was expressed in E. coli BL21. The GST hybrid protein was isolated from the bacterial lysate by affinity chromatography on a glutathione–agarose gel, and the purified protein was used for covalent cross-linking with CNBr-Sepharose. The purification of monospecific antibodies on the affinity column was carried out according to the standard protocol described in [1].

Production of embryos, separation of the lysate into the nuclear and cytoplasmic fractions, and sample preparation for Western blot assay. Xenopus laevis embryos were obtained by in vitro fertilization by the procedure described in [1]. The embryos were incubated in a modified Ringer’s solution for cultivation of amphibian embryos (0.1 MMR): 0.1 М NaCl, 2.0 μM KCl, 1 µM MgCl2 ∙ 6H2O, 2 μM CaCl2 ∙ 2H2O, 5 μM HEPES, pH 7.4) until the 32 blastomere stage, gastrula stage (12th stage), and mobile tadpole stage (26th stage). At each stage, 20 embryos were selected, half of the embryos were used to prepare lysate in a coimmunoprecipitation buffer (1× pH 7.2–7.4 buffer): 137 μM NaCl, 2.7 μM KCl, 8.1 μM Na2HPO4 ∙ 7H2O, 1.5 μM KH2PO4, 1% Triton-X100).

The embryos were homogenized by pipetting, the resulting crude lysate was centrifuged for 30 min at 15000 g, and the supernatant was collected and used to prepare samples for electrophoresis by the standard procedure. The other half of the embryos was used to separate the lysate into the nuclear and cytoplasmic fractions according by the procedure described in [21] with a minor modification: after purification in 0.8 M sucrose, the nuclei were dissolved in the coimmunoprecipitation buffer and centrifuged for 30 min at 16000 g and 4°C. The supernatant was used to prepare samples for electrophoresis.

SDS-PAGE. The samples were analyzed by SDSPAGE on 10% Laemmli gels and electroblotted onto a PVDF membrane (Millipore, France). Monospecific rabbit polyclonal antibodies to C- or N-zyxin were used as primary antibodies, and monoclonal antibodies to α-tubulin (Sigma, USA) were used as reference antibodies. The goat anti-rabbit Fab fragment of the antibody conjugated with alkaline phosphatase (Sigma, USA) and anti-mouse Fab fragment of the antibody conjugated with alkaline phosphatase (Sigma, USA) were used as secondary antibodies. For detection, a stabilized substrate for alkaline phosphatase Western Blue (Promega, USA) was used. The results of electrophoresis and blotting were assessed visually by the color of the colorimetric substrate. Experiments to analyze changes in the expression and localization of zyxin isoforms on PVDF membranes developed with specific antibodies were carried out in more than three repetitions.

CONCLUSIONS

In the present work, we discovered that zyxin from X. laevis has a few isoforms: along with the full-length modified protein with a molecular weight of ~105 kDa, an unmodified form of zyxin with a molecular weight of about 70 kDa and two truncated 45 and 37 kDa forms were detected. We found that the number and intracellular distribution of the truncated forms depend on the stage of embryo development: at the initial stages, the number of the 45 and 37 kDa is increased. However, in the process of gastrulation and neurulation, when cell layers begin to move and mechanical stress fields arise, the cytoplasmic form of zyxin 105 kDa and nuclear forms 70 and 37 kDa predominate. As a result of this work, an important body of evidence was obtained for the first time about changes in the level and localization of isoforms of the mechanosensitive protein zyxin in the course of embryonic development, which may serve as a connecting link in the transmission to the genetic apparatus of mechanical stresses that arise in the ectoderm and mesoderm of the embryo during the formation of its axial structures.