The Lim‐domain protein Zyxin was initially identified as a minor actin cytoskeleton protein that regulates the assembly and repair of actin filaments. At the same time, additional functions revealed ...for Zyxin in recent decades indicate that this protein can also play an important role in regulating gene expression and cell differentiation. In this review, we analysed the data in the literature pointing to Zyxin as one of the possible molecular hubs linking morphogenetic cell movements with gene expression, stem cell status regulation and pattern formation during the most complex processes in organism life, embryogenesis.
In this review, we analysed the data in the literature pointing to Zyxin as one of the possible molecular hubs linking morphogenetic cell movements with gene expression, stem cell status regulation and pattern formation in the most complex process in organism life, embryogenesis.
Zyxin is an LIM-domain-containing protein that regulates the assembly of F-actin filaments in cell contacts. Additionally, as a result of mechanical stress, Zyxin can enter nuclei and regulate gene ...expression. Previously, we found that Zyxin could affect mRNA stability of the maternally derived stemness factors of Pou5f3 family in Xenopus laevis embryos through binding to Y-box factor1. In the present work, we demonstrate that Zyxin can also affect mRNA stability of the maternally derived retinoid receptor Rxrγ through the same mechanism. Moreover, we confirmed the functional link between Zyxin and Rxrγ-dependent gene expression. As a result, Zyxin appears to play an essential role in the regulation of the retinoic acid signal pathway during early embryonic development. Besides, our research indicates that the mechanism based on the mRNA destabilization by Zyxin may take part in the control of the expression of a fairly wide range of maternal genes.
Zyxin is a cytoskeletal LIM-domain protein that regulates actin cytoskeleton assembly and gene expression. In the present work, we find that zyxin downregulation in Xenopus laevis embryos reduces the ...expression of numerous genes that regulate cell differentiation, but it enhances that of several genes responsible for embryonic stem cell status, specifically klf4, pou5f3.1, pou5f3.2, pou5f3.3, and vent2.1/2. For pou5f3 family genes (mammalian POU5F1/OCT4 homologs), we show that this effect is the result of mRNA stabilization due to complex formation with the Y-box protein Ybx1. When bound to Ybx1, zyxin interferes with the formation of these complexes, thereby stimulating pou5f3 mRNA degradation. In addition, in zebrafish embryos and human HEK293 cells, zyxin downregulation increases mRNA levels of the pluripotency genes KLF4, NANOG, and POU5F1/OCT4. Our findings indicate that zyxin may play a role as a switch among morphogenetic cell movement, differentiation, and embryonic stem cell status.
Display omitted
•Zyxin inhibits the activity of pou5f3 genes responsible for embryonic stem cell status•Ybx1 binds pou5f3 mRNA, thus increasing its stability•Zyxin directly binds to Ybx1 and holds it in the cytoplasm•Zyxin reduces stability of pou5f3 mRNA by interfering with its binding to Ybx1
Parshina et al. disclose a mechanism by which the cytoskeletal protein zyxin, which has known effects on cell movement and gene expression when it shuttles into the cell nucleus, reduces the mRNA stability of genes, maintaining embryonic stem cell status through its interaction with the mRNA-stabilizing protein Ybx1.
How embryos scale patterning according to size is still not fully understood. Through in silico screening and analysis of reaction-diffusion systems that could be responsible for scaling, we ...predicted the existence of genes whose expression is sensitive to embryo size and which regulate the scaling of embryonic patterning. To find these scalers, we identified genes with strongly altered expression in half-size Xenopus laevis embryos compared with full-size siblings at the gastrula stage. Among found genes, we investigated the role of matrix metalloproteinase-3 (mmp3), which was most strongly downregulated in half-size embryos. We show that Mmp3 scales dorsal-ventral patterning by degrading the slowly diffusing embryonic inducers Noggin1 and Noggin2 but preventing cleavage of the more rapidly diffusing inducer Chordin via degradation of a Tolloid-type proteinase. In addition to unraveling the mechanism underlying the scaling of dorsal-ventral patterning, this work provides proof of principal for scalers identification in embryos of other species.
Display omitted
•Scalers are patterning genes whose expression is sensitive to embryo size•Search for scalers using model of Xenopus wild-type and half-size embryos•Mmp3 scales dorsal-ventral patterning to match embryo size•Mmp3 protects Chordin from cleavage by Tolloid but promotes Noggin1/2 degradation
Orlov, Nesterenko et al. investigate embryonic scaling, the mechanism ensuring that embryonic structures are proportional to size. In a screen for “scalers” — genes with size-dependent expression — they identify matrix metalloproteinase 3 (mmp3) and show that it scales dorsal-ventral patterning in frog embryos by oppositely regulating the Chordin and Noggin1/2 morphogens.
This protocol is developed for identifying mRNAs that form complexes with mRNA-binding proteins (mRBPs) in Xenopus laevis embryos at different developmental stages. Here, we describe the use of the ...Ybx1 mRBP for immunoprecipitation-based mRNA isolation. This protocol features the translation of the mRBP of interest directly in living embryos following injection of synthetic mRNA templates encoding a hybrid of this protein with a specific tag. This approach allows precipitation of mRNA-protein complexes from embryonic lysates using commercially available anti-tag antibodies.
For complete details on the use and execution of this protocol, please refer to Parshina et al. (2020).
Display omitted
•Studying the repertoire of mRNA-binding proteins at different stages of development•Immunoprecipitation-based isolation of mRNAs from Xenopus laevis embryos•Precipitation of mRNA-protein complexes on commercially available carriers•Studying effects of the agents of interest upon the formation of mRNA-protein complexes
This protocol is developed for identifying mRNAs that form complexes with mRNA-binding proteins (mRBPs) in Xenopus laevis embryos at different developmental stages. Here, we describe the use of the Ybx1 mRBP for immunoprecipitation-based mRNA isolation. This protocol features the translation of the mRBP of interest directly in living embryos following injection of synthetic mRNA templates encoding a hybrid of this protein with a specific tag. This approach allows precipitation of mRNA-protein complexes from embryonic lysates using commercially available anti-tag antibodies.
Summary
The Agr family genes, Ag1, Agr2, and Agr3, encode for the thioredoxin domain containing secreted proteins and are specific only for vertebrates. These proteins are attracting increasing ...attention due to their involvement in many physiological and pathological processes, including exocrine secretion, cancer, regeneration of the body appendages, and the early brain development. At the same time, the mode by which Agrs regulate intracellular processes are poorly understood. Despite that the receptor to Agr2, the membrane anchored protein Prod1, has been firstly discovered in Urodeles, and it has been shown to interact with Agr2 in the regenerating limb, no functional homologs of Prod1 were identified in other vertebrates. This raises the question of the mechanisms by which Agrs can regulate regeneration in other lower vertebrates. Recently, we have identified that Tfp4 (three‐fingers Protein 4), the structural and functional homolog of Prod1 in Anurans, interacts with Agr2 in Xenopus laevis embryos. In the present work we show by several methods that the activity of Tfp4 is essential for the tadpole tail regeneration as well as for the early eye and forebrain development during embryogenesis. These data show for the first time the common molecular mechanism of regeneration regulation in amphibians by interaction of Prod1 and Agr2 proteins.
This protocol for the separation of nuclear and cytoplasmic fractions of cells of Xenopus laevis embryos was developed to study changes in the intracellular localization of the Zyxin and Ybx1 ...proteins, which are capable of changing localization in response to certain stimuli. Western blot analysis allows the quantification of changes in the distribution of these proteins between the cytoplasm and nucleus, whereas the posttranslational modifications specific to each compartment can be identified by changes in electrophoretic mobility.
For complete details on the use and execution of this protocol, please refer to Parshina et al. (2020).
Display omitted
•A simple way to obtain tagged proteins in Xenopus embryos•Rapid separation of Xenopus embryonic cells into nuclear, cytoplasmic fractions•Quantifying the distribution of shuttle proteins between the cytoplasm and nucleus•Protein posttranslational modifications specific to the cytoplasm and nucleus can be studied
This protocol for the separation of nuclear and cytoplasmic fractions of cells of Xenopus laevis embryos was developed to study changes in the intracellular localization of the Zyxin and Ybx1 proteins, which are capable of changing localization in response to certain stimuli. Western blot analysis allows the quantification of changes in the distribution of these proteins between the cytoplasm and nucleus, while the posttranslational modifications specific to each compartment can be identified by changes in electrophoretic mobility.
Carotenoids are potent antioxidants with a wide range of biomedical applications. However, their delivery into human cells is challenging and relatively inefficient. While the use of natural ...water-soluble carotenoproteins capable to reversibly bind carotenoids and transfer them into membranes is promising, the quantitative estimation of the delivery remains unclear. In the present work, we studied echinenone (ECN) delivery by cyanobacterial carotenoprotein AnaCTDH (C-terminal domain homolog of the Orange Carotenoid Protein from Anabaena), into liposome membranes labelled with BODIPY fluorescent probe. We observed that addition of AnaCTDH-ECN to liposomes led to the significant changes in the fast-kinetic component of the fluorescence decay curve, pointing on the dipole-dipole interactions between the probe and ECN within the membrane. It may serve as an indirect evidence of ECN delivery into membrane. To study the delivery in detail, we carried out molecular dynamics modeling of the localization of ECN within the lipid bilayer and calculate its orientation factor. Next, we exploited FRET to assess concentration of ECN delivered by AnaCTDH. Finally, we used time-resolved fluorescence anisotropy to assess changes in microviscosity of liposomal membranes. Incorporation of liposomes with β-carotene increased membrane microviscosity while the effect of astaxanthin and its mono- and diester forms was less pronounced. At temperatures below 30 °C addition of AnaCTDH-ECN increased membrane microviscosity in a concentration-dependent manner, supporting the protein-mediated carotenoid delivery mechanism. Combining all data, we propose FRET-based analysis and assessment of membrane microviscosity as potent approaches to characterize the efficiency of carotenoids delivery into membranes.
Abstract
The aim of this work was a comparative study of biohydrogen production from cheese whey and confectionary wastewater by a newly isolated thermophilic microbial strain
Thermoanaerobacterium ...thermosaccharolyticum
SP-H2. Experimental results showed that the fermentative hydrogen was successfully produced with the highest hydrogen yield of 3.9 mL H
2
/mL cheese whey or 80 mL H
2
/g chemical oxygen demand. The profile of soluble metabolite products showed that hydrogen generation by a new isolate was mainly acetate-type fermentation in the case of confectionary wastewater and mixed ethanol-acetate-lactate type fermentation in the case of cheese whey. The more optimal metabolic pathway of confectionary wastewater fermentation was confirmed by the better kinetic characteristics according to the Gompertz model.
PDF
