Components of the Gs signaling cascade exhibit distinct changes in mobility and membrane domain localization upon ß2 -adrenergic receptor activation.

The G protein signaling cascade is a key player in cell signaling. Cascade activation leads to a redistribution of its members in various cellular compartments. These changes are likely related to the "second wave" of signaling from endosomes. Here, we set out to determine whether Gs signaling cascade members expressed at very low levels exhibit altered mobility and localize in clathrin-coated structures (CCSs) or caveolae upon activation by ß2 -adrenergic receptors (ß2 AR). Activated ß2 AR showed decreased mobility and sustained accumulation in CCSs but not in caveolae. Arrestin 3 translocated to the plasma membrane after ß2 AR activation and showed very low mobility and pronounced accumulation in CCSs. In contrast, Gαs and Gγ2 exhibited a modest reduction in mobility but no detectable accumulation in or exclusion from CCSs or caveolae. The effector adenylyl cyclase 5 (AC5) showed a slight mobility increase upon ß2 AR stimulation, no redistribution to CCSs, and weak activation-independent accumulation in caveolae. Our findings show an overall decrease in the mobility of most activated Gs signaling cascade members and confirm that ß2 AR and arrestin 3 accumulate in CCSs, while Gαs , Gγ2 and AC5 can transiently enter CCSs and caveolae but do not accumulate in and are not excluded from these domains.

Designing a Scale to Assess Dialectical Thinking: Link to ECERS-R Items.

Dialectical thinking is gaining wide circulation as part of personal and social preschool child development in modern society, which makes all the more urgent the task of designing a tool to evaluate the extent to which the educational environment in a pre-school establishment supports the development of dialectical thinking in preschoolers. To implement this task, the researchers analyzed the ECERS-R as a means for assessing the quality of preschool education and concluded that this tool fails to focus on rating the environment in terms of the development of dialectical thinking. N.Ye. Veraksa and E.V. Sviridova designed a tool for assessing how well the educational environment supports dialectical thinking in preschoolers (the scale of dialectical thinking support (DTS scale). The research into the use of the DTS scale was conducted in 18 preparatory groups of three educational complexes in Moscow in February-April 2019. The comparison of the results on the DTS scale and those on the ECERS-R scale made it possible to conclude that the ECERS-R scale does not differentiate between the stimulation of dialectical thinking and formally logical thinking in preschool age children. The use of the newly designed tool was justified statistically. It is noted that the teacher activity in line with the ECERS-R scale scores of "Stimulating Communication with Children", "Books and Illustrations", "Using Speech to Develop Cognitive Skills" may be associated with decreased levels of support for children's dialectical thinking in preschool educational institutions. In addition, a positive relationship was found to exist between the ECERS-R score of "Care-Giver and Children Interaction" and DTS scale. The results obtained make it possible to hypothesize that there are interrelations between the development of dialectic thinking in children, on the one hand, and voluntariness and the emotional sphere, on the other.

Structural basis of the interaction between the putative adhesion-involved and iron-regulated FrpD and FrpC proteins of Neisseria meningitidis.

The iron-regulated protein FrpD from Neisseria meningitidis is an outer membrane lipoprotein that interacts with very high affinity (Kd ~ 0.2 nM) with the N-terminal domain of FrpC, a Type I-secreted protein from the Repeat in ToXin (RTX) protein family. In the presence of Ca2+, FrpC undergoes Ca2+ -dependent protein trans-splicing that includes an autocatalytic cleavage of the Asp414-Pro415 peptide bond and formation of an Asp414-Lys isopeptide bond. Here, we report the high-resolution structure of FrpD and describe the structure-function relationships underlying the interaction between FrpD and FrpC1-414. We identified FrpD residues involved in FrpC1-414 binding, which enabled localization of FrpD within the low-resolution SAXS model of the FrpD-FrpC1-414 complex. Moreover, the trans-splicing activity of FrpC resulted in covalent linkage of the FrpC1-414 fragment to plasma membrane proteins of epithelial cells in vitro, suggesting that formation of the FrpD-FrpC1-414 complex may be involved in the interaction of meningococci with the host cell surface.

Backbone resonance assignments of the outer membrane lipoprotein FrpD from Neisseria meningitidis.

The iron-regulated FrpD protein is a unique lipoprotein embedded into the outer membrane of the Gram-negative bacterium Neisseria meningitidis. The biological function of FrpD remains unknown but might consist in anchoring to the bacterial cell surface the Type I-secreted FrpC protein, which belongs to a Repeat in ToXins (RTX) protein family and binds FrpD with very high affinity (K(d) = 0.2 nM). Here, we report the backbone (1)H, (13)C, and (15)N chemical shift assignments for the FrpD(43-271) protein that allow us to characterize the intimate interaction between FrpD and the N-terminal domain of FrpC.

Crystallization and preliminary crystallographic characterization of the iron-regulated outer membrane lipoprotein FrpD from Neisseria meningitidis.

Fe-regulated protein D (FrpD) is a Neisseria meningitidis outer membrane lipoprotein that may be involved in the anchoring of the secreted repeat in toxins (RTX) protein FrpC to the outer bacterial membrane. However, the function and biological roles of the FrpD and FrpC proteins remain unknown. Native and selenomethionine-substituted variants of recombinant FrpD43-271 protein were crystallized using the sitting-drop vapour-diffusion method. Diffraction data were collected to a resolution of 2.25 A for native FrpD43-271 protein and to a resolution of 2.00 A for selenomethionine-substituted FrpD43-271 (SeMet FrpD43-271) protein. The crystals of native FrpD43-271 protein belonged to the hexagonal space group P6(2) or P6(4), while the crystals of SeMet FrpD43-271 protein belonged to the primitive orthorhombic space group P2(1)2(1)2(1).