The 14-3-3ζ Protein Binds to the Cell Adhesion Molecule L1, Promotes L1 Phosphorylation by CKII and Influences L1-Dependent Neurite Outgrowth

BACKGROUND: The cell adhesion molecule L1 is crucial for mammalian nervous system development. L1 acts as a mediator of signaling events through its intracellular domain, which comprises a putative binding site for 14-3-3 proteins. These regulators of diverse cellular processes are abundant in the brain and preferentially expressed by neurons. In this study, we investigated whether L1 interacts with 14-3-3 proteins, how this interaction is mediated, and whether 14-3-3 proteins influence the function of L1. METHODOLOGY/PRINCIPAL FINDINGS: By immunoprecipitation, we demonstrated that 14-3-3 proteins are associated with L1 in mouse brain. The site of 14-3-3 interaction in the L1 intracellular domain (L1ICD), which was identified by site-directed mutagenesis and direct binding assays, is phosphorylated by casein kinase II (CKII), and CKII phosphorylation of the L1ICD enhances binding of the 14-3-3 zeta isoform (14-3-3ζ). Interestingly, in an in vitro phosphorylation assay, 14-3-3ζ promoted CKII-dependent phosphorylation of the L1ICD. Given that L1 phosphorylation by CKII has been implicated in L1-triggered axonal elongation, we investigated the influence of 14-3-3ζ on L1-dependent neurite outgrowth. We found that expression of a mutated form of 14-3-3ζ, which impairs interactions of 14-3-3ζ with its binding partners, stimulated neurite elongation from cultured rat hippocampal neurons, supporting a functional connection between L1 and 14-3-3ζ. CONCLUSIONS/SIGNIFICANCE: Our results suggest that 14-3-3ζ, a novel direct binding partner of the L1ICD, promotes L1 phosphorylation by CKII in the central nervous system, and regulates neurite outgrowth, an important biological process triggered by L1

Heterodimerization of serotonin receptors 5-HT1A and 5-HT7 differentially regulates receptor signalling and trafficking.

Serotonin receptors 5-HT 1A and 5-HT 7 are highly coexpressed in brain regions implicated in depression. However, their functional interaction has not been established. In the present study we show that 5-HT 1A and 5-HT 7 receptors form heterodimers both in vitro and in vivo. Foerster resonance energy transfer-based assays revealed that, in addition to heterodimers, homodimers composed either of 5- HT 1A or 5-HT 7 receptors together with monomers coexist in cells. The highest affinity for complex formation was obtained for the 5-HT 7 –5-HT 7 homodimers, followed by the 5-HT 7 –5-HT 1A heterodimers and 5-HT 1A –5-HT 1A homodimers. Functionally, heterodimerization decreases 5-HT 1A -receptor-mediated activation of G i protein without affecting 5-HT 7 -receptor-mediated signalling. Moreover, heterodimerization markedly decreases the ability of the 5-HT 1A receptor to activate G-protein-gated inwardly rectifying potassium channels in a heterologous system. The inhibitory effect on such channels was also preserved in hippocampal neurons, demonstrating a physiological relevance of heteromerization in vivo. In addition, heterodimerization is crucially involved in initiation of the serotonin-mediated 5-HT 1A receptor internalization and also enhances the ability of the 5-HT 1A receptor to activate the mitogen-activated protein kinases. Finally, we found that production of 5-HT 7 receptors in the hippocampus continuously decreases during postnatal development, indicating that the relative concentration of 5-HT 1A –5-HT 7 heterodimers and, consequently, their functional importance undergoes pronounced developmental change

Electroporation: an arsenal of application

Electroporation is a way to induce nanometersized membrane pore for exogenous substances delivery into cytoplasm using an artificial electric field. Now it was widely used for molecules transfer especially in molecular experiments and genetic aspects. In recent years, modern electroporation on the embryo was developed, whose most important point is that it adopts low energy and rectangular pulse that could obtain high transfection efficiency and low damage to the embryo. This paper reviewed on the pool of application: from lab works to human clinical treatments