ABSTRACT
We recently reported, for the first time, the expression and regulation of the PDZ polarity proteins Scrib and Dlg1 in human APCs, and also described the viral targeting of these proteins by NS1 of influenza A virus in human dendritic cells (DCs). Scrib plays an important role in reactive oxygen species (ROS) production in MÏs and uropod formation and migration in T cells, while Dlg1 is important for T cell downstream activation after Ag recognition. Nevertheless, the functions of these proteins in human DCs remain unknown. Here, we knocked-down the expression of both Scrib and Dlg1 in human DCs and then evaluated the expression of co-stimulatory molecules and cytokine production during maturation. We demonstrated that Scrib is necessary for adequate CD86 expression, while Dlg1 is important for CD83 up-regulation and IL-6 production upon maturation, suggesting that Scrib and Dlg1 participate in separate pathways in DCs. Additionally, both proteins are required for adequate IL-12 production after maturation. Furthermore, we showed that the inefficient maturation of DCs induced by Scrib or Dlg1 depletion leads to impaired T cell activation. Our results revealed the previously unknown contribution of Scrib and Dlg1 in human DCs pivotal functions, which may be able to impact innate and adaptive immune response.
Subject(s)
Antigen Presentation , Dendritic Cells/immunology , Discs Large Homolog 1 Protein/physiology , Membrane Proteins/physiology , Tumor Suppressor Proteins/physiology , Adaptive Immunity , Antigens, CD/biosynthesis , Antigens, CD/genetics , B7-2 Antigen/biosynthesis , B7-2 Antigen/genetics , Cells, Cultured , Cytokines/biosynthesis , Cytokines/genetics , Discs Large Homolog 1 Protein/antagonists & inhibitors , Discs Large Homolog 1 Protein/genetics , Gene Knockdown Techniques , Humans , Immunity, Innate , Immunoglobulins/biosynthesis , Immunoglobulins/genetics , Interleukin-12/metabolism , Interleukin-6/biosynthesis , Interleukin-6/genetics , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Post-Synaptic Density/physiology , RNA Interference , RNA, Small Interfering/genetics , RNA, Small Interfering/pharmacology , Tumor Suppressor Proteins/antagonists & inhibitors , Tumor Suppressor Proteins/genetics , Up-Regulation , CD83 AntigenABSTRACT
AMPA receptors and interacting proteins are importantly involved in mediating stress-dependent plasticity. Previously we reported that GluA1-containing AMPA receptors and their interaction with PDZ-proteins are required for the experience-dependent expression of behavioral despair in the forced swim test. However, it is unclear if the expression of GluA1-containing AMPA receptors is affected by this type of behavior. Here we investigated in wild type mice, whether hippocampal gene or protein levels of GluA1 or associated PDZ proteins is altered following forced swim stress. We show that expression of Dlg4 (the gene coding for PSD-95) was strongly reduced after two days of forced swimming. In contrast, levels of Dlg1, Gria1, and Gria2 (coding for SAP97, GluA1, and GluA2 respectively) were not affected after one or two days of forced swimming. The changes in gene expression largely did not translate to the protein level. These findings indicate a limited acute effect of forced swim stress on the expression of the investigated targets and suggest that the acute involvement of GluA1-containing AMPA receptors tor forced swim behavior is a result of non-genomic mechanisms.
Subject(s)
Discs Large Homolog 1 Protein/metabolism , Disks Large Homolog 4 Protein/metabolism , Hippocampus/metabolism , Receptors, AMPA/metabolism , Animals , Blotting, Western , Discs Large Homolog 1 Protein/analysis , Discs Large Homolog 1 Protein/physiology , Disks Large Homolog 4 Protein/analysis , Disks Large Homolog 4 Protein/physiology , Female , Gene Expression Regulation , Hippocampus/chemistry , Hippocampus/physiology , Mice , Mice, Inbred C57BL , Real-Time Polymerase Chain Reaction , Receptors, AMPA/analysis , Receptors, AMPA/physiology , Stress, Physiological/physiology , SwimmingABSTRACT
Vertebrate embryogenesis and organogenesis are driven by cell biological processes, ranging from mitosis and migration to changes in cell size and polarity, but their control and causal relationships are not fully defined. Here, we use the developing limb skeleton to better define the relationships between mitosis and cell polarity. We combine protein-tagging and -perturbation reagents with advanced in vivo imaging to assess the role of Discs large 1 (Dlg1), a membrane-associated scaffolding protein, in mediating the spatiotemporal relationship between cytokinesis and cell polarity. Our results reveal that Dlg1 is enriched at the midbody during cytokinesis and that its multimerization is essential for the normal polarity of daughter cells. Defects in this process alter tissue dimensions without impacting other cellular processes. Our results extend the conventional view that division orientation is established at metaphase and anaphase and suggest that multiple mechanisms act at distinct phases of the cell cycle to transmit cell polarity. The approach employed can be used in other systems, as it offers a robust means to follow and to eliminate protein function and extends the Phasor approach for studying in vivo protein interactions by frequency-domain fluorescence lifetime imaging microscopy of Förster resonance energy transfer (FLIM-FRET) to organotypic explant culture.
