ABSTRACT
The vesicular glutamate transporter VGLUT1 loads synaptic vesicles with the neurotransmitter glutamate and thereby determines glutamate release at many synapses in the mammalian brain. Due to its function and selective localization, VGLUT1 is one of the most specific markers for glutamatergic synaptic vesicles. It has been used widely to identify glutamatergic synapses, and its expression levels are tightly correlated with changes in quantal size, modulations of synaptic plasticity, and corresponding behaviors. We generated a fluorescent VGLUT1(Venus) knock-in mouse for the analysis of VGLUT1 and glutamatergic synaptic vesicle trafficking. The mutation does not affect glutamatergic synapse function, and thus the new mouse model represents a universal tool for the analysis of glutamatergic transmitter systems in the forebrain. Previous studies demonstrated synaptic vesicle exchange between terminals in vitro. Using the VGLUT1(Venus) knock-in, we show that synaptic vesicles are dynamically shared among boutons in the cortex of mice in vivo. We provide a detailed analysis of synaptic vesicle sharing in vitro, and show that network homeostasis leads to dynamic scaling of synaptic VGLUT1 levels.
Subject(s)
Bacterial Proteins/metabolism , Luminescent Proteins/metabolism , Neurons/cytology , Presynaptic Terminals/physiology , Synapses/metabolism , Synaptic Vesicles/physiology , Animals , Bacterial Proteins/genetics , Cerebral Cortex/cytology , Cerebral Cortex/metabolism , Disks Large Homolog 4 Protein , Electric Stimulation , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/genetics , Fluorescence Recovery After Photobleaching/methods , Glutamic Acid/metabolism , Hippocampus/cytology , Hippocampus/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Luminescent Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Mutation/genetics , Nerve Tissue Proteins/metabolism , Neurons/physiology , Organ Culture Techniques , Patch-Clamp Techniques , Presynaptic Terminals/drug effects , Presynaptic Terminals/metabolism , Protein Transport/genetics , RNA, Messenger/metabolism , Subcellular Fractions/metabolism , Vesicular Glutamate Transport Protein 1/genetics , Vesicular Glutamate Transport Protein 1/metabolism , Vesicular Glutamate Transport Protein 2/metabolismABSTRACT
Activin-A is a transforming growth factor-beta (TGF-beta) superfamily member that plays a pivotal role in many developmental and reproductive processes. It is also involved in neuroprotection, apoptosis of tumor and some immune cells, wound healing, and cancer. Its role as an immune-regulating protein has not previously been described. Here we demonstrate for the first time that activin-A has potent autocrine effects on the capacity of human dendritic cells (DCs) to stimulate immune responses. Human monocyte-derived DCs (MoDCs) and the CD1c(+) and CD123(+) peripheral blood DC populations express both activin-A and the type I and II activin receptors. Furthermore, MoDCs and CD1c(+) myeloid DCs rapidly secrete high levels of activin-A after exposure to bacteria, specific toll-like receptor (TLR) ligands, or CD40 ligand (CD40L). Blocking autocrine activin-A signaling in DCs using its antagonist, follistatin, enhanced DC cytokine (IL-6, IL-10, IL-12p70, and tumor necrosis factor-alpha [TNF-alpha]) and chemokine (IL-8, IP-10, RANTES, and MCP-1) production during CD40L stimulation, but not TLR-4 ligation. Moreover, antagonizing DC-derived activin-A resulted in significantly enhanced expansion of viral antigen-specific effector CD8(+) T cells. These findings establish an immune-regulatory role for activin-A in DCs, highlighting the potential of antagonizing activin-A signaling in vivo to enhance vaccine immunogenicity.
Subject(s)
Activins/immunology , CD40 Ligand/immunology , Chemokines/biosynthesis , Dendritic Cells/immunology , Activins/genetics , Activins/metabolism , Bone Morphogenetic Protein 4 , Bone Morphogenetic Protein 7 , Bone Morphogenetic Proteins/genetics , Bone Morphogenetic Proteins/metabolism , CD40 Ligand/pharmacology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/immunology , Cell Proliferation/drug effects , Cell Separation , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Epitopes , Follistatin/pharmacology , Gene Expression Regulation/drug effects , Humans , Lipopolysaccharides/pharmacology , Myostatin , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolismABSTRACT
The integration of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membrane requires the integral thylakoid membrane protein ALB3, a homologue of the bacterial cytoplasmic membrane protein YidC. In bacteria, YidC is associated with the SecY-translocase and facilitates the integration of Sec-dependent proteins into the plasma membrane. In addition, it is also involved in the insertion of Sec-independent proteins. In the present study we demonstrate, in Arabidopsis thaliana, that most ALB3 is a constituent of an oligomeric complex of approx. 180 kDa. In addition, we detected ALB3 in several higher-molecular-mass complexes (up to 700 kDa). Furthermore, we show that most ALB3 co-fractionates with cpSecY during gel-filtration analysis and blue native gel electrophoresis, suggesting an association of ALB3 with the cpSecY complex. A direct interaction of ALB3 with the cpSecY complex was demonstrated by co-immunoprecipitation experiments using digitonin-solubilized thylakoid membrane proteins and anti-cpSecY or anti-ALB3 antibodies. This result was further confirmed by electron microscopic co-immunolocalization of ALB3 and cpSecY. In addition, an association of ALB3 with the cpSecY complex was demonstrated directly by cross-linking experiments using the chemical cross-linker disuccinimidyl suberate.
