RESUMO
Understanding the mechanisms that control synaptic efficacy through the availability of neurotransmitter receptors depends on uncovering their specific intracellular trafficking routes. gamma-Aminobutyric acid type B (GABA(B)) receptors (GABA(B)Rs) are obligatory heteromers present at dendritic excitatory and inhibitory postsynaptic sites. It is unknown whether synthesis and assembly of GABA(B)Rs occur in the somatic endoplasmic reticulum (ER) followed by vesicular transport to dendrites or whether somatic synthesis is followed by independent transport of the subunits for assembly and ER export throughout the somatodendritic compartment. To discriminate between these possibilities we studied the association of GABA(B)R subunits in dendrites of hippocampal neurons combining live fluorescence microscopy, biochemistry, quantitative colocalization, and bimolecular fluorescent complementation. We demonstrate that GABA(B)R subunits are segregated and differentially mobile in dendritic intracellular compartments and that a high proportion of non-associated intracellular subunits exist in the brain. Assembled heteromers are preferentially located at the plasma membrane, but blockade of ER exit results in their intracellular accumulation in the cell body and dendrites. We propose that GABA(B)R subunits assemble in the ER and are exported from the ER throughout the neuron prior to insertion at the plasma membrane. Our results are consistent with a bulk flow of segregated subunits through the ER and rule out a post-Golgi vesicular transport of preassembled GABA(B)Rs.
Assuntos
Dendritos/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Receptores de GABA-B/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Retículo Endoplasmático/metabolismo , Feminino , Imunofluorescência , Hipocampo/citologia , Imunoprecipitação , Hibridização in Situ Fluorescente , Neurônios/citologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/genéticaRESUMO
Staufen is a conserved double-stranded RNA-binding protein required for mRNA localization in Drosophila oocytes and embryos. The mammalian homologues Staufen 1 and Staufen 2 have been implicated in dendritic RNA targeting in neurons. Here we show that in rodent oligodendrocytes, these two proteins are present in two independent sets of RNA granules located at the distal myelinating processes. A third kind of RNA granules lacks Staufen and contains major myelin mRNAs. Myelin Staufen granules associate with microfilaments and microtubules, and their subcellular distribution is affected by polysome-disrupting drugs. Under oxidative stress, both Staufen 1 and Staufen 2 are recruited into stress granules (SGs), which are stress-induced organelles containing transiently silenced messengers. Staufen SGs contain the poly(A)-binding protein (PABP), the RNA-binding proteins HuR and TIAR, and small but not large ribosomal subunits. Staufen recruitment into perinuclear SGs is paralleled by a similar change in the overall localization of polyadenylated RNA. Under the same conditions, the distribution of recently transcribed and exported mRNAs is not affected. Our results indicate that Staufen 1 and Staufen 2 are novel and ubiquitous SG components and suggest that Staufen RNPs are involved in repositioning of most polysomal mRNAs, but not of recently synthesized transcripts, during the stress response.