Cellular and subcellular localization of EFA6C, a third member of the EFA6 family, in adult mouse Purkinje cells (original) (raw)
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European Journal of Neuroscience, 2004
EFA6A is a guanine nucleotide exchange protein (GEP) that can speci®cally activate ADP-ribosylation factor 6 (ARF6) in vitro. A recent study has demonstrated that ARF6 is involved in the dendritic formation of developing hippocampal neurons [Hernandez-Deviez et al. (2002) Nature Neurosci., 5, 623±624]. This study examined a potential role for EFA6A in hippocampal development in Wistar rats. Our results provided de®nitive evidence for somatodendritic localization of EFA6A mRNA in both cultured and in vivo hippocampal neurons by nonradioactive in situ hybridization. During postnatal development, EFA6A mRNA was dramatically increased and its dendritic localization was most evident between P7 and P14. In contrast, ARF6 mRNA was con®ned to the neuronal layers of the hippocampus throughout development. In addition, the overexpression of a GEP-defective mutant of EFA6A enhanced the dendritic formation of the primary hippocampal neurons. The present ®ndings suggest that EFA6A is intimately involved in the regulation of the dendritic development of hippocampal neurons. P < 0.001; Student's t-test, compared with the control vector. Scale bar, 10 mm (A).
European Journal of Neuroscience, 2007
EFA6A is a member of the guanine nucleotide exchange factors that can specifically activate ADP ribosylation factor 6 (ARF6). In this study, we identified a-actinin-1 as a possible interacting protein with EFA6A by the yeast two-hybrid screening with its C-terminal region as bait. The central region of a-actinin-1 containing a part of spectrin repeat 1 and spectrin repeats 2-3 is responsible for this interaction. In the hippocampal formation, EFA6A immunoreactivity occurred at a high level as numerous fine puncta in the strata oriens, radiatum, lacunosum-moleculare of the hippocampal CA1-3 subfields and the dentate molecular layer, whereas the immunoreactivity was faint in the neuronal cell layers and the stratum lucidum, the mossy fiber-recipient layer of the CA3 subfield. Double-immunofluorescent analyses revealed a partial overlapping of EFA6A and a-actinin at the dendritic spines of in vivo and cultured hippocampal neurons. Our present findings suggest that EFA6A may form a protein complex with a-actinin and activate ARF6 in close proximity of the actin cytoskeleton and membrane proteins in the dendritic spines.
Brain Research, 2006
The EFA6 family is a member of guanine nucleotide exchange factors (GEFs) that can activate ARF6 specifically in vitro. In this study, we determined the complete primary sequence of mouse EFA6D encoding a protein of 1004 amino acids with a calculated molecular weight of 111,207 Da. In ARF pull-down assay, EFA6D showed a preferential GEF activity toward ARF6. RT-PCR analysis revealed the widespread tissue distribution of EFA6D and the high expression of EFA6A, C and D in the brain. In situ hybridization analysis demonstrated a distinct spatiotemporal expression pattern of EFA6D from those of EFA6A and C in mouse brain. Furthermore, immunoblot analysis revealed that EFA6D was highly concentrated in the postsynaptic density fraction. These findings suggest differential spatiotemporal regulation of ARF6 by three members of the EFA6 family in the brain.
ARF6 and EFA6A Regulate the Development and Maintenance of Dendritic Spines
Journal of Neuroscience, 2006
The cellular and molecular mechanisms underlying the development and maintenance of dendritic spines are not fully understood. ADP-ribosylation factor 6 (ARF6) is a small GTPase known to regulate actin remodeling and membrane traffic. Here, we report involvement of ARF6 and exchange factor for ARF6 (EFA6A) in the regulation of spine development and maintenance. An active form of ARF6 promotes the formation of dendritic spines at the expense of filopodia. EFA6A promotes spine formation in an ARF6 activation-dependent manner. Knockdown of ARF6 and EFA6A by small interfering RNA decreases spine formation. Live imaging indicates that ARF6 knockdown decreases the conversion of filopodia to spines and the stability of early spines. The spine-promoting effect of ARF6 is partially blocked by Rac1. ARF6 and EFA6A protect mature spines from inactivity-induced destabilization. These results suggest that ARF6 and EFA6A may regulate the conversion of filopodia to spines and the stability of both early and mature spines.
Brain Research, 2008
EFA6A is a guanine nucleotide exchange factor that is highly expressed in the nervous system with the ability to activate ADP ribosylation factor 6 (ARF6). In this study, we demonstrated the immunohistochemical localization of EFA6A in the adult mouse retina. Strong immunoreactivity for EFA6A was detected predominantly in the outer plexiform layer (OPL), where EFA6A was partially overlapped with dystrophin and synaptophysin. Immunoelectron microscopic analysis revealed that EFA6A was localized predominantly at the perisynaptic processes of photoreceptor terminals without association with synaptic ribbons. These findings suggest that EFA6A-ARF6 pathway may play a specific role at a subcompartment of perisynaptic photoreceptor processes.
Molecular Brain Research, 2002
ADP-ribosylation factors (ARFs) play important roles in vesicular trafficking and cytoskeletal regulation and its activation depends on guanine nucleotide-exchange proteins (GEPs). By way of in situ hybridization histochemistry, the localization of mRNAs for subfamily members of low-molecular-weight ARF-GEPs in the rat brain was studied at embryonic and postnatal stages. In the embryonic brain, the gene expression for msec7-1 was distinct in the ventricular zone while that for msec7-1,-3 and EFA6 in the mantle zone. In early postnatal brain, the expression for msec7-1,-2,-3 and EFA6 was seen widely in various loci of the gray matter with different intensity, and the expression of msec7-1 and-2 mRNAs was evident in the cerebellar external granule cell layer. In the adult brain, the gene expression for the four ARF-GEPs decreased more or less in most gray matter and the distinct expression was maintained mainly in the hippocampal and dentate neuronal layers and cerebellar cortex. The expression of EFA6 mRNA was also evident in the molecular layer of the hippocampus and dentate gyrus. No obvious gene expression for cytohesin-4 and ARF-GEP100 was detected in the brain at any stages of development. The present findings suggest that ARF-GEPs are differentially involved in some processes essential to neuronal differentiation and maturation in association with ARFs.
Biochemical and Biophysical Research Communications, 2007
To identify neuron-specific genes, we performed gene expression profiling, cDNA microarray and in silico ESTs (expressed sequence tags) analyses. We identified a human neuron-specific gene, KIAA1110 (homologue of rat synArfGEF (Po)), that is a member of the guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF). RT-PCR analysis showed that the KIAA1110 gene was expressed specifically in the brain among adult human tissues, whereas no apparent expression was observed in immature neural tissues/cells, such as fetal brain, glioma tissues/cells, and neural stem/precursor cells (NSPCs). The KIAA1110 protein was shown to be expressed in mature neurons but not in undifferentiated NSPCs. Immunohistochemical analysis also showed that KIAA1110 was expressed in neurons of the human adult cerebral cortex. Furthermore, the pull-down assay revealed that KIAA1110 has a GEF activity toward ARF1 that regulates transport along the secretion pathway. These results suggest that KIAA1110 is expressed specifically in mature neurons and may play an important role in the secretion pathway as a GEF for ARF1.
Journal of Neurochemistry, 2004
We cloned from a rat brain cDNA library a novel cDNA and named it a potential synaptic guanine nucleotide exchange factor (GEF) for Arf (synArfGEF (Po)) (GenBank Accession no. AB057643) based on its domain structure and localization. The cloned gene was 7410 bases long with a 3585-bp coding sequence encoding a protein of 1194 amino acids. The deduced protein contained a coiled-coil structure in the N-terminal portion followed by Sec7 and Plekstrin homology (PH) domains. Thus, the protein was a member of the Sec7 family of proteins, GEFs. Conservation of the ADP-ribosylation factor (Arf)-binding sequence suggested that the protein was a GEF for Arf. The gene was expressed specifically in the brain, where it exhibited region-specific expression. The protein was highly enriched in the postsynaptic density (PSD) fraction prepared from the rat forebrain. Uniquely, the protein interacted with PSD-95, SAP97 and Homer/Vesl 1/PSD-Zip45 via its C-terminal PDZ-binding motif and co-localized with these proteins in cultured cortical neurons. These results supported its localization in the PSD. The postsynaptic localization was also supported by immunohistochemical examination of the rat brain. The mRNA for the synArfGEF was also localized to dendrites, as well as somas, of neuronal cells. Thus, both the mRNA and the protein were localized in the postsynaptic compartments. These results suggest a postsynaptic role of synArfGEF in the brain.
Molecular Brain Research, 2001
ADP-ribosylation factors (ARFs) play crucial roles in the intracellular vesicular transport and in regulation of phospholipid-modifying enzyme activities and cytoskeletons. Using in situ hybridization histochemistry, the gene expression for six isoforms of ARF was examined during normal development of rats and in the hypoglossal nucleus following its axotomy. In the embryonic brain, the expression for ARF-1,-4,-5,-6 mRNAs was distinct in the ventricular germinal zone while that for ARF-3,-4,-5 in the mantle zone. In early postnatal brain, the expression for six ARFs was seen widely in various loci of the gray matter with different intensity, and the expression of ARF-4,-5,-6 mRNAs was evident in the cerebellar external granule cell layer. In the adult brain, the gene expression for all ARF isoforms decreased more or less in most gray matters and the distinct expression was maintained mainly in the hippocampal and dentate neuronal layers and cerebellar cortex. The expression levels of ARF-2 and-4 mRNAs in affected hypoglossal nucleus increased after 24 h up to 7 days following axotomy of the hypoglossal nerve, while no such changes were seen in the expression levels for the other ARFs. The present findings suggest that ARFs are differentially involved in some processes essential to nerve regeneration as well as neuronal differentiation and maturation.
ARF6 Regulates Neuron Differentiation through Glucosylceramide Synthase
PLOS ONE, 2013
The small GTPase ADP ribosylation factor 6 (ARF6) mediates endocytosis and has in addition been shown to regulate neuron differentiation. Here we investigated whether ARF6 promotes differentiation of Neuro-2a neuronal cells by modifying the cellular lipid composition. We showed that knockdown of ARF6 by siRNA in Neuro-2a cells increased neuronal outgrowth as expected. ARF6 knockdown also resulted in increased glucosylceramide levels and decreased sphingomyelin levels, but did not affect the levels of ceramide or phospholipids. We speculated that the ARF6 knockdown-induced increase in glucosylceramide was caused by an effect on glucosylceramide synthase and, in agreement, showed that ARF6 knockdown increased the mRNA levels and activity of glucosylceramide synthase. Finally, we showed that incubation of Neuro-2a cells with the glucosylceramide synthase inhibitor D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) normalized the increased neuronal outgrowth induced by ARF6 knockdown. Our results thus show that ARF6 regulates neuronal differentiation through an effect on glucosylceramide synthase and glucosylceramide levels.