ADP Ribosylation Factor 4 (Arf4) Regulates Radial Migration through N-Cadherin Trafficking during Cerebral Cortical Development.

During the development of the cerebral cortex, N-cadherin plays a crucial role in facilitating radial migration by enabling cell-to-cell adhesion between migrating neurons and radial glial fibers or Cajar-Reztius cells. ADP ribosylation factor 4 (Arf4) and Arf5, which belong to the Class II Arf small GTPase subfamily, control membrane trafficking in the endocytic and secretory pathways. However, their specific contribution to cerebral cortex development remains unclear. In this study, we sought to investigate the functional involvement of Class II Arfs in radial migration during the layer formation of the cerebral cortex using mouse embryos and pups. Our findings indicate that knock-down of Arf4, but not Arf5, resulted in the stalling of transfected neurons with disorientation of the Golgi in the upper intermediate zone (IZ) and reduction in the migration speed in both the IZ and cortical plate (CP). Migrating neurons with Arf4 knock-down exhibited cytoplasmic accumulation of N-cadherin, along with disturbed organelle morphology and distribution. Furthermore, supplementation of exogenous N-cadherin partially rescued the migration defect caused by Arf4 knock-down. In conclusion, our results suggest that Arf4 plays a crucial role in regulating radial migration via N-cadherin trafficking during cerebral cortical development.

Cytohesin-2 mediates group I metabotropic glutamate receptor-dependent mechanical allodynia through the activation of ADP ribosylation factor 6 in the spinal cord.

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, in the spinal cord are implicated in nociceptive transmission and plasticity through G protein-mediated second messenger cascades leading to the activation of various protein kinases such as extracellular signal-regulated kinase (ERK). In this study, we demonstrated that cytohesin-2, a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), is abundantly expressed in subsets of excitatory interneurons and projection neurons in the superficial dorsal horn. Cytohesin-2 is enriched in the perisynapse on the postsynaptic membrane of dorsal horn neurons and forms a protein complex with mGluR5 in the spinal cord. Central nervous system-specific cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia in inflammatory and neuropathic pain models. Pharmacological blockade of cytohesin catalytic activity with SecinH3 similarly reduced mechanical allodynia and inhibited the spinal activation of Arf6, but not Arf1, in both pain models. Furthermore, cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia and ERK1/2 activation following the pharmacological activation of spinal mGluR1/5 with 3,5-dihydroxylphenylglycine (DHPG). The present study suggests that cytothesin-2 is functionally associated with mGluR5 during the development of mechanical allodynia through the activation of Arf6 in spinal dorsal horn neurons.

Canagliflozin Suppresses Atrial Remodeling in a Canine Atrial Fibrillation Model.

Background Recent clinical trials have demonstrated the possible pleiotropic effects of SGLT2 (sodium-glucose cotransporter 2) inhibitors in clinical cardiovascular diseases. Atrial electrical and structural remodeling is important as an atrial fibrillation (AF) substrate. Methods and Results The present study assessed the effect of canagliflozin (CAN), an SGLT2 inhibitor, on atrial remodeling in a canine AF model. The study included 12 beagle dogs, with 10 receiving continuous rapid atrial pacing and 2 acting as the nonpacing group. The 10 dogs that received continuous rapid atrial pacing for 3 weeks were subdivided as follows: pacing control group (n=5) and pacing+CAN (3 mg/kg per day) group (n=5). The atrial effective refractory period, conduction velocity, and AF inducibility were evaluated weekly through atrial epicardial wires. After the protocol, atrial tissues were sampled for histological examination. The degree of reactive oxygen species expression was evaluated by dihydroethidium staining. The atrial effective refractory period reduction was smaller (P=0.06) and the degree of conduction velocity decrease was smaller in the pacing+CAN group compared with the pacing control group (P=0.009). The AF inducibility gradually increased in the pacing control group, but such an increase was suppressed in the pacing+CAN group (P=0.011). The pacing control group exhibited interstitial fibrosis and enhanced oxidative stress, which were suppressed in the pacing+CAN group. Conclusions CAN and possibly other SGLT2 inhibitors might be useful for preventing AF and suppressing the promotion of atrial remodeling as an AF substrate.

The Vps52 subunit of the GARP and EARP complexes is a novel Arf6-interacting protein that negatively regulates neurite outgrowth of hippocampal neurons.

ADP ribosylation factor 6 (Arf6) is a small GTP-binding protein implicated in neuronal morphogenesis through endosomal trafficking and actin remodeling. In this study, we identified Vps52, a core subunit of the Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes, as a novel Arf6-binding protein by yeast two-hybrid screening. Vps52 interacted specifically with GTP-bound Arf6 among the Arf family. Immunohistochemical analyses of hippocampal pyramidal cells revealed that fine punctate immunolabeling for Vps52 was distributed throughout neuronal compartments, most densely in the cell body and dendritic shafts, and was largely associated with trans-Golgi network and vesicular endomembranes. In cultured hippocampal neurons, knockdown of Vps52 increased total length of axons and dendrites; these phenotypes were completely restored by co-expression of shRNA-resistant full-length Vps52. However, co-expression of a Vps52 mutant lacking the ability to interact with Arf6 restored only the Vps52-knockdown phenotype of the dendritic length. The present findings suggest that Vps52 is a novel Arf6-interacting protein that regulates neurite outgrowth in hippocampal neurons.

BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3.

Brefeldin A-resistant ArfGEF 2 (BRAG2) [or Iqsec1 (IQ motif and Sec7 domain-containing protein 1)] is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes. BRAG2 regulates Arf6-dependent endocytosis of AMPA receptors (AMPARs) through the direct interaction during the hippocampal long-term depression. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Herein, using mouse brains of both sexes, we demonstrated that BRAG2a, an alternative isoform with a long C-terminal insert containing a proline-rich domain and type I PDZ-binding motif, was selectively localized to the excitatory postsynaptic density (PSD). Using yeast two-hybrid screening, we identified PSD-95 and endophilin 1/3 as BRAG2a-binding partners in the brain. The interaction with PSD-95 was required for synaptic targeting of BRAG2a. In cultured hippocampal neurons, stimulation of group I metabotropic glutamate receptors (mGluRs) increased the interaction of BRAG2a with endophilin 3 and concomitant Arf6 activation in a time-dependent manner. Knockdown of BRAG2 in cultured hippocampal neurons blocked the mGluR-dependent decrease in surface AMPAR levels, which was rescued by introducing wild-type BRAG2a, but not wild-type BRAG2b or BRAG2a mutants lacking the ability to activate Arf6 or to interact with endophilin 3 or PSD-95. Further postembedding immunoelectron microscopic analysis revealed the preorganized lateral distribution of BRAG2a, Arf6, and endophilin 3 for efficient endocytosis at the postsynaptic membrane. Together, the present findings unveiled a novel molecular mechanism by which BRAG2a links AMPARs to the clathrin-dependent endocytic pathway through its interaction with PSD-95 and endophilin 3.SIGNIFICANCE STATEMENT BRAG2/Iqsec1 is a GDP/GTP exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes, and regulates Arf6-dependent endocytosis of AMPARs through direct interaction during hippocampal long-term depression, one of the mechanisms of synaptic plasticity related to learning and memory. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Here, we identified isoform-specific mechanisms of BRAG2-mediated AMPAR internalization. We demonstrated that the interaction of BRAG2a isoform with PSD-95 and endophilin 3 was required for the mGluR-dependent decrease in surface AMPARs in hippocampal neurons. These results unveiled a novel molecular mechanism by which BRAG2 links AMPARs to the clathrin-mediated endocytic machinery at postsynaptic sites.

Mice lacking EFA6C/Psd2, a guanine nucleotide exchange factor for Arf6, exhibit lower Purkinje cell synaptic density but normal cerebellar motor functions.

ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates various neuronal events including formation of the axon, dendrites and dendritic spines, and synaptic plasticity through actin cytoskeleton remodeling and endosomal trafficking. EFA6C, also known as Psd2, is a guanine nucleotide exchange factor for Arf6 that is preferentially expressed in the cerebellar cortex of adult mice, particularly in Purkinje cells. However, the roles of EFA6C in cerebellar development and functions remain unknown. In this study, we generated global EFA6C knockout (KO) mice using the CRISPR/Cas9 system and investigated their cerebellar phenotypes by histological and behavioral analyses. Histological analyses revealed that EFA6C KO mice exhibited normal gross anatomy of the cerebellar cortex, in terms of the thickness and cellularity of each layer, morphology of Purkinje cells, and distribution patterns of parallel fibers, climbing fibers, and inhibitory synapses. Electron microscopic observation of the cerebellar molecular layer revealed that the density of asymmetric synapses of Purkinje cells was significantly lower in EFA6C KO mice compared with wild-type control mice. However, behavioral analyses using accelerating rotarod and horizontal optokinetic response tests failed to detect any differences in motor coordination, learning or adaptation between the control and EFA6C KO mice. These results suggest that EFA6C plays ancillary roles in cerebellar development and motor functions.

FIP4/Arfophilin-2 plays overlapping but distinct roles from FIP3/Arfophilin-1 in neuronal migration during cortical layer formation.

The class II Rab11 family-interacting proteins, FIP3 and FIP4, also termed Arfophilin-1 and Arfophilin-2, respectively, are endosomal proteins that function as dual effector proteins for Rab11 and ADP ribosylation factor (Arf) small GTPases. In the present study, we examined the expression and role of FIP4 in neuronal migration during cerebral layer formation. FIP4 mRNA was first weakly detected in post-mitotic migrating neurons in the upper intermediate zone, and expression was markedly increased in the cortical layer. Exogenously expressed FIP4 protein was localized to subpopulations of EEA1- and syntaxin 12-positive endosomes in migrating neurons, and was partially colocalized with FIP3. Knockdown of FIP4 by in utero electroporation significantly stalled transfected neurons in the lower cortical layer and decreased the speed of neuronal migration in the upper intermediate zone and in the cortical plate compared with control small hairpin RNA (shRNA)-transfected neurons. Furthermore, co-transfection of shRNA-resistant wild-type FIP4, but not wild type FIP3 or FIP4 mutants lacking the binding region for Rab11 or Arf, significantly improved the disturbed cortical layer formation caused by FIP4 knockdown. Collectively, our findings suggest that FIP4 and FIP3 play overlapping but distinct roles in neuronal migration downstream of Arf and Rab11 during cortical layer formation.

Pallidin is a novel interacting protein for cytohesin-2 and regulates the early endosomal pathway and dendritic formation in neurons.

Cytohesin-2 is a member of the guanine nucleotide exchange factors for ADP ribosylation factor 1 (Arf1) and Arf6, which are small GTPases that regulate membrane traffic and actin dynamics. In this study, we first demonstrated that cytohesin-2 localized to the plasma membrane and vesicles in various subcellular compartment in hippocampal neurons by immunoelectron microscopy. Next, to understand the molecular network of cytohesin-2 in neurons, we conducted yeast two-hybrid screening of brain cDNA libraries using cytohesin-2 as bait and isolated pallidin, a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) involved in endosomal trafficking. Pallidin interacted specifically with cytohesin-2 among cytohesin family members. Glutathione S-transferase pull-down and immunoprecipitation assays further confirmed the formation of a protein complex between cytohesin-2 and pallidin. Immunofluorescence demonstrated that cytohesin-2 and pallidin partially colocalized in various subsets of endosomes immunopositive for EEA1, syntaxin 12, and LAMP2 in hippocampal neurons. Knockdown of pallidin or cytohesin-2 reduced cytoplasmic EEA1-positive early endosomes. Furthermore, knockdown of pallidin increased the total dendritic length of cultured hippocampal neurons, which was rescued by co-expression of wild-type pallidin but not a mutant lacking the ability to interact with cytohesin-2. In contrast, knockdown of cytohesin-2 had the opposite effect on total dendritic length. The present results suggested that the interaction between pallidin and cytohesin-2 may participate in various neuronal functions such as endosomal trafficking and dendritic formation in hippocampal neurons. Cover Image for this issue: doi: 10.1111/jnc.14197.

BRAG2a, a Guanine Nucleotide Exchange Factor for Arf6, Is a Component of the Dystrophin-Associated Glycoprotein Complex at the Photoreceptor Terminal.

Purpose: Mutations in genes encoding the dystrophin-associated glycoprotein complex (DGC) can cause muscular dystrophy and disturb synaptic transmission in the photoreceptor ribbon synapse. However, the molecular composition and specific functions of the photoreceptor DGC remain unknown. Brefeldin A-resistant Arf-GEF 2 (BRAG2), also known as IQSEC1, is a guanine nucleotide exchange factor for ADP-ribosylation factor 6 (Arf6), a critical GTPase that regulates endosomal trafficking and actin cytoskeleton remodeling. In the present study, we characterized the expression of BRAG2a, an alternative splicing isoform of BRAG2, in the adult mouse photoreceptor. Methods: Immunofluorescence and immunoelectron microscopic analyses of adult mouse retinas were performed using a novel anti-BRAG2a antibody. Pull-down, immunoprecipitation, and in situ proximity ligation assays were performed to examine the interaction between BRAG2a and the DGC in vivo. Results: Immunofluorescence demonstrated punctate colocalization of BRAG2a with ß-dystroglycan in the outer plexiform layer. Immunoelectron microscopy revealed the localization of BRAG2a at the plasma membrane of lateral walls and processes of photoreceptor terminals within the synaptic cavity. Pull-down and immunoprecipitation assays using retinal lysates demonstrated the protein complex formation between BRAG2a with the DGC. In situ proximity ligation assays further detected a close spatial relationship between BRAG2a and ß-dystroglycan in the outer plexiform layer. Conclusions: The present study provided evidence that BRAG2a is a novel component of the photoreceptor DGC, suggesting functional involvement of the BRAG2a-Arf6 pathway downstream of the DGC.

ADP Ribosylation Factor 6 Regulates Neuronal Migration in the Developing Cerebral Cortex through FIP3/Arfophilin-1-dependent Endosomal Trafficking of N-cadherin.

During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell-cell and cell-extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking.

Interaction of serologically defined colon cancer antigen-3 with Arf6 and its predominant expression in the mouse testis.

ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates endosomal trafficking and actin cytoskeleton remodeling. Here, we identified the serologically defined colon antigen-3 (SDCCAG3) as an Arf6-interacting protein by yeast two-hybrid screening with a constitutively active Arf6 mutant. SDCCAG3 interacts specifically with Arf6 among the Arf family members through its 101  C-terminal amino acids. SDCCAG3 is expressed most intensely in the testis at the mRNA and protein levels. In the testis, SDCCAG3 is expressed in spermatocytes and spermatids. We also show that full-length SDCCAG3, but not a mutant lacking the ability to interact with Arf6, is recruited to the midbody during cytokinesis when expressed exogenously in HeLa cells. These findings suggest that SDCCAG3 might function in endosomal trafficking downstream of Arf6.

Distinct subcellular localization of alternative splicing variants of EFA6D, a guanine nucleotide exchange factor for Arf6, in the mouse brain.

EFA6D (guanine nucleotide exchange factor for ADP-ribosylation factor 6 [Arf6]D) is also known as EFA6R, Psd3, and HCA67. It is the fourth member of the EFA6 family with guanine nucleotide exchange activity for Arf6, a small guanosine triphosphatase (GTPase) that regulates endosomal trafficking and actin cytoskeleton remodeling. We propose a classification and nomenclature of 10 EFA6D variants deposited in the GenBank database as EFA6D1a, 1b, 1c, 1d, 1s, 2a, 2b, 2c, 2d, and 2s based on the combination of N-terminal and C-terminal insertions. Polymerase chain reaction analysis showed the expression of all EFA6D variants except for variants a and d in the adult mouse brain. Immunoblotting analysis with novel variant-specific antibodies showed the endogenous expression of EFA6D1b, EFA6D1c, and EFA6D1s at the protein level, with the highest expression being EFA6D1s, in the brain. Immunoblotting analysis of forebrain subcellular fractions showed the distinct subcellular distribution of EFA6D1b/c and EFA6D1s. The immunohistochemical analysis revealed distinct but overlapping immunoreactive patterns between EFA6D1b/c and EFA6D1s in the mouse brain. In immunoelectron microscopic analyses of the hippocampal CA3 region, EFA6D1b/c was present predominantly in the mossy fiber axons of dentate granule cells, whereas EFA6D1s was present abundantly in the cell bodies, dendritic shafts, and spines of hippocampal pyramidal cells. These results provide the first anatomical evidence suggesting the functional diversity of EFA6D variants, particularly EFA6D1b/c and EFA6D1s, in neurons. J. Comp. Neurol. 524:2531-2552, 2016. © 2016 Wiley Periodicals, Inc.

Polyhydramnios in Lrp4 knockout mice with bilateral kidney agenesis: Defects in the pathways of amniotic fluid clearance.

Amniotic fluid volume during mid-to-late gestation depends mainly on the urine excretion from the foetal kidneys and partly on the fluid secretion from the foetal lungs during foetal breathing-like movements. Urine is necessary for foetal breathing-like movements, which is critical for foetal lung development. Bilateral renal agenesis and/or obstruction of the urinary tract lead to oligohydramnios, which causes infant death within a short period after birth due to pulmonary hypoplasia. Lrp4, which functions as an agrin receptor, is essential for the formation of neuromuscular junctions. Herein, we report novel phenotypes of Lrp4 knockout (Lrp4(-/-)) mice. Most Lrp4(-/-) foetuses showed unilateral or bilateral kidney agenesis, and Lrp4 knockout resulted in polyhydramnios. The loss of Lrp4 compromised foetal swallowing and breathing-like movements and downregulated the expression of aquaporin-9 in the foetal membrane and aquaporin-1 in the placenta, which possibly affected the amniotic fluid clearance. These results suggest that amniotic fluid removal was compromised in Lrp4(-/-) foetuses, resulting in polyhydramnios despite the impairment of urine production. Our findings indicate that amniotic fluid removal plays an essential role in regulating the amniotic fluid volume.

Preferential localization of type I phosphatidylinositol 4-phosphate 5-kinase γ at the periactive zone of mouse photoreceptor ribbon synapses.

Type I phosphatidylinositol 4-phosphate 5 kinase γ (PIP5KIγ) constitutes a major pathway for the generation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) that regulates a variety of neuronal functions at both presynaptic and postsynaptic compartments. In this study, we examined the expression and localization of PIP5KIγ in the adult mouse retina. RT-PCR analysis revealed that PIP5KIγ_v2 was predominantly expressed in the retina while PIP5KIγ_v3 was also expressed faintly. Immunostaining of the adult mouse retina revealed intense PIP5KIγ-immunoreactivity in the inner and outer plexiform layers in a punctate manner. In the photoreceptor ribbon synapse, PIP5KIγ was highly concentrated at the periactive zone. These findings suggest that PIP5KIγ, especially PIP5KIγ_i2, is localized at the periactive zone, a functionally suitable compartment for the endocytosis of synaptic vesicles in photoreceptor ribbon synapses.

Endosomal localization of FIP3/Arfophilin-1 and its involvement in dendritic formation of mouse hippocampal neurons.

Endosomal trafficking mediated by Rab11 and Arf6 small GTPases is essential for various neuronal functions. Family of Rab11-interacting protein 3 (FIP3)/Arfophilin-1, also termed Eferin, is a dual effector for Rab11 and Arf6 and implicated in endosomal trafficking during cytokinesis. To understand the neuronal functions of FIP3, we first showed the widespread neuronal expression of FIP3 mRNA in adult mouse brain by in situ hybridization. Immunohistochemical analysis showed the association of FIP3 with a subpopulation of endosomes labeled with EEA1 and syntaxin 12 in hippocampal neurons. Immunoblot analysis showed the progressive increase of FIP3 with a peak around postnatal day 15 during hippocampal development. Furthermore, knockdown of endogenous FIP3 decreased the total dendritic length of cultured hippocampal neurons with a concomitant increase in the number of short (<40µm) primary dendrites. Together, FIP3 is suggested to regulate dendritic formation possibly through Rab11- and Arf6-mediated endosomal trafficking.

EFA6A, a guanine nucleotide exchange factor for Arf6, interacts with sorting nexin-1 and regulates neurite outgrowth.

The membrane trafficking and actin cytoskeleton remodeling mediated by ADP ribosylation factor 6 (Arf6) are functionally linked to various neuronal processes including neurite formation and maintenance, neurotransmitter release, and receptor internalization. EFA6A is an Arf6-specific guanine nucleotide exchange factor that is abundantly expressed in the brain. In this study, we identified sorting nexin-1 (SNX1), a retromer component that is implicated in endosomal sorting and trafficking, as a novel interacting partner for EFA6A by yeast two-hybrid screening. The interaction was mediated by the C-terminal region of EFA6A and a BAR domain of SNX1, and further confirmed by pull-down assay and immunoprecipitation from mouse brain lysates. In situ hybridization analysis demonstrated the widespread expression of SNX1 in the mouse brain, which overlapped with the expression of EFA6A in the forebrain. Immunofluorescent analysis revealed the partial colocalization of EFA6A and SNX1 in the dendritic fields of the hippocampus. Immunoelectron microscopic analysis revealed the overlapping subcellular localization of EFA6A and SNX1 at the post-synaptic density and endosomes in dendritic spines. In Neuro-2a neuroblastoma cells, expression of either EFA6A or SNX1 induced neurite outgrowth, which was further enhanced by co-expression of EFA6A and SNX1. The present findings suggest a novel mechanism by which EFA6A regulates Arf6-mediated neurite formation through the interaction with SNX1.

Type I phosphatidylinositol 4-phosphate 5-kinase γ is required for neuronal migration in the mouse developing cerebral cortex.

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI)γ is one of the phosphoinositide kinases that produce phosphatidylinositol 4,5-bisphosphate, which is a critical regulator of cell adhesion formation, actin dynamics and membrane trafficking. Here, we examined the functional roles of PIP5KIγ in radial neuronal migration during cortical formation. Reverse transcription-polymerase chain reaction analysis revealed that PIP5KIγ_v2/v6 and PIP5KIγ_v3 were expressed throughout cortical development with distinct expression patterns. In situ hybridisation analysis showed that PIP5KIγ mRNA was expressed throughout the cortical layers. Immunohistochemical analysis revealed that PIP5KIγ was localised in a punctate manner in the radial glia and migrating neuroblasts. Knockdown of PIP5KIγ using in utero electroporation disturbed the radial neuronal migration and recruitment of talin and focal adhesion kinase to puncta beneath the plasma membrane. The same inhibitory effect on neuronal migration was observed by overexpression of a catalytically inactive mutant of PIP5KIγ_v2 but not PIP5KIγ_v1 or PIP5KIγ_v3. These findings suggest an essential role of PIP5KIγ, particularly PIP5KIγ_i2, in neuronal migration, possibly through recruitment of adhesion components to the plasma membrane.

Distinct synaptic localization patterns of brefeldin A-resistant guanine nucleotide exchange factors BRAG2 and BRAG3 in the mouse retina.

The BRAG/IQSEC is a family of guanine nucleotide exchange factors for ADP ribosylation factors, small GTPases that regulate membrane trafficking and actin cytoskeleton, and comprises three structurally related members (BRAG1-3) generated from different genes. In the mouse retina, BRAG1 (also known as IQSEC2) was previously shown to localize at synaptic ribbons of photoreceptor terminals and to form a protein complex with RIBEYE. In this study, we examined the immunohistochemical localization of BRAG2 (IQSEC1) and BRAG3 (IQSEC3) in the adult mouse retina at the light and electron microscopic levels. In the outer plexiform layer, BRAG2 showed a punctate distribution in intimate association with dystrophin and ß-dystroglycan. Immunoelectron microscopic analysis revealed that BRAG2 localized at specific subcompartments of photoreceptor terminals in both rod spherules and cone pedicles. In the inner plexiform layer, immunolabeling for both BRAG2 and BRAG3 had a punctate appearance, suggestive of synaptic labeling. Double immunostaining demonstrated that BRAG2 colocalized preferentially with PSD-95 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptors (AMPARs). By contrast, BRAG3 colocalized with gephyrin and a subpopulation of inhibitory synapses expressing glycine receptors or γ-aminobutyric acid type A receptors (GABA(A) Rs). Immunoelectron microscopic analysis revealed that BRAG2 localized to postsynaptic processes at bipolar dyads, while BRAG3 localized to postsynaptic components at conventional synapses. These findings suggest that BRAG/IQSEC family members have key roles in the function and organization of distinct excitatory and inhibitory synapses in the retina.

Pilt is a coiled-coil domain-containing protein that localizes at the trans-Golgi complex and regulates its structure.

Protein incorporated later into tight junctions (Pilt), also termed tight junction-associated protein 1 or tight junction protein 4, is a coiled-coil domain-containing protein that was originally identified as a human discs large-interacting protein. In this study, we identified Pilt as an Arf6-binding protein by yeast two-hybrid screening. By immunocytochemical analysis, Pilt was shown to be predominantly localized at the trans-Golgi complex and to exhibit diffuse cytoplasmic distribution in association with endosomes and plasma membrane in NIH3T3 cells. Silencing of endogenous Pilt disrupted the Golgi structure. The present findings suggest the functional involvement of Pilt in the maintenance of the Golgi structure.

SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses.

SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both ß-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.