Proliferation in the developing intestine is regulated by the endosomal protein Endotubin.

During postnatal intestinal development, the intestinal epithelium is highly proliferative, and this proliferation is regulated by signaling in the intervillous and crypt regions. This signaling is primarily mediated by Wnt, and requires membrane trafficking. However, the mechanisms by which membrane trafficking regulates signaling during this developmental phase are largely unknown. Endotubin (EDTB, MAMDC4) is an endosomal protein that is highly expressed in the apical endocytic complex (AEC) of villus enterocytes during fetal and postnatal development, and knockout of EDTB results in defective formation of the AEC and giant lysosome. Further, knockout of EDTB in cell lines results in decreased proliferation. However, the role of EDTB in proliferation during the development of the intestine is unknown. Using Villin-CreERT2 in EDTBfl/fl mice, we deleted EDTB in the intestine in the early postnatal period, or in enteroids in vitro after isolation of intervillous cells. Loss of EDTB results in decreased proliferation in the developing intestinal epithelium and decreased ability to form enteroids. EDTB is present in cells that contain the stem cell markers LGR5 and OLFM4, indicating that it is expressed in the proliferative compartment. Further, using immunoblot analysis and TCF/LEF-GFP mice as a reporter of Wnt activity, we find that knockout of EDTB results in decreased Wnt signaling. Our results show that EDTB is essential for normal proliferation during the early stages of intestinal development and suggest that this effect is through modulation of Wnt signaling.

Cell type-specific biogenesis of novel vesicles containing viral products in human cytomegalovirus infection.

Human cytomegalovirus (HCMV), while highly restricted for the human species, infects an diverse array of cell types in the host. Patterns of infection are dictated by the cell type infected, but cell type-specific factors and how they impact tropism for specific cell types is poorly understood. Previous studies in primary endothelial cells showed that HCMV infection induces large multivesicular-like bodies (MVBs) that incorporate viral products, including dense bodies (DBs) and virions. Here we define the nature of these large vesicles using a recombinant virus where UL32, encoding the pp150 tegument protein, is fused in frame with green fluorescent protein (GFP, TB40/E-UL32-GFP). In fibroblasts, UL32-GFP-positive vesicles were marked with classical markers of MVBs, including CD63 and lysobisphosphatidic acid (LBPA), both classical MVB markers, as well as the clathrin and LAMP1. Unexpectedly, UL32-GFP-positive vesicles in primary human microvascular endothelial cells (HMVECs) were not labeled by CD63, and LBPA was completely lost from infected cells. We defined these UL32-positive vesicles in endothelial cells using markers for the cis-Golgi (GM130), lysosome (LAMP1), and autophagy (LC3B). These findings suggest that UL32-GFP containing MVBs in fibroblasts are derived from the canonical endocytic pathway and takeover classical exosomal release pathway. However, UL32-GFP containing MVBs in HMVECs are derived from the early biosynthetic pathway and exploit a less characterized early Golgi-LAMP1-associated non- canonical secretory autophagy pathway. These results reveal striking cell-type specific membrane trafficking differences in host pathways that are exploited by HCMV, which may reflect distinct pathways for virus egress.ImportanceHuman cytomegalovirus (HCMV) is a herpesvirus that, like all herpesvirus, that establishes a life-long infection. HCMV remains a significant cause of morbidity and mortality in the immunocompromised and HCMV seropositivity is associated with age-related pathology. HCMV infects many cells in the human host and the biology underlying the different patterns of infection in different cell types is poorly understood. Endothelial cells are important target of infection that contribute to hematogenous spread of the virus to tissues. Here we define striking differences in the biogenesis of large vesicles that incorporate virions in fibroblasts and endothelial cells. In fibroblasts, HCMV is incorporated into canonical MVBs derived from an endocytic pathway, whereas HCMV matures through vesicles derived from the biosynthetic pathway in endothelial cells. This work defines basic biological differences between these cell types that may impact how progeny virus is trafficked out of infected cells.

Rab22a regulates the establishment of epithelial polarity.

Membrane trafficking establishes and maintains epithelial polarity. Rab22a has a polarized distribution in activated T-cells, but its role in epithelial polarity has not been investigated. We showed previously that Rab14 acts upstream of Arf6 to establish the apical membrane initiation site (AMIS), but its interaction with Rab22a is unknown. Here we show that Rab14 and Rab22a colocalize in endosomes of both unpolarized and polarized MDCK cells and Rab22a localizes to the cell:cell interface of polarizing cell pairs. Knockdown of Rab22a results in a multi-lumen phenotype in three-dimensional culture. Further, overexpression of Rab22a in Rab14 knockdown cells rescues the multi-lumen phenotype observed with Rab14 knockdown, suggesting that Rab22a is downstream of Rab14. Because of the relationship between Rab14 and Arf6, we investigated the effect of Rab22a knockdown on Arf6. We find that Rab22a knockdown results in decreased active Arf6 and that Rab22a co-immunoprecipitates with the Arf6 GEF EFA6. In addition, EFA6 is retained in intracellular puncta in Rab22a KD cells. These results suggest that Rab22a acts downstream of Rab14 to traffic EFA6 to the AMIS to regulate Arf6 in the establishment of polarity.

Intestinal Epithelial Expression of MHCII Determines Severity of Chemical, T-Cell-Induced, and Infectious Colitis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis. METHODS: We disrupted the histocompatibility 2, class II antigen A, beta 1 gene (H2-Ab1) in IECs of C57BL/6 mice (I-AbΔIEC) or Rag1-/- mice (Rag1-/-I-AbΔIEC); we used I-AbWT mice as controls. Colitis was induced by administration of DSS, transfer of CD4+CD45RBhi T cells, or infection with Citrobacter rodentium. Colon tissues were collected and analyzed by histology, immunofluorescence, xMAP, and reverse-transcription polymerase chain reaction and organoids were generated. Microbiota (total and immunoglobulin [Ig]A-coated) in intestinal samples were analyzed by16S amplicon profiling. IgA+CD138+ plasma cells from Peyer's patches and lamina propria were analyzed by flow cytometry and IgA repertoire was determined by next-generation sequencing. RESULTS: Mice with IEC-specific loss of MHCII (I-AbΔIEC mice) developed less severe DSS- or T-cell transfer-induced colitis than control mice. Intestinal tissues from I-AbΔIEC mice had a lower proportion of IgA-coated bacteria compared with control mice, and a reduced luminal concentration of secretory IgA (SIgA) following infection with C rodentium. There was no significant difference in the mucosal IgA repertoire of I-AbΔIEC vs control mice, but opsonization of cultured C rodentium by SIgA isolated from I-AbΔIEC mice was 50% lower than that of SIgA from mAbWT mice. Fifty percent of I-AbΔIEC mice died after infection with C rodentium, compared with none of the control mice. We observed a transient but significant expansion of the pathogen in the feces of I-AbΔIEC mice compared with I-AbWT mice. CONCLUSIONS: In mice with DSS or T-cell-induced colitis, loss of MHCII from IECs reduces but does not eliminate mucosal inflammation. However, in mice with C rodentium-induced colitis, loss of MHCII reduces bacterial clearance by decreasing binding of IgA to commensal and pathogenic bacteria.

Loss of myosin Vb promotes apical bulk endocytosis in neonatal enterocytes.

In patients with inactivating mutations in myosin Vb (Myo5B), enterocytes show large inclusions lined by microvilli. The origin of inclusions in small-intestinal enterocytes in microvillus inclusion disease is currently unclear. We postulated that inclusions in Myo5b KO mouse enterocytes form through invagination of the apical brush border membrane. 70-kD FITC-dextran added apically to Myo5b KO intestinal explants accumulated in intracellular inclusions. Live imaging of Myo5b KO-derived enteroids confirmed the formation of inclusions from the apical membrane. Treatment of intestinal explants and enteroids with Dyngo resulted in accumulation of inclusions at the apical membrane. Inclusions in Myo5b KO enterocytes contained VAMP4 and Pacsin 2 (Syndapin 2). Myo5b;Pacsin 2 double-KO mice showed a significant decrease in inclusion formation. Our results suggest that apical bulk endocytosis in Myo5b KO enterocytes resembles activity-dependent bulk endocytosis, the primary mechanism for synaptic vesicle uptake during intense neuronal stimulation. Thus, apical bulk endocytosis mediates the formation of inclusions in neonatal Myo5b KO enterocytes.

Rapid Downregulation of DAB2 by Toll-Like Receptor Activation Contributes to a Pro-Inflammatory Switch in Activated Dendritic Cells.

Dendritic cells (DCs) are pivotal in regulating tolerogenic as well as immunogenic responses against microorganisms by directing both the innate and adaptive immune response. In health, phenotypically different DC subsets found in the gut mucosa are maintained in their tolerogenic state but switch to a pro-inflammatory phenotype during infection or chronic autoinflammatory conditions such as inflammatory bowel disease (IBD). The mechanisms that promote the switch among the mucosal DCs from a tolerogenic to an immunogenic, pro-inflammatory phenotype are incompletely understood. We hypothesized that disabled homolog 2 (DAB2), recently described as a negative regulator of DC immunogenicity during their development, is regulated during intestinal inflammation and modulates mucosal DC function. We show that DAB2 is highly expressed in colonic CD11b+CD103- DCs, a subset known for its capacity to induce inflammatory Th1/Th17 responses in the colon, and is downregulated predominantly in this DC subset during adoptive T cell transfer colitis. Administration of Dab2-deficient DCs (DC2.4 Dab2-/- cells) modulated the course of DSS colitis in wild-type mice, enhanced mucosal expression of Tnfa, Il6, and Il17a, and promoted neutrophil recruitment. In bone-marrow derived dendritic cells (BMDC), DAB2 expression correlated with CD11b levels and DAB2 was rapidly and profoundly inhibited by TLR ligands in a TRIF- and MyD88-dependent manner. The negative modulation of DAB2 was biphasic, initiated with a quick drop in DAB2 protein, followed by a sustained reduction in Dab2 mRNA. DAB2 downregulation promoted a more functional and activated DC phenotype, reduced phagocytosis, and increased CD40 expression after TLR activation. Furthermore, Dab2 knockout in DCs inhibited autophagy and promoted apoptotic cell death. Collectively, our results highlight the immunoregulatory role for DAB2 in the intestinal dendritic cells and suggest that DAB2 downregulation after microbial exposure promotes their switch to an inflammatory phenotype.

The Endosomal Protein Endotubin Is Required for Enterocyte Differentiation.

BACKGROUND & AIMS: During late embryonic development and through weaning, enterocytes of the ileum are highly endocytic. Defects in endocytosis and trafficking are implicated in neonatal disease, however, the mechanisms regulating trafficking during the developmental period are incompletely understood. The apical endosomal protein endotubin (EDTB) is highly expressed in the late embryonic and neonatal ileum. In epithelial cells in vitro, EDTB regulates both trafficking of tight junction proteins and proliferation through modulation of YAP activity. However, EDTB function during the endocytic stage of development of the intestine is unknown. METHODS: By using Villin-CreERT2, we induced knockout of EDTB during late gestation and analyzed the impact on endocytic compartments and enterocyte structure in neonates using immunofluorescence, immunocytochemistry, and transmission electron microscopy. RESULTS: Deletion of the apical endosomal protein EDTB in the small intestine during development impairs enterocyte morphogenesis, including loss of the apical endocytic complex, defective formation of the lysosomal compartment, and some cells had large microvillus-rich inclusions similar to those observed in microvillus inclusion disease. There also was a decrease in apical endocytosis and mislocalization of proteins involved in apical trafficking. CONCLUSIONS: Our results show that EDTB-mediated trafficking within the epithelial cells of the developing ileum is important for maintenance of endocytic compartments and enterocyte integrity during early stages of gut development.

Rabs set the stage for polarity.

Cell polarity refers to the asymmetric localization of cellular components that allows cells to carry out their specialized functions, be they epithelial barrier function, transmission of action potentials in nerve cells, or modulation of the immune response. The establishment and maintenance of cell polarity requires the directed trafficking of membrane proteins and lipids - essential processes that are mediated by Rab GTPases. Interestingly, several of the Rabs that impact polarity are present in the earliest eukaryotes, and the Rab polarity repertoire has expanded as cells have become more complex. There is a substantial conservation of Rab function across diverse cell types. Rabs act through an assortment of effector proteins that include scaffolding proteins, cytoskeletal motors, and other small GTPases. In this review we highlight the similarities and differences in Rab function for the instruction of polarity in diverse cell types.

Arf6 and Rab22 mediate T cell conjugate formation by regulating clathrin-independent endosomal membrane trafficking.

Endosomal trafficking can influence the composition of the plasma membrane and the ability of cells to polarize their membranes. Here, we examined whether trafficking through clathrin-independent endocytosis (CIE) affects the ability of T cells to form a cell-cell conjugate with antigen-presenting cells (APCs). We show that CIE occurs in both the Jurkat T cell line and primary human T cells. In Jurkat cells, the activities of two guanine nucleotide binding proteins, Arf6 and Rab22 (also known as Rab22a), influence CIE and conjugate formation. Expression of the constitutively active form of Arf6, Arf6Q67L, inhibits CIE and conjugate formation, and results in the accumulation of vacuoles containing lymphocyte function-associated antigen 1 (LFA-1) and CD4, molecules important for T cell interaction with the APC. Moreover, expression of the GTP-binding defective mutant of Rab22, Rab22S19N, inhibits CIE and conjugate formation, suggesting that Rab22 function is required for these activities. Furthermore, Jurkat cells expressing Rab22S19N were impaired in spreading onto coverslips coated with T cell receptor-activating antibodies. These observations support a role for CIE, Arf6 and Rab22 in conjugate formation between T cells and APCs.

Rab14 specifies the apical membrane through Arf6-mediated regulation of lipid domains and Cdc42.

The generation of cell polarity is essential for the development of multi-cellular organisms as well as for the function of epithelial organs in the mature animal. Small GTPases regulate the establishment and maintenance of polarity through effects on cytoskeleton, membrane trafficking, and signaling. Using short-term 3-dimensional culture of MDCK cells, we find that the small GTPase Rab14 is required for apical membrane specification. Rab14 knockdown results in disruption of polarized lipid domains and failure of the Par/aPKC/Cdc42 polarity complex to localize to the apical membrane. These effects are mediated through tight control of lipid localization, as overexpression of the phosphatidylinositol 4-phosphate 5-kinase α [PtdIns(4)P5K] activator Arf6 or PtdIns(4)P5K alone, or treatment with the phosphatidylinositol 3-kinase (PtdInsI3K) inhibitor wortmannin, rescued the multiple-apical domain phenotype observed after Rab14 knockdown. Rab14 also co-immunoprecipitates and colocalizes with the small GTPase Cdc42, and Rab14 knockdown results in increased Cdc42 activity. Furthermore, Rab14 regulates trafficking of vesicles to the apical domain, mitotic spindle orientation, and midbody position, consistent with Rab14's reported localization to the midbody as well as its effects upon Cdc42. These results position Rab14 at the top of a molecular cascade that regulates the establishment of cell polarity.

Endosomal regulation of contact inhibition through the AMOT:YAP pathway.

Contact-mediated inhibition of cell proliferation is an essential part of organ growth control; the transcription coactivator Yes-associated protein (YAP) plays a pivotal role in this process. In addition to phosphorylation-dependent regulation of YAP, the integral membrane protein angiomotin (AMOT) and AMOT family members control YAP through direct binding. Here we report that regulation of YAP activity occurs at the endosomal membrane through a dynamic interaction of AMOT with an endosomal integral membrane protein, endotubin (EDTB). EDTB interacts with both AMOT and occludin and preferentially associates with occludin in confluent cells but with AMOT family members in subconfluent cells. EDTB competes with YAP for binding to AMOT proteins in subconfluent cells. Overexpression of the cytoplasmic domain or full-length EDTB induces translocation of YAP to the nucleus, an overgrowth phenotype, and growth in soft agar. This increase in proliferation is dependent upon YAP activity and is complemented by overexpression of p130-AMOT. Furthermore, overexpression of EDTB inhibits the AMOT:YAP interaction. EDTB and AMOT have a greater association in subconfluent cells compared with confluent cells, and this association is regulated at the endosomal membrane. These data provide a link between the trafficking of tight junction proteins through endosomes and contact-inhibition-regulated cell growth.

PKCι interacts with Rab14 and modulates epithelial barrier function through regulation of claudin-2 levels.

PKCι is essential for the establishment of epithelial polarity and the normal assembly of tight junctions. We find that PKCι knockdown does not compromise the steady-state distribution of most tight junction proteins but results in increased transepithelial resistance (TER) and decreased paracellular permeability. Analysis of the levels of tight junction components demonstrates that claudin-2 protein levels are decreased. However, other tight junction proteins, such as claudin-1, ZO-1, and occludin, are unchanged. Incubation with an aPKC pseudosubstrate recapitulates the phenotype of PKCι knockdown, including increased TER and decreased levels of claudin-2. In addition, overexpression of PKCι results in increased claudin-2 levels. ELISA and coimmunoprecipitation show that the TGN/endosomal small GTPase Rab14 and PKCι interact directly. Immunolabeling shows that PKCι and Rab14 colocalize in both intracellular puncta and at the plasma membrane and that Rab14 expression is required for normal PKCι distribution in cysts in 3D culture. We showed previously that knockdown of Rab14 results in increased TER and decreased claudin-2. Our results suggest that Rab14 and aPKC interact to regulate trafficking of claudin-2 out of the lysosome-directed pathway.

Scaffolding protein GOPC regulates tight junction structure.

GOPC (FIG/PIST/CAL) is a PDZ-domain scaffolding protein that regulates the trafficking of a wide array of proteins, including small GTPases, receptors and cell surface molecules such as cadherin 23 and cystic fibrosis transmembrane regulator. In Madin-Darby canine kidney (MDCK) cells, we find that GOPC localizes to the trans-Golgi network (TGN) but not to the cis- or trans-Golgi cisternae. Colocalization occurs with the early endosome Rab GTPase Rab5 and a TGN/endosome marker Rab14 but not with Rab11, a marker of recycling endosomes. No localization of GOPC was detected to the lateral membranes or tight junctions. Knockdown of GOPC in MDCK cells results in decreased transepithelial resistance and increased paracellular flux. This might be attributable to the compromised trafficking of tight junction components from the TGN, as GOPC-knockdown cells have decreased lateral labeling of the tight junction protein claudin-1 and decreased protein levels of claudin-2. GOPC might mediate the trafficking of newly synthesized tight junction proteins from the TGN to the cell surface or the recycling of these proteins from specialized endosomal compartments.

Rab14 regulation of claudin-2 trafficking modulates epithelial permeability and lumen morphogenesis.

Regulation of epithelial barrier function requires targeted insertion of tight junction proteins that have distinct selectively permeable characteristics. The insertion of newly synthesized proteins and recycling of internalized tight junction components control both polarity and junction function. Here we show that the small GTPase Rab14 regulates tight junction structure. In Madin-Darby canine kidney (MDCK) II cells, Rab14 colocalizes with junctional proteins, and knockdown of Rab14 results in increased transepithelial resistance. In cells without Rab14, there are small changes in the trafficking of claudin-1 and occludin. In addition, there is substantial depletion of the leaky claudin, claudin-2, but not other tight junction components. The loss of claudin-2 is complemented by inhibition of lysosomal function, suggesting that Rab14 sorts claudin-2 out of the lysosome-directed pathway. MDCK I cells lack claudin-2 endogenously, and knockdown of Rab14 in these cells does not result in a change in transepithelial resistance, suggesting that the effect is specific to claudin-2 trafficking. Furthermore, leaky claudins have been shown to be required for epithelial morphogenesis, and knockdown of Rab14 results in failure to form normal single-lumen cysts in three-dimensional culture. These results implicate Rab14 in specialized trafficking of claudin-2 from the recycling endosome.

Competing molecular interactions of aPKC isoforms regulate neuronal polarity.

Atypical protein kinase C (aPKC) isoforms ζ and λ interact with polarity complex protein Par3 and are evolutionarily conserved regulators of cell polarity. Prkcz encodes aPKC-ζ and PKM-ζ, a truncated, neuron-specific alternative transcript, and Prkcl encodes aPKC-λ. Here we show that, in embryonic hippocampal neurons, two aPKC isoforms, aPKC-λ and PKM-ζ, are expressed. The localization of these isoforms is spatially distinct in a polarized neuron. aPKC-λ, as well as Par3, localizes at the presumptive axon, whereas PKM-ζ and Par3 are distributed at non-axon-forming neurites. PKM-ζ competes with aPKC-λ for binding to Par3 and disrupts the aPKC-λ-Par3 complex. Silencing of PKM-ζ or overexpression of aPKC-λ in hippocampal neurons alters neuronal polarity, resulting in neurons with supernumerary axons. In contrast, the overexpression of PKM-ζ prevents axon specification. Our studies suggest a molecular model wherein mutually antagonistic intermolecular competition between aPKC isoforms directs the establishment of neuronal polarity.

Regulation of tight junction assembly and epithelial polarity by a resident protein of apical endosomes.

The establishment of tight junctions and cell polarity is an essential process in all epithelia. Endotubin is an integral membrane protein found in apical endosomes of developing epithelia when tight junctions and epithelial polarity first arise. We found that the disruption of endotubin function in cells in culture by siRNA or overexpression of the C-terminal cytoplasmic domain of endotubin causes defects in organization and function of tight junctions. We observe defects in localization of tight junction proteins, reduced transepithelial resistance, increased lanthanum penetration between cells and reduced ability of cells to form cysts in three-dimensional culture. In addition, in cells overexpressing the C-terminal domain of endotubin, we observe a delay in re-establishing the normal distribution of endosomes after calcium switch. These results suggest that endotubin regulates trafficking of polarity proteins and tight junction components out of the endosomal compartment, thereby providing a critical link between a resident protein of apical endosomes and tight junctions.

Rab14 regulates apical targeting in polarized epithelial cells.

Epithelial cells display distinct apical and basolateral membrane domains, and maintenance of this asymmetry is essential to the function of epithelial tissues. Polarized delivery of apical and basolateral membrane proteins from the trans Golgi network (TGN) and/or endosomes to the correct domain requires specific cytoplasmic machinery to control the sorting, budding and fission of vesicles. However, the molecular machinery that regulates polarized delivery of apical proteins remains poorly understood. In this study, we show that the small guanosine triphosphatase Rab14 is involved in the apical targeting pathway. Using yeast two-hybrid analysis and glutathione S-transferase pull down, we show that Rab14 interacts with apical membrane proteins and localizes to the TGN and apical endosomes. Overexpression of the GDP mutant form of Rab14 (S25N) induces an enlargement of the TGN and vesicle accumulation around Golgi membranes. Moreover, expression of Rab14-S25N results in mislocalization of the apical raft-associated protein vasoactive intestinal peptide/MAL to the basolateral domain but does not disrupt basolateral targeting or recycling. These data suggest that Rab14 specifically regulates delivery of cargo from the TGN to the apical domain.

A Role for ARF6 and ARNO in the regulation of endosomal dynamics in neurons.

During development, neuronal processes extend, branch and navigate to ultimately synapse with target tissue. We have shown a regulatory role for ARNO and ARF6 in dendritic branching and axonal elongation and branching during neuritogenesis, particularly with respect to cytoskeletal dynamics. Here, we have examined the role of ARF6 and the ARF GEF ARNO in endosomal dynamics during neurite elongation in hippocampal neurons. Axonal and dendritic endosomes were labeled by expression of the endosomal marker, endotubin. Expression of endotubin-green fluorescent protein resulted in targeting to tubular-vesicular structures throughout the somatodendritic and axonal domains. These endosomal structures did not colocalize with conventional early or late endosomal markers or with the synaptic vesicle marker, SV2. However, they did label with internalized lectin, indicating that they are endosomal structures. Expression of catalytically inactive ARNO (ARNO-E156K) or inactive ARF6 (ARF6-T27N) caused a redistribution of endotubin to the cell surface of the axons and dendrites. In contrast, expression of these constructs had no effect upon the distribution of SV2-positive structures. Furthermore, expression of inactive ARF1 (ARF1-T31N) did not change endotubin distribution. These results suggest that endotubin labels a distinct endosomal structure in neurons and that ARNO and ARF6 mediate neurite extension through the regulation of this compartment.

Functional assay of ARNO and ARF6 in neurite elongation and branching.

During development of the nervous system, neurite outgrowth is necessary for the formation of connections between nerve cells. Neurons are highly polarized cells that send out distinct processes, axons, and dendrites; however, the molecular regulation of the differential growth of these processes remains incompletely understood. Primary cultures of rat hippocampal neurons have been used to study many aspects of neuronal cell biology, including neurite extension, establishment of polarity, biogenesis of synapses, and membrane trafficking. After attachment to the substrate, hippocampal neurons begin sending out multiple processes by approximately 12 h after plating. The axonal process is derived from one of these processes, and is evident after 48 h in culture. Complete polarity of axons and dendrites is established after 7 days in culture. The establishment of these cultures and the ability to transfect them with potential regulatory genes allows the researcher to dissect out the pathways relevant to neurite extension. To study the role of small GTPases in neurite extension and branching, we describe methods for culture of hippocampal neurons, for transfection of these cells, and assessment of neurite extension and branching.

ARNO and ARF6 regulate axonal elongation and branching through downstream activation of phosphatidylinositol 4-phosphate 5-kinase alpha.

In the developing nervous system, controlled neurite extension and branching are critical for the establishment of connections between neurons and their targets. Although much is known about the regulation of axonal development, many of the molecular events that regulate axonal extension remain unknown. ADP-ribosylation factor nucleotide-binding site opener (ARNO) and ADP-ribosylation factor (ARF)6 have important roles in the regulation of the cytoskeleton as well as membrane trafficking. To investigate the role of these molecules in axonogenesis, we expressed ARNO and ARF6 in cultured rat hippocampal neurons. Expression of catalytically inactive ARNO or dominant negative ARF6 resulted in enhanced axonal extension and branching and this effect was abrogated by coexpression of constitutively active ARF6. We sought to identify the downstream effectors of ARF6 during neurite extension by coexpressing phosphatidyl-inositol-4-phosphate 5-Kinase alpha [PI(4)P 5-Kinase alpha] with catalytically inactive ARNO and dominant negative ARF6. We found that PI(4)P 5-Kinase alpha plays a role in neurite extension and branching downstream of ARF6. Also, expression of inactive ARNO/ARF6 depleted the actin binding protein mammalian ena (Mena) from the growth cone leading edge, indicating that these effects on axonogenesis may be mediated by changes in cytoskeletal dynamics. These results suggest that ARNO and ARF6, through PI(4)P 5-Kinase alpha, regulate axonal elongation and branching during neuronal development.