Rab6-Mediated Polarized Transport of Synaptic Vesicle Precursors Is Essential for the Establishment of Neuronal Polarity and Brain Formation.

Neurons are highly polarized cells that are composed of a single axon and multiple dendrites. Axon-dendrite polarity is essential for proper tissue formation and brain functions. Intracellular protein transport plays an important role in the establishment of neuronal polarity. However, the regulatory mechanism of polarized transport remains unclear. Here, we show that Rab6, a small GTPase that acts on the regulation of intracellular vesicular trafficking, plays key roles in neuronal polarization and brain development. Central nervous system-specific Rab6a/b double knock-out (Rab6 DKO) mice of both sexes exhibit severe dysplasia of the neocortex and the cerebellum. In the Rab6 DKO neocortex, impaired axonal extension of neurons results in hypoplasia of the intermediate zone. In vitro, deletion of Rab6a and Rab6b in cultured neurons from both sexes causes the abnormal accumulation of synaptic vesicle precursors (SVPs) adjacent to the Golgi apparatus, which leads to defects in axonal extension and the loss of axon-dendrite polarity. Moreover, Rab6 DKO causes significant expansion of lysosomes in the soma in neurons. Overall, our results reveal that Rab6-mediated polarized transport of SVPs is crucial for neuronal polarization and subsequent brain formation.

The Rab GTPase-binding protein EHBP1L1 and its interactors CD2AP/CIN85 negatively regulate the length of primary cilia via actin remodeling.

Primary cilia are organelles consisting of axonemal microtubules and plasma membranes, and they protrude from the cell surface to the extracellular region and function in signal sensing and transduction. The integrity of cilia, including the length and structure, is associated with signaling functions; however, factors involved in regulating the integrity of cilia have not been fully elucidated. Here, we showed that the Rab GTPase-binding protein EHBP1L1 and its newly identified interactors CD2AP and CIN85, known as adaptor proteins of actin regulators, are involved in ciliary length control. Immunofluorescence microscopy showed that EHBP1L1 and CD2AP/CIN85 are localized to the ciliary sheath. EHBP1L1 depletion caused mislocalization of CD2AP/CIN85, suggesting that CD2AP/CIN85 localization to the ciliary sheath is dependent on EHBP1L1. Additionally, we determined that EHBP1L1- and CD2AP/CIN85-depleted cells had elongated cilia. The aberrantly elongated cilia phenotype and the ciliary localization defect of CD2AP/CIN85 in EHBP1L1-depleted cells were rescued by the expression of WT EHBP1L1, although this was not observed in the CD2AP/CIN85-binding-deficient mutant, indicating that the EHBP1L1-CD2AP/CIN85 interaction is crucial for controlling ciliary length. Furthermore, EHBP1L1- and CD2AP/CIN85-depleted cells exhibited actin nucleation and branching defects around the ciliary base. Taken together, our data demonstrate that the EHBP1L1-CD2AP/CIN85 axis negatively regulates ciliary length via actin network remodeling around the basal body.

Functional Cooperation of α-Synuclein and Tau Is Essential for Proper Corticogenesis.

Alpha-synuclein (αSyn) and tau are abundant multifunctional neuronal proteins, and their intracellular deposits have been linked to many neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Despite the disease relevance, their physiological roles remain elusive, as mice with knock-out of either of these genes do not exhibit overt phenotypes. To reveal functional cooperation, we generated αSyn-/-tau-/- double-knock-out mice and characterized the functional cross talk between these proteins during brain development. Intriguingly, deletion of αSyn and tau reduced Notch signaling and accelerated interkinetic nuclear migration of G2 phase at early embryonic stage. This significantly altered the balance between the proliferative and neurogenic divisions of progenitor cells, resulting in an overproduction of early born neurons and enhanced neurogenesis, by which the brain size was enlarged during the embryonic stage in both sexes. On the other hand, a reduction in the number of neural progenitor cells in the middle stage of corticogenesis diminished subsequent gliogenesis in the αSyn-/-tau-/- cortex. Additionally, the expansion and maturation of macroglial cells (astrocytes and oligodendrocytes) were suppressed in the αSyn-/-tau-/- postnatal brain, which in turn reduced the male αSyn-/-tau-/- brain size and cortical thickness to less than the control values. Our study identifies important functional cooperation of αSyn and tau during corticogenesis.SIGNIFICANCE STATEMENT Correct understanding of the physiological functions of αSyn and tau in CNS is critical to elucidate pathogenesis involved in the etiology of neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. We show here that αSyn and tau are cooperatively involved in brain development via maintenance of progenitor cells. αSyn and tau double-knock-out mice exhibited an overproduction of early born neurons and accelerated neurogenesis at early corticogenesis. Furthermore, loss of αSyn and tau also perturbed gliogenesis at later embryonic stage, as well as the subsequent glial expansion and maturation at postnatal brain. Our findings provide new mechanistic insights and extend therapeutic opportunities for neurodegenerative diseases caused by aberrant αSyn and tau.

Rab11-mediated post-Golgi transport of the sialyltransferase ST3GAL4 suggests a new mechanism for regulating glycosylation.

Glycosylation, the most common posttranslational modification of proteins, is a stepwise process that relies on tight regulation of subcellular glycosyltransferase location to control the addition of each monosaccharide. Glycosyltransferases primarily reside and function in the endoplasmic reticulum (ER) and the Golgi apparatus; whether and how they traffic beyond the Golgi, how this trafficking is controlled, and how it impacts glycosylation remain unclear. Our previous work identified a connection between N-glycosylation and Rab11, a key player in the post-Golgi transport that connects recycling endosomes and other compartments. To learn more about the specific role of Rab11, we knocked down Rab11 in HeLa cells. Our findings indicate that Rab11 knockdown results in a dramatic enhancement in the sialylation of N-glycans. Structural analyses of glycans using lectins and LC-MS revealed that α2,3-sialylation is selectively enhanced, suggesting that an α2,3-sialyltransferase that catalyzes the sialyation of glycoproteins is activated or upregulated as the result of Rab11 knockdown. ST3GAL4 is the major α2,3-sialyltransferase that acts on N-glycans; we demonstrated that the localization of ST3GAL4, but not the levels of its mRNA, protein, or donor substrate, was altered by Rab11 depletion. In knockdown cells, ST3GAL4 is densely distributed in the trans-Golgi network, compared with the wider distribution in the Golgi and in other peripheral puncta in control cells, whereas the α2,6-sialyltransferase ST6GAL1 is predominantly localized to the Golgi regardless of Rab11 knockdown. This indicates that Rab11 may negatively regulate α2,3-sialylation by transporting ST3GAL4 to post-Golgi compartments (PGCs), which is a novel mechanism of glycosyltransferase regulation.

Palmitoylated CKAP4 regulates mitochondrial functions through an interaction with VDAC2 at ER-mitochondria contact sites.

Cytoskeleton-associated protein 4 (CKAP4) is a palmitoylated type II transmembrane protein localized to the endoplasmic reticulum (ER). Here, we found that knockout (KO) of CKAP4 in HeLaS3 cells induces the alteration of mitochondrial structures and increases the number of ER-mitochondria contact sites. To understand the involvement of CKAP4 in mitochondrial functions, the binding proteins of CKAP4 were explored, enabling identification of the mitochondrial porin voltage-dependent anion-selective channel protein 2 (VDAC2), which is localized to the outer mitochondrial membrane. Palmitoylation at Cys100 of CKAP4 was required for the binding between CKAP4 and VDAC2. In CKAP4 KO cells, the binding of inositol trisphosphate receptor (IP3R) and VDAC2 was enhanced, the intramitochondrial Ca2+ concentration increased and the mitochondrial membrane potential decreased. In addition, CKAP4 KO decreased the oxidative consumption rate, in vitro cancer cell proliferation under low-glucose conditions and in vivo xenograft tumor formation. The phenotypes were not rescued by expression of a palmitoylation-deficient CKAP4 mutant. These results suggest that CKAP4 plays a role in maintaining mitochondrial functions through the binding to VDAC2 at ER-mitochondria contact sites and that palmitoylation is required for this novel function of CKAP4.This article has an associated First Person interview with the first author of the paper.

Loss of Rab6a in the small intestine causes lipid accumulation and epithelial cell death from lactation.

Intestinal epithelial cells (IECs) are not only responsible for the digestion and absorption of dietary substrates but also function as a first line of host defense against commensal and pathogenic luminal bacteria. Disruption of the epithelial layer causes malnutrition and enteritis. Rab6 is a small GTPase localized to the Golgi, where it regulates anterograde and retrograde transport by interacting with various effector proteins. Here, we generated mice with IEC-specific deletion of Rab6a (Rab6a∆IEC mice). While Rab6aΔIEC mice were born at the Mendelian ratio, they started to show IEC death, inflammation, and bleeding in the small intestine shortly after birth, and these changes culminated in early postnatal death. We further found massive lipid accumulation in the IECs of Rab6a∆IEC neonates. In contrast to Rab6a∆IEC neonates, knockout embryos did not show any of these abnormalities. Lipid accumulation and IEC death became evident when Rab6a∆IEC embryos were nursed by a foster mother, suggesting that dietary milk-derived lipids accumulated in Rab6a-deficient IECs and triggered IEC death. These results indicate that Rab6a plays a crucial role in regulating the lipid transport and maintaining tissue integrity.

LRRK2 and its substrate Rab GTPases are sequentially targeted onto stressed lysosomes and maintain their homeostasis.

Leucine-rich repeat kinase 2 (LRRK2) has been associated with a variety of human diseases, including Parkinson's disease and Crohn's disease, whereas LRRK2 deficiency leads to accumulation of abnormal lysosomes in aged animals. However, the cellular roles and mechanisms of LRRK2-mediated lysosomal regulation have remained elusive. Here, we reveal a mechanism of stress-induced lysosomal response by LRRK2 and its target Rab GTPases. Lysosomal overload stress induced the recruitment of endogenous LRRK2 onto lysosomal membranes and activated LRRK2. An upstream adaptor Rab7L1 (Rab29) promoted the lysosomal recruitment of LRRK2. Subsequent family-wide screening of Rab GTPases that may act downstream of LRRK2 translocation revealed that Rab8a and Rab10 were specifically accumulated on overloaded lysosomes dependent on their phosphorylation by LRRK2. Rab7L1-mediated lysosomal targeting of LRRK2 attenuated the stress-induced lysosomal enlargement and promoted lysosomal secretion, whereas Rab8 stabilized by LRRK2 on stressed lysosomes suppressed lysosomal enlargement and Rab10 promoted lysosomal secretion, respectively. These effects were mediated by the recruitment of Rab8/10 effectors EHBP1 and EHBP1L1. LRRK2 deficiency augmented the chloroquine-induced lysosomal vacuolation of renal tubules in vivo. These results implicate the stress-responsive machinery composed of Rab7L1, LRRK2, phosphorylated Rab8/10, and their downstream effectors in the maintenance of lysosomal homeostasis.

Mutations in the pH-Sensing G-protein-Coupled Receptor GPR68 Cause Amelogenesis Imperfecta.

Amelogenesis is the process of dental enamel formation, leading to the deposition of the hardest tissue in the human body. This process requires the intricate regulation of ion transport and controlled changes to the pH of the developing enamel matrix. The means by which the enamel organ regulates pH during amelogenesis is largely unknown. We identified rare homozygous variants in GPR68 in three families with amelogenesis imperfecta, a genetically and phenotypically heterogeneous group of inherited conditions associated with abnormal enamel formation. Each of these homozygous variants (a large in-frame deletion, a frameshift deletion, and a missense variant) were predicted to result in loss of function. GPR68 encodes a proton-sensing G-protein-coupled receptor with sensitivity in the pH range that occurs in the developing enamel matrix during amelogenesis. Immunohistochemistry of rat mandibles confirmed localization of GPR68 in the enamel organ at all stages of amelogenesis. Our data identify a role for GPR68 as a proton sensor that is required for proper enamel formation.

Vesicular and non-vesicular transport feed distinct glycosylation pathways in the Golgi.

Newly synthesized proteins and lipids are transported across the Golgi complex via different mechanisms whose respective roles are not completely clear. We previously identified a non-vesicular intra-Golgi transport pathway for glucosylceramide (GlcCer)--the common precursor of the different series of glycosphingolipids-that is operated by the cytosolic GlcCer-transfer protein FAPP2 (also known as PLEKHA8) (ref. 1). However, the molecular determinants of the FAPP2-mediated transfer of GlcCer from the cis-Golgi to the trans-Golgi network, as well as the physiological relevance of maintaining two parallel transport pathways of GlcCer--vesicular and non-vesicular--through the Golgi, remain poorly defined. Here, using mouse and cell models, we clarify the molecular mechanisms underlying the intra-Golgi vectorial transfer of GlcCer by FAPP2 and show that GlcCer is channelled by vesicular and non-vesicular transport to two topologically distinct glycosylation tracks in the Golgi cisternae and the trans-Golgi network, respectively. Our results indicate that the transport modality across the Golgi complex is a key determinant for the glycosylation pattern of a cargo and establish a new paradigm for the branching of the glycosphingolipid synthetic pathway.

Rab8a vesicles regulate Wnt ligand delivery and Paneth cell maturation at the intestinal stem cell niche.

Communication between stem and niche supporting cells maintains the homeostasis of adult tissues. Wnt signaling is a crucial regulator of the stem cell niche, but the mechanism that governs Wnt ligand delivery in this compartment has not been fully investigated. We identified that Wnt secretion is partly dependent on Rab8a-mediated anterograde transport of Gpr177 (wntless), a Wnt-specific transmembrane transporter. Gpr177 binds to Rab8a, depletion of which compromises Gpr177 traffic, thereby weakening the secretion of multiple Wnts. Analyses of generic Wnt/ß-catenin targets in Rab8a knockout mouse intestinal crypts indicate reduced signaling activities; maturation of Paneth cells - a Wnt-dependent cell type - is severely affected. Rab8a knockout crypts show an expansion of Lgr5(+) and Hopx(+) cells in vivo. However, in vitro, the knockout enteroids exhibit significantly weakened growth that can be partly restored by exogenous Wnts or Gsk3ß inhibitors. Immunogold labeling and surface protein isolation identified decreased plasma membrane localization of Gpr177 in Rab8a knockout Paneth cells and fibroblasts. Upon stimulation by exogenous Wnts, Rab8a-deficient cells show ligand-induced Lrp6 phosphorylation and transcriptional reporter activation. Rab8a thus controls Wnt delivery in producing cells and is crucial for Paneth cell maturation. Our data highlight the profound tissue plasticity that occurs in response to stress induced by depletion of a stem cell niche signal.

Rab8b Regulates Transport of West Nile Virus Particles from Recycling Endosomes.

West Nile virus (WNV) particles assemble at and bud into the endoplasmic reticulum (ER) and are secreted from infected cells through the secretory pathway. However, the host factor related to these steps is not fully understood. Rab proteins, belonging to the Ras superfamily, play essential roles in regulating many aspects of vesicular trafficking. In this study, we sought to determine which Rab proteins are involved in intracellular trafficking of nascent WNV particles. RNAi analysis revealed that Rab8b plays a role in WNV particle release. We found that Rab8 and WNV antigen were colocalized in WNV-infected human neuroblastoma cells, and that WNV infection enhanced Rab8 expression in the cells. In addition, the amount of WNV particles in the supernatant of Rab8b-deficient cells was significantly decreased compared with that of wild-type cells. We also demonstrated that WNV particles accumulated in the recycling endosomes in WNV-infected cells. In summary, these results suggest that Rab8b is involved in trafficking of WNV particles from recycling endosomes to the plasma membrane.

Drebrin E depletion in human intestinal epithelial cells mimics Rab8a loss of function.

Intestinal epithelial cells are highly polarized and exhibit a complex architecture with a columnar shape and a specialized apical surface supporting microvilli organized in a brush border. These microvilli are rooted in a dense meshwork of acto-myosin called the terminal web. We have shown recently that Drebrin E, an F-actin-binding protein, is a key protein for the organization of the terminal web and the brush border. Drebrin E is also required for the columnar cell shape of Caco2 cells (human colonic cells). Here, we found that the subcellular localization of several apical markers including dipeptidyl peptidase IV (DPPIV) was strikingly modified in Drebrin E-depleted Caco2 cells. Instead of being mostly present at the apical surface, these proteins are accumulated in an enlarged subapical compartment. Using known intracellular markers, we show by both confocal and electron microscopy that this compartment is related to lysosomes. We also demonstrate that the enrichment of DPPIV in this compartment originates from apical endocytosis and that depletion of Rab8a induces an accumulation of apical proteins in a similar compartment. Consistent with this, the phenotype observed in Drebrin E knock-down Caco2 cells shares some features with a pathology called microvillar inclusion disease (MVID) involving both Myosin Vb and Rab8a. Taken together, these results suggest that Drebrin E redirects the apical recycling pathway in intestinal epithelial cells to the lysosomes, demonstrating that Drebrin E is a key regulator in apical trafficking in Caco2 cells.

Rab8a and Rab8b are essential for several apical transport pathways but insufficient for ciliogenesis.

The small GTP-binding protein Rab8 is known to play an essential role in intracellular transport and cilia formation. We have previously demonstrated that Rab8a is required for localising apical markers in various organisms. Rab8a has a closely related isoform, Rab8b. To determine whether Rab8b can compensate for Rab8a, we generated Rab8b-knockout mice. Although the Rab8b-knockout mice did not display an overt phenotype, Rab8a and Rab8b double-knockout mice exhibited mislocalisation of apical markers and died earlier than Rab8a-knockout mice. The apical markers accumulated in three intracellular patterns in the double-knockout mice. However, the localisation of basolateral and/or dendritic markers of the double-knockout mice seemed normal. The morphology and the length of various primary and/or motile cilia, and the frequency of ciliated cells appeared to be identical in control and double-knockout mice. However, an additional knockdown of Rab10 in double-knockout cells greatly reduced the percentage of ciliated cells. Our results highlight the compensatory effect of Rab8a and Rab8b in apical transport, and the complexity of the apical transport process. In addition, neither Rab8a nor Rab8b are required for basolateral and/or dendritic transport. However, simultaneous loss of Rab8a and Rab8b has little effect on ciliogenesis, whereas additional loss of Rab10 greatly affects ciliogenesis.

Critical roles of type III phosphatidylinositol phosphate kinase in murine embryonic visceral endoderm and adult intestine.

The metabolism of membrane phosphoinositides is critical for a variety of cellular processes. Phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P(2)] controls multiple steps of the intracellular membrane trafficking system in both yeast and mammalian cells. However, other than in neuronal tissues, little is known about the physiological functions of PtdIns(3,5)P(2) in mammals. Here, we provide genetic evidence that type III phosphatidylinositol phosphate kinase (PIPKIII), which produces PtdIns(3,5)P(2), is essential for the functions of polarized epithelial cells. PIPKIII-null mouse embryos die by embryonic day 8.5 because of a failure of the visceral endoderm to supply the epiblast with maternal nutrients. Similarly, although intestine-specific PIPKIII-deficient mice are born, they fail to thrive and eventually die of malnutrition. At the mechanistic level, we show that PIPKIII regulates the trafficking of proteins to a cell's apical membrane domain. Importantly, mice with intestine-specific deletion of PIPKIII exhibit diarrhea and bloody stool, and their gut epithelial layers show inflammation and fibrosis, making our mutants an improved model for inflammatory bowel diseases. In summary, our data demonstrate that PIPKIII is required for the structural and functional integrity of two different types of polarized epithelial cells and suggest that PtdIns(3,5)P(2) metabolism is an unexpected and critical link between membrane trafficking in intestinal epithelial cells and the pathogenesis of inflammatory bowel disease.

CLAC-P/collagen type XXV is required for the intramuscular innervation of motoneurons during neuromuscular development.

Formation of proper neuromuscular connections is a process coordinated by both motoneuron-intrinsic and target-dependent programs. Under these programs, motoneurons innervate target muscles, escape programmed cell death during fetal development, and form neuromuscular junctions (NMJ). Although a number of studies have revealed molecules involved in axon guidance to target muscles and NMJ formation, little is known about the molecular mechanisms linking intramuscular innervation and target-derived trophic factor-dependent prevention of motoneuron apoptosis. Here we studied the physiological function of CLAC-P/collagen XXV, a transmembrane-type collagen originally identified as a component of senile plaque amyloid of Alzheimer's disease brains, by means of generating Col25a1-deficient (KO) mice. Col25a1 KO mice died immediately after birth of respiratory failure. In Col25a1 KO mice, motor axons projected properly toward the target muscles but failed to elongate and branch within the muscle, followed by degeneration of axons. Failure of muscular innervation in Col25a1 KO mice led to excessive apoptosis during development, resulting in almost complete and exclusive loss of spinal motoneurons and immaturity in skeletal muscle development. Bax deletion in Col25a1 KO mice rescued motoneurons from apoptosis, although motor axons remained halted around the muscle entry site. Furthermore, these motoneurons were positive for phosphorylated c-Jun, an indicator of insufficient supply of target-derived survival signals. Together, these observations indicate that CLAC-P/collagen XXV is a novel essential factor that regulates the initial phase of intramuscular motor innervation, which is required for subsequent target-dependent motoneuron survival and NMJ formation during development.

Hypothalamic SIRT1 prevents age-associated weight gain by improving leptin sensitivity in mice.

AIMS/HYPOTHESIS: Obesity is associated with ageing and increased energy intake, while restriction of energy intake improves health and longevity in multiple organisms; the NAD(+)-dependent deacetylase sirtuin 1 (SIRT1) is implicated in this process. Pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in the arcuate nucleus (ARC) of the hypothalamus are critical for energy balance regulation, and the level of SIRT1 protein decreases with age in the ARC. In the current study we tested whether conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevents age-associated weight gain and diet-induced obesity. METHODS: We targeted Sirt1 cDNA sequence into the Rosa26 locus and generated conditional Sirt1 knock-in mice. These mice were crossed with mice harbouring either Pomc-Cre or Agrp-Cre and the metabolic variables, food intake, energy expenditure and sympathetic activity in adipose tissue of the resultant mice were analysed. We also used a hypothalamic cell line to investigate the molecular mechanism by which Sirt1 overexpression modulates leptin signalling. RESULTS: Conditional Sirt1 overexpression in mouse POMC or AgRP neurons prevented age-associated weight gain; overexpression in POMC neurons stimulated energy expenditure via increased sympathetic activity in adipose tissue, whereas overexpression in AgRP neurons suppressed food intake. SIRT1 improved leptin sensitivity in hypothalamic neurons in vitro and in vivo by downregulating protein-tyrosine phosphatase 1B, T cell protein-tyrosine phosphatase and suppressor of cytokine signalling 3. However, these phenotypes were absent in mice consuming a high-fat, high-sucrose diet due to decreases in ARC SIRT1 protein and hypothalamic NAD(+) levels. CONCLUSIONS/INTERPRETATION: ARC SIRT1 is a negative regulator of energy balance, and decline in ARC SIRT1 function contributes to disruption of energy homeostasis by ageing and diet-induced obesity.

The role of PKD in cell polarity, biosynthetic pathways, and organelle/F-actin distribution.

Protein Kinase D (PKD) 1, 2, and 3 are members of the PKD family. PKDs influence many cellular processes, including cell polarity, structure of the Golgi, polarized transport from the Golgi to the basolateral plasma membrane, and actin polymerization. However, the role of the PKD family in cell polarity has not yet been elucidated in vivo. Here, we show that KO mice displayed similar localization of the apical and basolateral proteins, transport of VSV-G and a GPI-anchored protein, and similar localization of actin filaments. As DKO mice were embryonic lethal, we generated MEFs that lacked all PKD isoforms from the PKD1 and PKD2 double floxed mice using Cre recombinase and PKD3 siRNA. We observed a similar localization of various organelles, a similar time course in the transport of VSV-G and a GPI-anchored protein, and a similar distribution of F-actin in the PKD-null MEFs. Collectively, our results demonstrate that the complete deletion of PKDs does not affect the transport of VSV-G or a GPI-anchored protein, and the distribution of F-actin. However, simultaneous deletion of PKD1 and PKD2 affect embryonic development, demonstrating their functional redundancy during development.

Dynamic movement of the Golgi unit and its glycosylation enzyme zones.

Knowledge on the distribution and dynamics of glycosylation enzymes in the Golgi is essential for better understanding this modification. Here, using a combination of CRISPR/Cas9 knockin technology and super-resolution microscopy, we show that the Golgi complex is assembled by a number of small 'Golgi units' that have 1-3 µm in diameter. Each Golgi unit contains small domains of glycosylation enzymes which we call 'zones'. The zones of N- and O-glycosylation enzymes are colocalised. However, they are less colocalised with the zones of a glycosaminoglycan synthesizing enzyme. Golgi units change shapes dynamically and the zones of glycosylation enzymes rapidly move near the rim of the unit. Photobleaching analysis indicates that a glycosaminoglycan synthesizing enzyme moves between units. Depletion of giantin dissociates units and prevents the movement of glycosaminoglycan synthesizing enzymes, which leads to insufficient glycosaminoglycan synthesis. Thus, we show the structure-function relationship of the Golgi and its implications in human pathogenesis.

The role of VAMP7/TI-VAMP in cell polarity and lysosomal exocytosis in vivo.

VAMP7 or tetanus neurotoxin-insensitive vesicle- associated membrane protein (TI-VAMP) has been proposed to regulate apical transport in polarized epithelial cells, axonal transport in neurons and lysosomal exocytosis. To investigate the function of VAMP7 in vivo, we generated VAMP7 knockout mice. Here, we show that VAMP7 knockout mice are indistinguishable from control mice and display a similar localization of apical proteins in the kidney and small intestine and a similar localization of axonal proteins in the nervous system. Neurite outgrowth of cultured mutant hippocampal neurons was reduced in mutant neurons. However, lysosomal exocytosis was not affected in mutant fibroblasts. Our results show that VAMP7 is required in neurons to extend axons to the full extent. However, VAMP7 does not seem to be required for epithelial cell polarity and lysosomal exocytosis.

Uncovering genes required for neuronal morphology by morphology-based gene trap screening with a revertible retrovirus vector.

The molecular mechanisms of neuronal morphology and synaptic vesicle transport have been largely elusive, and only a few of the molecules involved in these processes have been identified. Here, we developed a novel morphology-based gene trap method, which is theoretically applicable to all cell lines, to easily and rapidly identify the responsible genes. Using this method, we selected several gene-trapped clones of rat pheochromocytoma PC12 cells, which displayed abnormal morphology and distribution of synaptic vesicle-like microvesicles (SLMVs). We identified several genes responsible for the phenotypes and analyzed three genes in more detail. The first gene was BTB/POZ domain-containing protein 9 (Btbd9), which is associated with restless legs syndrome. The second gene was cytokine receptor-like factor 3 (Crlf3), whose involvement in the nervous system remains unknown. The third gene was single-stranded DNA-binding protein 3 (Ssbp3), a gene known to regulate head morphogenesis. These results suggest that Btbd9, Crlf3, and Ssbp3 regulate neuronal morphology and the biogenesis/transport of synaptic vesicles. Because our novel morphology-based gene trap method is generally applicable, this method is promising for uncovering novel genes involved in the function of interest in any cell lines.