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.

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.

EHBP1L1, an apicobasal polarity regulator, is critical for nuclear polarization during enucleation of erythroblasts.

Cell polarity, the asymmetric distribution of proteins and organelles, is permanently or transiently established in various cell types and plays an important role in many physiological events. epidermal growth factor receptor substrate 15 homology domain-binding protein 1-like 1 (EHBP1L1) is an adapter protein that is localized on recycling endosomes and regulates apical-directed transport in polarized epithelial cells. However, the role of EHBP1L1 in nonepithelial cells, remains unknown. Here, Ehbp1l1-/- mice showed impaired erythroblast enucleation. Further analyses showed that nuclear polarization before enucleation was impaired in Ehbp1l1-/- erythroblasts. It was also revealed that EHBP1L1 interactors Rab10, Bin1, and dynamin were involved in erythroblast enucleation. In addition, Ehbp1l1-/- erythrocytes exhibited stomatocytic morphology and dehydration. These defects in erythroid cells culminated in early postnatal anemic lethality in Ehbp1l1-/- mice. Moreover, we found the mislocalization of nuclei and mitochondria in the skeletal muscle cells of Ehbp1l1-/- mice, as observed in patients with centronuclear myopathy with genetic mutations in Bin1 or dynamin 2. Taken together, our findings indicate that the Rab8/10-EHBP1L1-Bin1-dynamin axis plays an important role in multiple cell polarity systems in epithelial and nonepithelial cells.

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.

The lysosomal Ragulator complex activates NLRP3 inflammasome in vivo via HDAC6.

The cellular activation of the NLRP3 inflammasome is spatiotemporally orchestrated by various organelles, but whether lysosomes contribute to this process remains unclear. Here, we show the vital role of the lysosomal membrane-tethered Ragulator complex in NLRP3 inflammasome activation. Deficiency of Lamtor1, an essential component of the Ragulator complex, abrogated NLRP3 inflammasome activation in murine macrophages and human monocytic cells. Myeloid-specific Lamtor1-deficient mice showed marked attenuation of NLRP3-associated inflammatory disease severity, including LPS-induced sepsis, alum-induced peritonitis, and monosodium urate (MSU)-induced arthritis. Mechanistically, Lamtor1 interacted with both NLRP3 and histone deacetylase 6 (HDAC6). HDAC6 enhances the interaction between Lamtor1 and NLRP3, resulting in NLRP3 inflammasome activation. DL-all-rac-α-tocopherol, a synthetic form of vitamin E, inhibited the Lamtor1-HDAC6 interaction, resulting in diminished NLRP3 inflammasome activation. Further, DL-all-rac-α-tocopherol alleviated acute gouty arthritis and MSU-induced peritonitis. These results provide novel insights into the role of lysosomes in the activation of NLRP3 inflammasomes by the Ragulator complex.

PACSIN1 is indispensable for amphisome-lysosome fusion during basal autophagy and subsets of selective autophagy.

Autophagy is an indispensable process that degrades cytoplasmic materials to maintain cellular homeostasis. During autophagy, double-membrane autophagosomes surround cytoplasmic materials and either fuse with endosomes (called amphisomes) and then lysosomes, or directly fuse with lysosomes, in both cases generating autolysosomes that degrade their contents by lysosomal hydrolases. However, it remains unclear if there are specific mechanisms and/or conditions which distinguish these alternate routes. Here, we identified PACSIN1 as a novel autophagy regulator. PACSIN1 deletion markedly decreased autophagic activity under basal nutrient-rich conditions but not starvation conditions, and led to amphisome accumulation as demonstrated by electron microscopic and co-localization analysis, indicating inhibition of lysosome fusion. PACSIN1 interacted with SNAP29, an autophagic SNARE, and was required for proper assembly of the STX17 and YKT6 complexes. Moreover, PACSIN1 was required for lysophagy, aggrephagy but not mitophagy, suggesting cargo-specific fusion mechanisms. In C. elegans, deletion of sdpn-1, a homolog of PACSINs, inhibited basal autophagy and impaired clearance of aggregated protein, implying a conserved role of PACSIN1. Taken together, our results demonstrate the amphisome-lysosome fusion process is preferentially regulated in response to nutrient state and stress, and PACSIN1 is a key to specificity during autophagy.

SNAP23 deficiency causes severe brain dysplasia through the loss of radial glial cell polarity.

In the developing brain, the polarity of neural progenitor cells, termed radial glial cells (RGCs), is important for neurogenesis. Intercellular adhesions, termed apical junctional complexes (AJCs), at the apical surface between RGCs are necessary for cell polarization. However, the mechanism by which AJCs are established remains unclear. Here, we show that a SNARE complex composed of SNAP23, VAMP8, and Syntaxin1B has crucial roles in AJC formation and RGC polarization. Central nervous system (CNS)-specific ablation of SNAP23 (NcKO) results in mice with severe hypoplasia of the neocortex and no hippocampus or cerebellum. In the developing NcKO brain, RGCs lose their polarity following the disruption of AJCs and exhibit reduced proliferation, increased differentiation, and increased apoptosis. SNAP23 and its partner SNAREs, VAMP8 and Syntaxin1B, are important for the localization of an AJC protein, N-cadherin, to the apical plasma membrane of RGCs. Altogether, SNARE-mediated localization of N-cadherin is essential for AJC formation and RGC polarization during brain development.

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.

Extremely short setting of optimal sensed atrioventricular interval in patients after Fontan procedure with implanted dual-chamber pacemaker.

BACKGROUND: Atrioventricular interval optimisation is important in patients with dual-chamber pacing, especially with heart failure. In patients with CHD, especially in those with Fontan circulation, the systemic atrial contraction is supposed to be more important than in patients without structural heart disease. METHODS: We retrospectively evaluated two patients after Fontan procedure with dual-chamber pacemaker with a unique setting of optimal sensed atrioventricular interval. RESULTS: The optimal sensed atrioventricular interval determined by echocardiogram was extremely short sensed atrioventricular interval at 25 and 30 ms in both cases; however, the actual P wave and ventricular pacing interval showed 180 and 140 ms, respectively. In both cases, the atrial epicardial leads were implanted on the opposite site of the origin of their own atrial rhythm. The time differences between sensed atrioventricular interval and actual P wave and ventricular pacing interval occurred because of the site of the epicardial atrial pacing leads and the intra-atrial conduction delay. CONCLUSION: We need to consider the origin of the atrial rhythm, the site of the epicardial atrial lead, and the atrial conduction delay by using electrocardiogram and X-ray when we set the optimal sensed atrioventricular interval in complicated CHD.

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.

Continuous infusion of lipo-prostaglandin E1 for Takayasu's arteritis with heart failure in an 11-month-old baby: a case report.

BACKGROUND: Takayasu's arteritis is extremely rare in children aged below 6 years. At the onset of Takayasu's arteritis in children, symptoms are varied but differ from those in adults. Corticosteroids are the mainstay of treatment for preventing irreversible vascular damage but there is no standard treatment for progressive vascular stenosis. CASE PRESENTATION: A Japanese 11-month-old baby boy presented with Takayasu's arteritis and heart failure, possibly due to afterload mismatch caused by high blood pressure. Computed tomography was performed and revealed thoracic and abdominal aortic aneurysms. It also revealed severe celiac artery stenosis and bilateral renal artery stenosis. Prednisolone was initiated as first-line therapy. The fever resolved, and C-reactive protein levels returned to normal. Although his general condition improved, deterioration of vascular lesions was evident. Celiac artery occlusion, severe right renal artery stenosis, and new superior mesenteric artery stenosis were observed. We decided to use a continuous infusion of lipo-prostaglandin E1 for prevention of branch stenosis of his abdominal aorta. The progression of vascular stenosis was stopped and our patient's cardiac function gradually improved. CONCLUSIONS: A differential diagnosis of heart failure with high blood pressure should be considered in babies. The progression of vascular stenosis may be suppressed by lipo-prostaglandin E1.

The Rab11-binding protein RELCH/KIAA1468 controls intracellular cholesterol distribution.

Cholesterol, which is endocytosed to the late endosome (LE)/lysosome, is delivered to other organelles through vesicular and nonvesicular transport mechanisms. In this study, we discuss a novel mechanism of cholesterol transport from recycling endosomes (REs) to the trans-Golgi network (TGN) through RELCH/KIAA1468, which is newly identified in this study as a Rab11-GTP- and OSBP-binding protein. After treating cells with 25-hydroxycholesterol to induce OSBP relocation from the cytoplasm to the TGN, REs accumulated around the TGN area, but this accumulation was diminished in RELCH- or OSBP-depleted cells. Cholesterol content in the TGN was decreased in Rab11-, RELCH-, and OSBP-depleted cells and increased in the LE/lysosome. According to in vitro reconstitution experiments, RELCH tethers Rab11-bound RE-like and OSBP-bound TGN-like liposomes and promotes OSBP-dependent cholesterol transfer from RE-like to TGN-like liposomes. These data suggest that RELCH promotes nonvesicular cholesterol transport from REs to the TGN through membrane tethering.

BIG1 is required for the survival of deep layer neurons, neuronal polarity, and the formation of axonal tracts between the thalamus and neocortex in developing brain.

BIG1, an activator protein of the small GTPase, Arf, and encoded by the Arfgef1 gene, is one of candidate genes for epileptic encephalopathy. To know the involvement of BIG1 in epileptic encephalopathy, we analyzed BIG1-deficient mice and found that BIG1 regulates neurite outgrowth and brain development in vitro and in vivo. The loss of BIG1 decreased the size of the neocortex and hippocampus. In BIG1-deficient mice, the neuronal progenitor cells (NPCs) and the interneurons were unaffected. However, Tbr1+ and Ctip2+ deep layer (DL) neurons showed spatial-temporal dependent apoptosis. This apoptosis gradually progressed from the piriform cortex (PIR), peaked in the neocortex, and then progressed into the hippocampus from embryonic day 13.5 (E13.5) to E17.5. The upper layer (UL) and DL order in the neocortex was maintained in BIG1-deficient mice, but the excitatory neurons tended to accumulate before their destination layers. Further pulse-chase migration assay showed that the migration defect was non-cell autonomous and secondary to the progression of apoptosis into the BIG1-deficient neocortex after E15.5. In BIG1-deficient mice, we observed an ectopic projection of corticothalamic axons from the primary somatosensory cortex (S1) into the dorsal lateral geniculate nucleus (dLGN). The thalamocortical axons were unable to cross the diencephalon-telencephalon boundary (DTB). In vitro, BIG1-deficient neurons showed a delay in neuronal polarization. BIG1-deficient neurons were also hypersensitive to low dose glutamate (5 µM), and died via apoptosis. This study showed the role of BIG1 in the survival of DL neurons in developing embryonic brain and in the generation of neuronal polarity.

Opposing roles for SNAP23 in secretion in exocrine and endocrine pancreatic cells.

The membrane fusion of secretory granules with plasma membranes is crucial for the exocytosis of hormones and enzymes. Secretion disorders can cause various diseases such as diabetes or pancreatitis. Synaptosomal-associated protein 23 (SNAP23), a soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE) molecule, is essential for secretory granule fusion in several cell lines. However, the in vivo functions of SNAP23 in endocrine and exocrine tissues remain unclear. In this study, we show opposing roles for SNAP23 in secretion in pancreatic exocrine and endocrine cells. The loss of SNAP23 in the exocrine and endocrine pancreas resulted in decreased and increased fusion of granules to the plasma membrane after stimulation, respectively. Furthermore, we identified a low molecular weight compound, MF286, that binds specifically to SNAP23 and promotes insulin secretion in mice. Our results demonstrate opposing roles for SNAP23 in the secretion mechanisms of the endocrine and exocrine pancreas and reveal that the SNAP23-binding compound MF286 may be a promising drug for diabetes treatment.

EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells.

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

Functional redundancy of protein kinase D1 and protein kinase D2 in neuronal polarity.

Mammalian protein kinase D (PKD) isoforms have been proposed to regulate diverse biological processes, including the establishment and maintenance of neuronal polarity. To investigate the function of PKD in neuronal polarization in vivo, we generated PKD knockout (KO) mice. Here, we show that the brain, particularly the hippocampus, of both PKD1 KO and PKD2 KO mice was similar to that of control animals. Neurite length in cultured PKD1 KO and PKD2 KO hippocampal neurons was similar to that of wild-type neurons. However, hippocampal neurons deficient in both PKD1 and PKD2 genes showed a reduction in axonal elongation and an increase in the percentage of neurons with multiple axons relative to control neurons. These results reveal that whereas PKD1 and PKD2 are essential for neuronal polarity, there exists a functional redundancy between the two proteins.

Rab11a is required for apical protein localisation in the intestine.

The small GTPase Rab11 plays an important role in the recycling of proteins to the plasma membrane as well as in polarised transport in epithelial cells and neurons. We generated conditional knockout mice deficient in Rab11a. Rab11a-deficient mice are embryonic lethal, and brain-specific Rab11a knockout mice show no overt abnormalities in brain architecture. In contrast, intestine-specific Rab11a knockout mice begin dying approximately 1 week after birth. Apical proteins in the intestines of knockout mice accumulate in the cytoplasm and mislocalise to the basolateral plasma membrane, whereas the localisation of basolateral proteins is unaffected. Shorter microvilli and microvillus inclusion bodies are also observed in the knockout mice. Elevation of a serum starvation marker was also observed, likely caused by the mislocalisation of apical proteins and reduced nutrient uptake. In addition, Rab8a is mislocalised in Rab11a knockout mice. Conversely, Rab11a is mislocalised in Rab8a knockout mice and in a microvillus atrophy patient, which has a mutation in the myosin Vb gene. Our data show an essential role for Rab11a in the localisation of apical proteins in the intestine and demonstrate functional relationships between Rab11a, Rab8a and myosin Vb in vivo.

Rab18 and a Rab18 GEF complex are required for normal ER structure.

The ancestral Rab GTPase Rab18 and both subunits of the Rab3GAP complex are mutated in the human neurological and developmental disorder Warburg Micro syndrome. Here, we demonstrate that the Rab3GAP complex is a specific Rab18 guanine nucleotide exchange factor (GEF). The Rab3GAP complex localizes to the endoplasmic reticulum (ER) and is necessary for ER targeting of Rab18. It is also sufficient to promote membrane recruitment of Rab18. Disease-associated point mutations of conserved residues in either the Rab3GAP1 (T18P and E24V) or Rab3GAP2 (R426C) subunits result in loss of the Rab18 GEF and membrane-targeting activities. Supporting the view that Rab18 activity is important for ER structure, in the absence of either Rab3GAP subunit or Rab18 function, ER tubular networks marked by reticulon 4 were disrupted, and ER sheets defined by CLIMP-63 spread out into the cell periphery. Micro syndrome is therefore a disease characterized by direct loss of Rab18 function or loss of Rab18 activation at the ER by its GEF Rab3GAP.

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.

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.