Masataka Kunii

Rab11a is required for apical protein localisation in the intestine.

ABSTRACT 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.

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.

Generation of cortactin floxed mice and cellular analysis of motility in fibroblasts

Cortactin is an F‐actin binding protein that has been suggested to play key roles in various cellular functions. Here, we generated mice carrying floxed alleles of the cortactin (Cttn) gene (Cttnflox/flox mice). Expression of Cre recombinase in mouse embryonic fibroblasts (MEFs) isolated from Cttnflox/flox embryos depleted cortactin within days, without disturbing F‐actin distribution and localization of multiple actin‐binding proteins. Cre‐mediated deletion of Cttn also did not affect cell migration. To obtain mice with a Cttn null allele, we next crossed Cttnflox/flox mice with transgenic mice that express Cre recombinase ubiquitously. Western blot and immunocytochemical analysis confirmed complete elimination of cortactin expression in MEFs carrying homozygously Cttn null alleles. However, we found no marked alteration of F‐actin organization and cell migration in Cttn null‐MEFs. Thus, our results indicate that depletion of cortactin in MEFs does not profoundly influence actin‐dependent cell motility. genesis 47:638–646, 2009.

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

The novel protein EHBP1L1 links Rab8 to Bin1and dynamin to regulate apical transport in epithelial cells.

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.—Hashimoto, Y., Muramatsu, K., Kunii, M., Yoshimura, S., Yamada, M., Sato, T., Ishida, Y., Harada, R., Harada, A. Uncovering genes required for neuronal morphology by morphology‐based gene trap screening with a revertible retrovirus vector. FASEB J. 26, 4662–4674 (2012). www.fasebj.org

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

Kunii et al. reveal that the SNARE protein SNAP23 plays distinct roles in the secretion of amylase in exocrine cells and of insulin in endocrine cells the pancreas and show that MF286, a novel inhibitor of SNAP23, may be a new drug candidate for diabetes.

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.

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.