Oxysterol-binding protein-related protein (ORP) 6 localizes to the ER and ER-plasma membrane contact sites and is involved in the turnover of PI4P in cerebellar granule neurons.

Oxysterol-binding protein (OSBP)-related proteins (ORPs) are conserved lipid binding proteins found in organisms ranging from yeast to mammals. Recent findings have indicated that these proteins mainly localize to contact sites of 2 different membranous organelles. ORP6, a member of the ORP subfamily III, is one of the least studied ORPs. Using approaches in molecular cell biology, we attempted to study the characteristics of ORP6 and found that ORP6 is abundantly expressed in mouse cultured neurons. Deconvolution microscopy of cultured cerebellar granular cells revealed that ORP6 is localized to the endoplasmic reticulum (ER) and ER-plasma membrane (PM) contact sites, where it co-localized with extended synaptotagmin2 (E-Syt2), a well-known ER-PM contact site marker. E-Syt2 also co-localized with ORP3, another subfamily III member, and ORP5, a subfamily IV member. However, ORP5 does not distribute to the same ER-PM contact sites as subfamily III members. Also, the co-expression of ORP3 but not ORP5 altered the distribution of ORP6 into the processes of cerebellar neurons. Immunoprecipitation demonstrated binding between the intermediate region of ORP6 and ORP3 or ORP6 itself. Additionally, the localization of ORP6 in the PM decreased when co-expressed with the intermediate region of ORP6, in which the pleckstrin homology (PH) domain and OSBP-related ligand binding domain (ORD) are deleted. Over-expression of this intermediate region shifted the location of a phophtidylinositol-4-phosphate (PI4P) marker from the Golgi to the PM. Knockdown of ORP6 resulted in the same shift of the PI4P marker. Collectively, our data suggests that the recruitment of ORP6 to ER-PM contact sites is involved in the turnover of PI4P in cerebellar granular neurons.

Mechanism of the Dendritic Translation and Localization of Brain-derived Neurotrophic Factor.

Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor critical for synaptic plasticity, neuronal development and neurite extension. BDNF mRNA is transported to dendrites and axons, where it is expressed locally. We previously reported that dendritic targeting elements in the BDNF 3' UTR are necessary for dendritic transport and interact with cytoplasmic polyadenylation element binding protein 1. Here, we demonstrated that the short 3' UTR directs local translation of BDNF and that locally synthesized BDNF exists in a novel compartment that does not co-localize with markers of endosomes, endoplasmic reticulum, Golgi or the trans-Golgi network. Further, locally synthesized BDNF vesicles co-localized with Bicaudal-D2 (BicD2), a member of dynein motor complex proteins. Silencing BicD2 significantly reduced BDNF local synthesis in dendrites. These new findings may underlie the mechanism of local neuronal response to environmental stimuli.

The involvement of oxysterol-binding protein related protein (ORP) 6 in the counter-transport of phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) in neurons.

Oxysterol-binding protein (OSBP)-related protein (ORP) 6, a member of subfamily III in the ORP family, localizes to membrane contact sites between the endoplasmic reticulum (ER) and other organelles and functions in non-vesicular exchange of lipids including phosphatidylinositol-4-phosphate (PI4P) in neurons. In this study, we searched for the lipid counter-transported in exchange for PI4P by using molecular cell biology techniques. Deconvolution microscopy revealed that knockdown of ORP6 partially shifted localization of a phosphatidylserine (PS) marker but not filipin in primary cultured cerebellar neurons. Overexpression of ORP6 constructs lacking the OSBP-related ligand binding domain (ORD) resulted in the same shift of the PS marker. A PI4KⅢα inhibitor specifically inhibiting the synthesis and plasma membrane (PM) localization of PI4P, suppressed the localization of ORP6 and the PS marker at the PM. Overexpression of mutant PS synthase 1 (PSS1) inhibited transport of the PS marker to the PM and relocated the PI4P marker to the PM in Neuro-2A cells. Introduction of ORP6 but not the dominant negative ORP6 constructs, shifted the localization of PS back to the PM. These data collectively suggest the involvement of ORP6 in the counter-transport of PI4P and PS.

Effect of long-term confinement on metabolic and physiological parameters in mice.

Long-term and mild confinement or isolation in an enclosed environment can occur in situations such as disasters, specific political, economic or social events, nuclear shelters, seabed exploration, polar expeditions, and space travel. To investigate the effects of stress caused by long-term confinement in an enclosed environment in mammals, we divided 8-week-old C57BL/6J mice into four groups that were housed in a closed environment with a narrow metabolic cage (stress group), normal metabolic cage (control group), conventional cage (conventional group) or conventional cage with wire mesh floor (wire mesh group). The phenotypes of the mice were examined for four weeks, followed by behavioral tests. Weight gain suppression was observed in the stress group. Continuous analysis of these mice every two minutes for four weeks using an implanted measuring device showed a significantly decreased amount of spontaneous activity and subcutaneous temperature in the stress group. After housing in each environment for four weeks, the behavioral tests of mice in the stress group also revealed a shorter latency to fall off in the rotarod test and shorter stride length and interstep distance in the footprint test. Interestingly, the lower spontaneous activity of mice in the stress group was rescued by housing in conventional cages. These results suggest a temporary effect of long-term confinement in an enclosed environment as a chronic and mild stress on homeostasis in mammals.

Analysis of the kinesin superfamily: insights into structure and function.

Kinesin superfamily proteins (KIFs) are key players or 'hub' proteins in the intracellular transport system, which is essential for cellular function and morphology. The KIF superfamily is also the first large protein family in mammals whose constituents have been completely identified and confirmed both in silico and in vivo. Numerous studies have revealed the structures and functions of individual family members; however, the relationships between members or a perspective of the whole superfamily structure until recently remained elusive. Here, we present a comprehensive summary based on a large, systematic phylogenetic analysis of the kinesin superfamily. All available sequences in public databases, including genomic information from all model organisms, were analyzed to yield the most complete phylogenetic kinesin tree thus far, comprising 14 families. This comprehensive classification builds on the recently proposed standardized nomenclature for kinesins and allows systematic analysis of the structural and functional relationships within the kinesin superfamily.

A standardized kinesin nomenclature.

In recent years the kinesin superfamily has become so large that several different naming schemes have emerged, leading to confusion and miscommunication. Here, we set forth a standardized kinesin nomenclature based on 14 family designations. The scheme unifies all previous phylogenies and nomenclature proposals, while allowing individual sequence names to remain the same, and for expansion to occur as new sequences are discovered.

Demethylation of the MafB promoter in a compromised ß-cell model.

Recent studies suggest that dedifferentiation of pancreatic ß-cells is involved in compromised ß-cell function in diabetes mellitus. We have previously shown that the promoter activity of MafB, which is expressed in α-cells of adult islets and immature ß-cells in embryonic pancreas but not in mature ß-cells in mice, is increased in compromised ß-cells of diabetic model mice. Here, we investigated a rat ß-cell line of INS1 cells with late-passage numbers, which showed extremely low expression of MafA and insulin, as an in vitro model of compromised ß-cells. In these INS1 cells, the mRNA expression and the promoter activity of MafB were upregulated compared with the early-passage ('conventional') INS1 cells. Analysis of the MafB promoter in these late-passage INS1 cells revealed that specific CpG sites in the MafB promoter were partially demethylated. The reporter assay revealed that the unmethylated promoter activity of the 373 bp region containing these CpG sites was higher than the in vitro methylated promoter activity. These results suggest that the chronic culture of the rat ß-cell line resulted in partial DNA demethylation of the MafB promoter, which may have a role in MafB promoter activation and possible dedifferentiation in our compromised ß-cell model.

KIF19A is a microtubule-depolymerizing kinesin for ciliary length control.

Cilia control homeostasis of the mammalian body by generating fluid flow. It has long been assumed that ciliary length-control mechanisms are essential for proper flow generation, because fluid flow generation is a function of ciliary length. However, the molecular mechanisms of ciliary length control in mammals remain elusive. Here, we suggest that KIF19A, a member of the kinesin superfamily, regulates ciliary length by depolymerizing microtubules at the tips of cilia. Kif19a(-/-) mice displayed hydrocephalus and female infertility phenotypes due to abnormally elongated cilia that cannot generate proper fluid flow. KIF19A localized to cilia tips, and recombinant KIF19A controlled the length of microtubules polymerized from axonemes in vitro. KIF19A had ATP-dependent microtubule-depolymerizing activity mainly at the plus end of microtubules. Our results indicated a molecular mechanism of ciliary length regulation in mammals, which plays an important role in the maintenance of the mammalian body.

Kinesin superfamily proteins (KIFs) in the mouse transcriptome.

In the post genomic era where virtually all the genes and the proteins are known, an important task is to provide a comprehensive analysis of the expression of important classes of genes, such as those that are required for intracellular transport. We report the comprehensive analysis of the Kinesin Superfamily, which is the first and only large protein family whose constituents have been completely identified and confirmed in silico and at the cDNA, mRNA level. In FANTOM2, we have found 90 clones from 33 Kinesin Superfamily Protein (KIF) gene loci. The clones were analyzed in reference to sequence state, library of origin, detection methods, and alternative splicing. More than half of the representative transcriptional units (TU) were full length. The FANTOM2 library also contains novel splice variants previously unreported. We have compared and evaluated various protein classification tools and protein search methods using this data set. This report provides a foundation for future research of the intracellular transport along microtubules and proves the significance of intracellular transport protein transcripts as part of the transcriptome.

Analysis of the mouse transcriptome for genes involved in the function of the nervous system.

We analyzed the mouse Representative Transcript and Protein Set for molecules involved in brain function. We found full-length cDNAs of many known brain genes and discovered new members of known brain gene families, including Family 3 G-protein coupled receptors, voltage-gated channels, and connexins. We also identified previously unknown candidates for secreted neuroactive molecules. The existence of a large number of unique brain ESTs suggests an additional molecular complexity that remains to be explored.A list of genes containing CAG stretches in the coding region represents a first step in the potential identification of candidates for hereditary neurological disorders.