Publications by authors named "Naoko Kajimura"

23 Publications

  • Page 1 of 1

Functional analysis of Samd11, a retinal photoreceptor PRC1 component, in establishing rod photoreceptor identity.

Sci Rep 2021 Feb 18;11(1):4180. Epub 2021 Feb 18.

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Establishing correct neuronal cell identity is essential to build intricate neural tissue architecture and acquire precise neural function during vertebrate development. While it is known that transcription factors play important roles in retinal cell differentiation, the contribution of epigenetic factors to establishing cell identity during retinal development remains unclear. We previously reported that Samd7, a rod photoreceptor cell-specific sterile alpha motif (SAM) domain protein, functions as a Polycomb repressive complex 1 component (PRC1) that is essential for establishing rod identity. In the current study, we analyzed a functional role of Samd11, another photoreceptor-enriched SAM-domain protein, in photoreceptor differentiation and maturation. We observed that Samd11 interacts with Phc2 and Samd7, suggesting that Samd11 is a component of PRC1 in photoreceptor cells. We generated Samd11-null allele and established Samd7/11 double knock-out (DKO) mouse. The Samd7/11 DKO retina exhibits shortened photoreceptor outer segments by electron microscopy analysis. Microarray analysis revealed that Samd7/11 DKO up-regulated more retinal genes than Samd7 alone, partial functional redundancy of Samd7 and Samd11. Taken together, the current results suggest that Samd7 and Samd11 are PRC1 components and that Samd7 is the major regulator while Samd11 is an accessory factor used for the establishment of precise rod photoreceptor identity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-83781-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892874PMC
February 2021

Sequence- and seed-structure-dependent polymorphic fibrils of alpha-synuclein.

Biochim Biophys Acta Mol Basis Dis 2019 06 18;1865(6):1410-1420. Epub 2019 Feb 18.

Laboratory of Structural Neuropathology, Doshisha University Graduate School of Brain Science, 1-3 Miyakodanitatara, Kyotanabe-shi, Kyoto 610-0394, Japan. Electronic address:

Synucleinopathies comprise a diverse group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. These share a common pathological feature, the deposition of alpha-synuclein (a-syn) in neurons or oligodendroglia. A-syn is highly conserved in vertebrates, but the primary sequence of mouse a-syn differs from that of human at seven positions. However, structural differences of their aggregates remain to be fully characterized. In this study, we found that human and mouse a-syn aggregated in vitro formed morphologically distinct amyloid fibrils exhibiting twisted and straight structures, respectively. Furthermore, we identified different protease-resistant core regions, long and short, in human and mouse a-syn aggregates. Interestingly, among the seven unconserved amino acids, only A53T substitution, one of the familial PD mutations, was responsible for structural conversion to the straight-type. Finally, we checked whether the structural differences are transmissible by seeding and found that human a-syn seeded with A53T aggregates formed straight-type fibrils with short protease-resistant cores. These results suggest that a-syn aggregates form sequence-dependent polymorphic fibrils upon spontaneous aggregation but become seed structure-dependent upon seeding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbadis.2019.02.013DOI Listing
June 2019

Biochemical and morphological classification of disease-associated alpha-synuclein mutants aggregates.

Biochem Biophys Res Commun 2019 01 6;508(3):729-734. Epub 2018 Dec 6.

Laboratory of Structural Neuropathology, Doshisha University Graduate School of Brain Science, 1-3 Miyakodanitatara, Kyotanabe-shi, Kyoto, 610-0394, Japan. Electronic address:

Alpha-synuclein (a-syn) aggregation in brain is implicated in several synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Until date, at least six disease-associated mutations in a-syn (namely A30P, E46K, H50Q, G51D, A53T, and A53E) are known to cause dominantly inherited familial forms of synucleinopathies. Previous studies using recombinant proteins have reported that a subset of disease-associated mutants show higher aggregation propensities and form spectroscopically distinguishable aggregates compared to wild-type (WT). However, morphological and biochemical comparison of the aggregates for all disease-associated a-syn mutants have not yet been performed. In this study, we performed electron microscopic examination, guanidinium hydrochloride (GdnHCl) denaturation, and protease digestion to classify the aggregates from their respective point mutations. Using electron microscopy we observed variations of amyloid fibrillar morphologies among the aggregates of a-syn mutants, mainly categorized into two groups: twisted fibrils observed for both WT and E46K while straight fibrils for the other mutants. GdnHCl denaturation experiments revealed the a-syn mutants except for E46K were more resistant than WT against the denaturation. Mass spectrometry analysis of protease-treated aggregates showed a variety of protease-resistant cores, which may correspond to their morphological properties. The difference of their properties could be implicated in the clinicopathological difference of synucleinopathies with those mutations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2018.11.200DOI Listing
January 2019

Putative Neural Network Within an Olfactory Sensory Unit for Nestmate and Non-nestmate Discrimination in the Japanese Carpenter Ant: The Ultra-structures and Mathematical Simulation.

Front Cell Neurosci 2018 19;12:310. Epub 2018 Sep 19.

Department of Biology, Graduate School of Science, Kobe University, Kobe, Japan.

Ants are known to use a colony-specific blend of cuticular hydrocarbons (CHCs) as a pheromone to discriminate between nestmates and non-nestmates and the CHCs were sensed in the basiconic type of antennal sensilla (). To investigate the functional design of this type of antennal sensilla, we observed the ultra-structures at 2D and 3D in the Japanese carpenter ant, , using a serial block-face scanning electron microscope (SBF-SEM), and conventional and high-voltage transmission electron microscopes. Based on the serial images of 352 cross sections of SBF-SEM, we reconstructed a 3D model of the sensillum revealing that each houses > 100 unbranched dendritic processes, which extend from the same number of olfactory receptor neurons (ORNs). The dendritic processes had characteristic beaded-structures and formed a twisted bundle within the sensillum. At the "beads," the cell membranes of the processes were closely adjacent in the interdigitated profiles, suggesting functional interactions via gap junctions (GJs). Immunohistochemistry with anti-innexin (invertebrate GJ protein) antisera revealed positive labeling in the antennae of . Innexin 3, one of the five antennal innexin subtypes, was detected as a dotted signal within the as a sensory organ for nestmate recognition. These morphological results suggest that ORNs form an electrical network via GJs between dendritic processes. We were unable to functionally certify the electric connections in an olfactory sensory unit comprising such multiple ORNs; however, with the aid of simulation of a mathematical model, we examined the putative function of this novel chemosensory information network, which possibly contributes to the distinct discrimination of colony-specific blends of CHCs or other odor detection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2018.00310DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6157317PMC
September 2018

Lrit1, a Retinal Transmembrane Protein, Regulates Selective Synapse Formation in Cone Photoreceptor Cells and Visual Acuity.

Cell Rep 2018 03;22(13):3548-3561

Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address:

In the vertebrate retina, cone photoreceptors play crucial roles in photopic vision by transmitting light-evoked signals to ON- and/or OFF-bipolar cells. However, the mechanisms underlying selective synapse formation in the cone photoreceptor pathway remain poorly understood. Here, we found that Lrit1, a leucine-rich transmembrane protein, localizes to the photoreceptor synaptic terminal and regulates the synaptic connection between cone photoreceptors and cone ON-bipolar cells. Lrit1-deficient retinas exhibit an aberrant morphology of cone photoreceptor pedicles, as well as an impairment of signal transmission from cone photoreceptors to cone ON-bipolar cells. Furthermore, we demonstrated that Lrit1 interacts with Frmpd2, a photoreceptor scaffold protein, and with mGluR6, an ON-bipolar cell-specific glutamate receptor. Additionally, Lrit1-null mice showed visual acuity impairments in their optokinetic responses. These results suggest that the Frmpd2-Lrit1-mGluR6 axis regulates selective synapse formation in cone photoreceptors and is essential for normal visual function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.03.007DOI Listing
March 2018

The Architecture of the Cytoplasmic Region of Type III Secretion Systems.

Sci Rep 2016 Sep 30;6:33341. Epub 2016 Sep 30.

Schools of Cellular &Molecular Medicine and Biochemistry, Faculty of Biomedical Sciences, University of Bristol, University Walk, United Kingdom.

Type III secretion systems (T3SSs) are essential devices in the virulence of many Gram-negative bacterial pathogens. They mediate injection of protein effectors of virulence from bacteria into eukaryotic host cells to manipulate them during infection. T3SSs involved in virulence (vT3SSs) are evolutionarily related to bacterial flagellar protein export apparatuses (fT3SSs), which are essential for flagellar assembly and cell motility. The structure of the external and transmembrane parts of both fT3SS and vT3SS is increasingly well-defined. However, the arrangement of their cytoplasmic and inner membrane export apparatuses is much less clear. Here we compare the architecture of the cytoplasmic regions of the vT3SSs of Shigella flexneri and the vT3SS and fT3SS of Salmonella enterica serovar Typhimurium at ~5 and ~4 nm resolution using electron cryotomography and subtomogram averaging. We show that the cytoplasmic regions of vT3SSs display conserved six-fold symmetric features including pods, linkers and an ATPase complex, while fT3SSs probably only display six-fold symmetry in their ATPase region. We also identify other morphological differences between vT3SSs and fT3SSs, such as relative disposition of their inner membrane-attached export platform, C-ring/pods and ATPase complex. Finally, using classification, we find that both types of apparatuses can loose elements of their cytoplasmic region, which may therefore be dynamic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5043178PMC
http://dx.doi.org/10.1038/srep33341DOI Listing
September 2016

Loss of ift122, a Retrograde Intraflagellar Transport (IFT) Complex Component, Leads to Slow, Progressive Photoreceptor Degeneration Due to Inefficient Opsin Transport.

J Biol Chem 2016 Nov 28;291(47):24465-24474. Epub 2016 Sep 28.

From the Laboratory for Molecular and Developmental Biology, Institute for Protein Research, Osaka University, and; PRESTO, Japan Science and Technology Agency (JST), 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan,. Electronic address:

In the retina, aberrant opsin transport from cell bodies to outer segments leads to retinal degenerative diseases such as retinitis pigmentosa. Opsin transport is facilitated by the intraflagellar transport (IFT) system that mediates the bidirectional movement of proteins within cilia. In contrast to functions of the anterograde transport executed by IFT complex B (IFT-B), the precise functions of the retrograde transport mediated by IFT complex A (IFT-A) have not been well studied in photoreceptor cilia. Here, we analyzed developing zebrafish larvae carrying a null mutation in ift122 encoding a component of IFT-A. ift122 mutant larvae show unexpectedly mild phenotypes, compared with those of mutants defective in IFT-B. ift122 mutants exhibit a slow onset of progressive photoreceptor degeneration mainly after 7 days post-fertilization. ift122 mutant larvae also develop cystic kidney but not curly body, both of which are typically observed in various ciliary mutants. ift122 mutants display a loss of cilia in the inner ear hair cells and nasal pit epithelia. Loss of ift122 causes disorganization of outer segment discs. Ectopic accumulation of an IFT-B component, ift88, is observed in the ift122 mutant photoreceptor cilia. In addition, pulse-chase experiments using GFP-opsin fusion proteins revealed that ift122 is required for the efficient transport of opsin and the distal elongation of outer segments. These results show that IFT-A is essential for the efficient transport of outer segment proteins, including opsin, and for the survival of retinal photoreceptor cells, rendering the ift122 mutant a unique model for human retinal degenerative diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M116.738658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5114401PMC
November 2016

Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells.

Mol Biol Cell 2016 06 6;27(11):1809-20. Epub 2016 Apr 6.

Graduate School of Life Science, University of Hyogo, Himeji 671-2201, Japan

The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1091/mbc.E15-12-0820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4884071PMC
June 2016

Effects of C-terminal Truncation of Chaperonin GroEL on the Yield of In-cage Folding of the Green Fluorescent Protein.

J Biol Chem 2015 Jun 17;290(24):15042-51. Epub 2015 Apr 17.

From the Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi, Sakyo-ku, Kyoto 606-8501, Japan,

Chaperonin GroEL from Escherichia coli consists of two heptameric rings stacked back-to-back to form a cagelike structure. It assists in the folding of substrate proteins in concert with the co-chaperonin GroES by incorporating them into its large cavity. The mechanism underlying the incorporation of substrate proteins currently remains unclear. The flexible C-terminal residues of GroEL, which are invisible in the x-ray crystal structure, have recently been suggested to play a key role in the efficient encapsulation of substrates. These C-terminal regions have also been suggested to separate the double rings of GroEL at the bottom of the cavity. To elucidate the role of the C-terminal regions of GroEL on the efficient encapsulation of substrate proteins, we herein investigated the effects of C-terminal truncation on GroE-mediated folding using the green fluorescent protein (GFP) as a substrate. We demonstrated that the yield of in-cage folding mediated by a single ring GroEL (SR1) was markedly decreased by truncation, whereas that mediated by a double ring football-shaped complex was not affected. These results suggest that the C-terminal region of GroEL functions as a barrier between rings, preventing the leakage of GFP through the bottom space of the cage. We also found that once GFP folded into its native conformation within the cavity of SR1 it never escaped even in the absence of the C-terminal tails. This suggests that GFP molecules escaped through the pore only when they adopted a denatured conformation. Therefore, the folding and escape of GFP from C-terminally truncated SR1·GroES appeared to be competing with each other.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.633636DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4463448PMC
June 2015

A new mouse allele of glutamate receptor delta 2 with cerebellar atrophy and progressive ataxia.

PLoS One 2014 24;9(9):e107867. Epub 2014 Sep 24.

Department of Anatomy and Neurobiology, Kinki University School of Medicine, Osaka-Sayama, Osaka, Japan; Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), Kawaguchi, Saitama, Japan.

Spinocerebellar degenerations (SCDs) are a large class of sporadic or hereditary neurodegenerative disorders characterized by progressive motion defects and degenerative changes in the cerebellum and other parts of the CNS. Here we report the identification and establishment from a C57BL/6J mouse colony of a novel mouse line developing spontaneous progressive ataxia, which we refer to as ts3. Frequency of the phenotypic expression was consistent with an autosomal recessive Mendelian trait of inheritance, suggesting that a single gene mutation is responsible for the ataxic phenotype of this line. The onset of ataxia was observed at about three weeks of age, which slowly progressed until the hind limbs became entirely paralyzed in many cases. Micro-MRI study revealed significant cerebellar atrophy in all the ataxic mice, although individual variations were observed. Detailed histological analyses demonstrated significant atrophy of the anterior folia with reduced granule cells (GC) and abnormal morphology of cerebellar Purkinje cells (PC). Study by ultra-high voltage electron microscopy (UHVEM) further indicated aberrant morphology of PC dendrites and their spines, suggesting both morphological and functional abnormalities of the PC in the mutants. Immunohistochemical studies also revealed defects in parallel fiber (PF)-PC synapse formation and abnormal distal extension of climbing fibers (CF). Based on the phenotypic similarities of the ts3 mutant with other known ataxic mutants, we performed immunohistological analyses and found that expression levels of two genes and their products, glutamate receptor delta2 (grid2) and its ligand, cerebellin1 (Cbln1), are significantly reduced or undetectable. Finally, we sequenced the candidate genes and detected a large deletion in the coding region of the grid2 gene. Our present study suggests that ts3 is a new allele of the grid2 gene, which causes similar but different phenotypes as compared to other grid2 mutants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0107867PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176021PMC
June 2015

Electron tomographic analysis of cytokinesis in the brown alga Silvetia babingtonii (Fucales, Phaeophyceae).

Protoplasma 2014 Nov 27;251(6):1347-57. Epub 2014 Mar 27.

Muroran Marine Station, Field Science Center for Northern Biosphere, Hokkaido University, Muroran, 051-0013, Japan,

In brown algae, membrane resources for the new cell partition during cytokinesis are mainly flat cisternae (FCs) and Golgi-derived vesicles. We used electron tomography coupled with rapid freezing/freeze substitution of zygotes to clarify the structure of transient membrane compartments during cytokinesis in Silvetia zygotes. After mitosis, an amorphous membranous structure, considered to be an FC intermediate was observed near endoplasmic reticulum clusters, lying between two daughter nuclei. FCs were arrayed at the cytokinetic plane, and a tubular membranous network was formed around them. This network might be formed by the consecutive fusion of spherical vesicles that are linked to the edges of FCs to form a membranous network (MN). At the initial stage of the formation of a membranous sac (MS) from the MN, the MS had flat and swollen parts, with the latter showing membranous tunnels. Coated pits were detected with high frequency at the swollen parts of the MS. This observation indicated that membranous tunnels disappeared by recycling of excess membrane via endocytosis, and the swollen part became flat. The MN appeared at the edges of the growing MS. MN and the MN-MS complex were observed along the cytokinetic plane in several spaces. The MS expanded by the incorporation of MN or other MS in its neighborhood. With the maturation of the new cell partition membrane, the thickness of the MS became constant and the membrane cavity disappeared. The changes in the surface area and volume of the transient membrane compartment during cytokinesis were analyzed from the tomographic data.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00709-014-0635-yDOI Listing
November 2014

A method to achieve homogeneous dispersion of large transmembrane complexes within the holes of carbon films for electron cryomicroscopy.

J Struct Biol 2013 Apr 26;182(1):51-6. Epub 2013 Jan 26.

School of Cellular and Molecular Medicine, Medical Sciences Building, University of Bristol, University Walk BS8 1TD, United Kingdom.

Difficulties associated with using X-ray crystallography for structural studies of large macromolecular complexes have made single particle cryo-electron microscopy (cryoEM) a key technique in structural biology. The efficient application of the single particle cryoEM approach requires the sample to be vitrified within the holes of carbon films, with particles well dispersed throughout the ice and adopting multiple orientations. To achieve this, the carbon support film is first hydrophilised by glow discharge, which allows the sample to spread over the film. Unfortunately, for transmembrane complexes especially, this procedure can result in severe sample adsorption to the carbon support film, reducing the number of particles dispersed in the ice. This problem is rate-limiting in the single particle cryoEM approach and has hindered its widespread application to hydrophobic complexes. We describe a novel grid preparation technique that allows for good particle dispersion in the ice and minimal hydrophobic particle adhesion to the support film. This is achieved by hydrophilisation of the carbon support film by the use of selected detergents that interact with the support so as to achieve a hydrophilic and neutral or selectively charged surface.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsb.2013.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3698441PMC
April 2013

Morphofunctional changes of the astrocyte in rat hippocampus under different corticosteroid conditions.

Med Mol Morphol 2012 Dec 7;45(4):206-13. Epub 2012 Dec 7.

Department of Anatomy and Neurobiology, Nippon Medical School, 1-1-5 Sendagi, Tokyo 113-8602, Japan.

In the present study, we examined the changes in the morphofunction of astrocytes in rat hippocampus under different circulating corticosteroid conditions by immunohistochemistry analysis of glial fibrillary acidic protein (GFAP) and ultra-high-voltage electron microscopy. Each GFAP-immunoreactive cell showed a hypertrophic appearance with well-developed thicker fibrous processes, and the number and the density of GFAP-immunoreactive cells were increased 4 weeks after adrenalectomy, whereas the changes were restored to the sham-control level with corticosterone replacement. The morphometric changes were observed in particular around the pyramidal neurons of CA1 and in the subgranular layer of dentate gyrus. The quantitative analysis clearly showed a significant increase in the number and the density of GFAP-immunoreactive cells in the adrenalectomy group; following corticosterone replacement, these increases were returned to the sham-control level. These changes were also specifically revealed by stereo-observation with ultra-high-voltage electron microscopy. The astrocyte showed more complicated fine three-dimensional branching after adrenalectomy. These results suggested that both the structure and function of astrocytes were modulated by corticosteroids via glucocorticoid receptor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00795-011-0561-4DOI Listing
December 2012

Presynaptic dystroglycan-pikachurin complex regulates the proper synaptic connection between retinal photoreceptor and bipolar cells.

J Neurosci 2012 May;32(18):6126-37

Department of Developmental Biology, Osaka Bioscience Institute, Osaka, 565-0874, Japan.

Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex (DGC) at the neuromuscular junction postsynapse. In the mouse retina, the DGC is localized at the presynapse of photoreceptor cells, however, the function of presynaptic DGC is poorly understood. Here, we developed and analyzed retinal photoreceptor-specific DG conditional knock-out (DG CKO) mice. We found that the DG CKO retina showed a reduced amplitude and a prolonged implicit time of the ERG b-wave. Electron microscopic analysis revealed that bipolar dendrite invagination into the photoreceptor terminus is perturbed in the DG CKO retina. In the DG CKO retina, pikachurin, a DG ligand in the retina, is markedly decreased at photoreceptor synapses. Interestingly, in the Pikachurin(-/-) retina, the DG signal at the ribbon synaptic terminus was severely reduced, suggesting that pikachurin is required for the presynaptic accumulation of DG at the photoreceptor synaptic terminus, and conversely DG is required for pikachurin accumulation. Furthermore, we found that overexpression of pikachurin induces formation and clustering of a DG-pikachurin complex on the cell surface. The Laminin G repeats of pikachurin, which are critical for its oligomerization and interaction with DG, were essential for the clustering of the DG-pikachurin complex as well. These results suggest that oligomerization of pikachurin and its interaction with DG causes DG assembly on the synapse surface of the photoreceptor synaptic terminals. Our results reveal that the presynaptic interaction of pikachurin with DG at photoreceptor terminals is essential for both the formation of proper photoreceptor ribbon synaptic structures and normal retinal electrophysiology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.0322-12.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6622127PMC
May 2012

Ultrastructural study of plasmodesmata in the brown alga Dictyota dichotoma (Dictyotales, Phaeophyceae).

Planta 2012 Oct 1;236(4):1013-26. Epub 2012 May 1.

Graduate School of Environmental Science, Hokkaido University, Sapporo 060-0810, Japan.

Plasmodesmata are intercellular bridges that directly connect the cytoplasm of neighboring cells and play a crucial role in cell-to-cell communication and cell development in multicellular plants. Although brown algae (Phaeophyceae, Heterokontophyta) are phylogenetically distant to land plants, they nevertheless possess a complex multicellular organization that includes plasmodesmata. In this study, the ultrastructure and formation of plasmodesmata in the brown alga Dictyota dichotoma were studied using transmission electron microscopy and electron tomography with rapid freezing and freeze substitution. D. dichotoma possesses plasma membrane-lined, simple plasmodesmata without internal endoplasmic reticulum (desmotubule). This structure differs from those in land plants. Plasmodesmata were clustered in regions with thin cell walls and formed pit fields. Fine proteinaceous "internal bridges" were observed in the cavity. Ultrastructural observations of cytokinesis in D. dichotoma showed that plasmodesmata formation began at an early stage of cell division with the formation of tubular pre-plasmodesmata within membranous sacs of the cytokinetic diaphragm. Clusters of pre-plasmodesmata formed the future pit field. As cytokinesis proceeded, electron-dense material extended from the outer surface of the mid region of the pre-plasmodesmata and finally formed the nascent cell wall. From these results, we suggest that pre-plasmodesmata are associated with cell wall development during cytokinesis in D. dichotoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00425-012-1656-4DOI Listing
October 2012

Negative regulation of ciliary length by ciliary male germ cell-associated kinase (Mak) is required for retinal photoreceptor survival.

Proc Natl Acad Sci U S A 2010 Dec 8;107(52):22671-6. Epub 2010 Dec 8.

Department of Developmental Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka 565-0874, Japan.

Cilia function as cell sensors in many organs, and their disorders are referred to as "ciliopathies." Although ciliary components and transport machinery have been well studied, regulatory mechanisms of ciliary formation and maintenance are poorly understood. Here we show that male germ cell-associated kinase (Mak) regulates retinal photoreceptor ciliary length and subcompartmentalization. Mak was localized both in the connecting cilia and outer-segment axonemes of photoreceptor cells. In the Mak-null retina, photoreceptors exhibit elongated cilia and progressive degeneration. We observed accumulation of intraflagellar transport 88 (IFT88) and IFT57, expansion of kinesin family member 3A (Kif3a), and acetylated α-tubulin signals in the Mak-null photoreceptor cilia. We found abnormal rhodopsin accumulation in the Mak-null photoreceptor cell bodies at postnatal day 14. In addition, overexpression of retinitis pigmentosa 1 (RP1), a microtubule-associated protein localized in outer-segment axonemes, induced ciliary elongation, and Mak coexpression rescued excessive ciliary elongation by RP1. The RP1 N-terminal portion induces ciliary elongation and increased intensity of acetylated α-tubulin labeling in the cells and is phosphorylated by Mak. These results suggest that Mak is essential for the regulation of ciliary length and is required for the long-term survival of photoreceptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1009437108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3012466PMC
December 2010

A method for observing silver-stained osteocytes in situ in 3-microm sections using ultra-high voltage electron microscopy tomography.

Microsc Microanal 2009 Oct 27;15(5):377-83. Epub 2009 Aug 27.

Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan.

Osteocytes are surrounded by hard bone matrix, and it has not been possible previously to directly observe the in situ architecture of osteocyte morphology in bone. Electron microscope tomography, however, is a technique that has the unique potential to provide three-dimensional (3D) visualization of cellular ultrastructure. This approach is based on reconstruction of 3D volumes from a tilt series of electron micrographs of cells, and resolution at the nanometer level has been achieved. We applied electron microscope tomography to thick sections of silver-stained osteocytes in bone using a Hitachi H-3000 ultra-high voltage electron microscope equipped with a 360 degrees tilt specimen holder, at an accelerating voltage of 2 MeV. Osteocytes with numerous processes and branches were clearly seen in the serial tilt series acquired from 3-microm-thick sections. Reconstruction of young osteocytes showed the 3D topographic morphology of the cell body and processes at high resolution. This morphological data on osteocytes should provide useful information to those who study osteocyte physiology and the several models used to explain their mechanosensory properties.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1017/S1431927609990420DOI Listing
October 2009

Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation.

Nat Neurosci 2008 Aug 20;11(8):923-31. Epub 2008 Jul 20.

Department of Developmental Biology, Osaka Bioscience Institute, 6-2-4 Furuedai, Suita, Osaka, 565-0874, Japan.

Exquisitely precise synapse formation is crucial for the mammalian CNS to function correctly. Retinal photoreceptors transfer information to bipolar and horizontal cells at a specialized synapse, the ribbon synapse. We identified pikachurin, an extracellular matrix-like retinal protein, and observed that it localized to the synaptic cleft in the photoreceptor ribbon synapse. Pikachurin null-mutant mice showed improper apposition of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, resulting in alterations in synaptic signal transmission and visual function. Pikachurin colocalized with both dystrophin and dystroglycan at the ribbon synapses. Furthermore, we observed direct biochemical interactions between pikachurin and dystroglycan. Together, our results identify pikachurin as a dystroglycan-interacting protein and demonstrate that it has an essential role in the precise interactions between the photoreceptor ribbon synapse and the bipolar dendrites. This may also advance our understanding of the molecular mechanisms underlying the retinal electrophysiological abnormalities observed in muscular dystrophy patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nn.2160DOI Listing
August 2008

Alteration of the nucleosomal DNA path in the crystal structure of a human nucleosome core particle.

Nucleic Acids Res 2005 10;33(10):3424-34. Epub 2005 Jun 10.

Department of Structural Biology, Biomolecular Engineering Research Institute 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan.

Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 A crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location -5.0 to -2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA-DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn2+ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gki663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1150222PMC
June 2005

Upregulation of ghrelin expression in cachectic nude mice bearing human melanoma cells.

Metabolism 2004 Jan;53(1):84-8

Third Department of Internal Medicine, Miyazaki Medical College, Kiyotake, Japan.

Ghrelin is a gastrointestinal peptide that stimulates food intake and growth hormone (GH) secretion. We studied the biosynthesis and secretion of ghrelin in a cancer cachexia mouse model. G361, a human melanoma cell line, was inoculated into nude mice. The body weight was reduced and the plasma concentration of interleukin-1beta (IL-1beta) was markedly higher in tumor-inoculated mice compared with vehicle-treated mice. Furthermore, white adipose tissue (WAT) weight, blood sugar level, and plasma concentrations of leptin and nonesterified fatty acids (NEFA) were significantly lower in tumor-inoculated mice. The plasma concentration of ghrelin increased with the progression of cachexia. The levels of both ghrelin peptide and mRNA in the stomach were also upregulated in tumor-inoculated mice. This study demonstrates that both ghrelin biosynthesis and secretion are stimulated in the long-term negative energy balance of tumor-inoculated cachectic mice. These findings suggest the involvement of ghrelin in the regulation of energy homeostasis in cancer cachexia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.metabol.2003.06.004DOI Listing
January 2004

Mammalian Mcm2/4/6/7 complex forms a toroidal structure.

Genes Cells 2003 May;8(5):413-21

Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

Background: The Mcm proteins are a family of six homologous proteins (Mcm2-7) that play an important role in DNA replication. They form Mcm4/6/7 and Mcm2/4/6/7 complexes, but their structures are not known.

Results: We found that the human Mcm2/4/6/7 tetramer forms a toroidal structure, with a central cavity about 3-4 nm in diameter. Observations were made using electron microscopy, employing the image analysis of single particles. The most predominant averaged image displayed a toroid harbouring four bulges forming corners, one of which was larger than the others. This structure was very similar to the mouse Mcm2/4/6/7 tetramer that was independently prepared and analysed by electron microscopy. These toroidal structures are distinct from that of the Mcm4/6/7 hexamer, which was also examined by electron microscopy. GST(glutathione S-transferase)-pull down and two hybrid experiments suggest that a putative Mcm6-Mcm6 hinge contributes to the formation of the Mcm7/4/6/6/4/7 heterohexamer.

Conclusions: The Mcm2/4/6/7 tetramer forms a toroidal structure that is distinct from that of the Mcm4/6/7 hexamer in size and shape.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1046/j.1365-2443.2003.00645.xDOI Listing
May 2003

Anti-cachectic effect of ghrelin in nude mice bearing human melanoma cells.

Biochem Biophys Res Commun 2003 Feb;301(2):275-9

Third Department of Internal Medicine, Miyazaki Medical College, Miyazaki 889-1692, Japan.

Ghrelin is a novel brain-gut peptide that stimulates food intake and body weight gain. We studied the anabolic effect of ghrelin in a cancer cachexia mouse model. SEKI, a human melanoma cell line, was inoculated into nude mice to examine the effects of ghrelin on food intake and body weight. The intraperitoneal administration of ghrelin twice a day (6 nmol/mice/day) for 6 days suppressed weight loss in SEKI-inoculated mice and increased the rate of weight gain in vehicle-treated nude mice. Ghrelin administration also increased food intake in both SEKI- and vehicle-treated mice. Both the weight of white adipose tissue and the plasma leptin concentration were reduced in tumor-inoculated mice compared with vehicle-treated mice; these factors increased following ghrelin administration. The levels of both ghrelin peptide and mRNA in the stomach were upregulated in tumor-inoculated mice. The anabolic effect of ghrelin efficiently reverses the cachexia in mice bearing SEKI human melanoma. Ghrelin therefore may have a therapeutic ability to ameliorate cancer cachexia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s0006-291x(02)03028-0DOI Listing
February 2003

Three-dimensional structure of non-activated cGMP phosphodiesterase 6 and comparison of its image with those of activated forms.

J Struct Biol 2002 Jul;139(1):27-38

Biomolecular Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan.

Cyclic GMP phosphodiesterase (PDE6) in rod photoreceptors, a key enzyme in vertebrate phototransduction, consists of two homologous catalytic subunits (Palpha and Pbeta) and two identical regulatory subunits (Pgammas). Pgamma regulates the PDE activity through its direct interaction with transducin. Here, using electron microscopy and image analysis of single particles, we show the three-dimensional organization of the basic form of bovine PDE, Palphabetagammagamma, and compare its average image with those of Pgamma-released PDE. The structure of Palphabetagammagamma appears to be a flattened bell-shape, with dimensions of 150 x 108 x 60A, and with a handle-like protrusion attached to the top of the structure. Except for the protrusion, the organization consists of two homologous structures arranged side by side, with each structure having three distinct regions, showing pseudo twofold symmetry. These characteristics are consistent with a model in which the overall structure of Palphabetagammagamma is determined by hetero-dimerization of Palpha and Pbeta, with each subunit consisting of one catalytic and two GAF regions. A comparison of the average image of Palphabetagammagamma with those of Pgamma-released PDE suggests that Pgamma release does not affect the overall structure of Palphabeta, and that the Palphabeta C-terminus, but not Pgamma, is a determinant for the Palphabeta orientation on carbon-coated grids. These observations suggest that the basic structure of PDE does not change during its regulation, which implies that Palphabeta is regulated by its regional interaction with Pgamma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/s1047-8477(02)00502-6DOI Listing
July 2002