Miho Shimizu

Chiral blastomere arrangement dictates zygotic left–right asymmetry pathway in snails

Most animals display internal and/or external left–right asymmetry. Several mechanisms for left–right asymmetry determination have been proposed for vertebrates and invertebrates but they are still not well characterized, particularly at the early developmental stage. The gastropods Lymnaea stagnalis and the closely related Lymnaea peregra have both the sinistral (recessive) and the dextral (dominant) snails within a species and the chirality is hereditary, determined by a single locus that functions maternally. Intriguingly, the handedness-determining gene(s) and the mechanisms are not yet identified. Here we show that in L. stagnalis, the chiral blastomere arrangement at the eight-cell stage (but not the two- or four-cell stage) determines the left–right asymmetry throughout the developmental programme, and acts upstream of the Nodal signalling pathway. Thus, we could demonstrate that mechanical micromanipulation of the third cleavage chirality (from the four- to the eight-cell stage) leads to reversal of embryonic handedness. These manipulated embryos grew to ‘dextralized’ sinistral and ‘sinistralized’ dextral snails—that is, normal healthy fertile organisms with all the usual left–right asymmetries reversed to that encoded by the mothers’ genetic information. Moreover, manipulation reversed the embryonic nodal expression patterns. Using backcrossed F7 congenic animals, we could demonstrate a strong genetic linkage between the handedness-determining gene(s) and the chiral cytoskeletal dynamics at the third cleavage that promotes the dominant-type blastomere arrangement. These results establish the crucial importance of the maternally determined blastomere arrangement at the eight-cell stage in dictating zygotic signalling pathways in the organismal chiromorphogenesis. Similar chiral blastomere configuration mechanisms may also operate upstream of the Nodal pathway in left–right patterning of deuterostomes/vertebrates.

Body Handedness Is Directed by Genetically Determined Cytoskeletal Dynamics in the Early Embryo

Although substantial progress has been made recently in understanding the establishment of left-right asymmetry in several organisms, little is known about the initial step for any embryo. In gastropods, left-right body handedness is determined by an unknown maternally inherited single gene or genes at closely linked loci and is associated with the sense of spiral cleavage in early embryos. Contrary to what has been believed, we show that temporal and spatial cytoskeletal dynamics for the left- and right-handed snails within a species are not mirror images of each other. Thus, during the third cleavage of Lymnaea stagnalis, helical spindle inclination (SI) and spiral blastomere deformation (SD) are observed only in the dominant dextral embryos at metaphase-anaphase, whereas in the recessive sinistral embryos, helicity emerges during the furrow ingression. Actin depolymerization agents altered both cleavages to neutral. Further, we found a strong genetic linkage between the handedness-specific cytoskeletal organization and the organismal handedness, using backcrossed F4 congenic animals that inherit only 1/16 of dextral strain-derived genome either with or without the dextrality-determining gene(s). Physa acuta, a sinistral-only gastropod, exhibits substantial SD and SI levotropically. Thus, cytoskeletal dynamics have a crucial role in determination of body handedness with further molecular, cellular, and evolutionary implications.

Hydrolyzed eggshell membrane immobilized on phosphorylcholine polymer supplies extracellular matrix environment for human dermal fibroblasts

We have found that a water-soluble alkaline-digested form of eggshell membrane (ASESM) can provide an extracellular matrix (ECM) environment for human dermal fibroblast cells (HDF) in vitro. Avian eggshell membrane (ESM) has a fibrous-meshwork structure and has long been utilized as a Chinese medicine for recovery from burn injuries and wounds in Asian countries. Therefore, ESM is expected to provide an excellent natural material for biomedical use. However, such applications have been hampered by the insolubility of ESM proteins. We have used a recently developed artificial cell membrane biointerface, 2-methacryloyloxyethyl phosphorylcholine polymer (PMBN) to immobilize ASESM proteins. The surface shows a fibrous structure under the atomic force microscope, and adhesion of HDF to ASESM is ASESM-dose-dependent. Quantitative mRNA analysis has revealed that the expression of type III collagen, matrix metalloproteinase-2, and decorin mRNAs is more than two-fold higher when HDF come into contact with a lower dose ASESM proteins immobilized on PMBN surface. A particle-exclusion assay with fixed erythrocytes has visualized secreted water-binding molecules around the cells. Thus, HDF seems to possess an ECM environment on the newly designed PMBN-ASESM surface, and future applications of the ASESM-PMBN system for biomedical use should be of great interest.

Protective role of the ubiquitin binding protein Tollip against the toxicity of polyglutamine-expansion proteins

Huntington disease (HD) is caused by the expansion of polyglutamine (polyQ) repeats in the amino-terminal of hungtintin (htt). PolyQ-expanded htt forms intracellular ubiquitinated aggregates in neurons and causes neuronal cell death. Here, utilizing a HD cellular model, we report that Tollip, an ubiquitin binding protein that participates in intracellular transport via endosomes, co-localizes with and stimulates aggregation of polyQ-expanded amino-terminal htt. Furthermore, we demonstrate that Tollip protects cells against the toxicity of polyQ-expanded htt. We propose that association of Tollip with polyubiquitin accelerates aggregation of toxic htt species into inclusions and thus provides a cell protective role by sequestration.

Toxicodynamic analysis of cardiac effects induced by four cholinesterase inhibitors in rats.

The cardiac effect of edrophonium (2–20 μmol kg−1), pyridostigmine (0.5‐5 μmol kg−1), neostigmine (0.05‐0.5 μmol kg−1) and ambenonium (0.02‐0.3 μmol kg−1) was investigated after intravenous administration to rats.

Expression of exogenous fluorescent proteins in early freshwater pond snail embryos

We have for the first time succeeded in expressing in vitro-synthesized mRNAs in both the sinistral and the dextral Lymnaea stagnalis early embryos by microinjecting the mRNAs into the eggs before the first polar body stage. Translation of exogenous mRNA in developing embryos was confirmed by expressing various fluorescent proteins; mCherry, DsRed-Express, and enhanced green fluorescent protein. We have found that the protein expression derived from the introduced exogenous mRNA largely depends on the elapsed time after the microinjection and not on the developmental stage of injection, and also on the amount of injected mRNA. Developmental abnormalities were hardly observed. The first notable fluorescent signal was detected within 2–3xa0h after the injection while the embryos were still in uncleaved stage. Fluorescence gradually increased until 8–9xa0h and was stable up to 24xa0h. From these results, it is suggested that there is enough translation machinery necessary for early development and the translation of injected mRNA proceeds immediately and constantly in the early embryos. This is true for both the sinistral and dextral L. stagnalis embryos. Application of the developed method to other freshwater pond snails, dextral Lymnaea peregra, sinistral Physa acuta, and sinistral Indoplanorbis exustus revealed that their early expression mechanisms to be similar to that of L. stagnalis. Thus, in vitro-synthesized mRNA expression is expected to be important for the understanding of evolutional process and the molecular mechanism underlining the handedness determination in these freshwater snail embryos.

Toll-interacting protein pathway: degradation of an ubiquitin-binding protein.

The nine neurodegenerative disorders including Huntington disease (HD) are caused by the expansion of a trinucleotide CAG repeats (polyQ), which are located within the coding of the affected gene. Previous studies suggested that a gain of toxic function by polyQ repeats is widely thought to have a major role in pathogenesis. PolyQ-expanded htt induced ubiquitinated aggregates cause cell death in neuronal cells. Using a HD cellular model, we demonstrate that Tollip protects cells against the toxicity of polyQ-expanded htt and also protects cells from death (Oguro, Kubota, Shimizu, Ishiura, & Atomi, 2011). Tom1 which belongs to the VHS domain-containing protein family is also found to be directly binding to ubiquitin chains and Tollip (Katoh et al., 2004; Yamakami, Yoshimori, & Yokosawa, 2003). Tollip recruits misfolded protein to aggresome via late endosome. The cell system can be used to determine if your protein of interest is controlled under a part of Tollip pathway or not among other cell homeostatic systems: molecular chaperons, autophagy, and endoplasmic reticulum (ER)-associated degradation (ERAD). Tollip can be used for polyQ cell toxicity sensor by detecting microtubule-dependent trafficking and aggresome colocalization of aggregated protein.

Basis of Body-Mind Axis (I): Significance and specialty of trunk (soma) regulation in balance control of the standing human being, and its evaluation of abdomen muscle activities by ultrasound imaging

P3-h07 Basis of Body-Mind Axis (I): Significance and specialty of trunk (soma) regulation in balance control of the standing human being, and its evaluation of abdomen muscle activities by ultrasound imaging Yoriko Atomi 1 , Tomoaki Atomi 2, Noboru Hirose 2, Miho Shimizu 3, Muneko Ishimizu 4 1 Radioisotope Center, The Univ. of Tokyo, Tokyo, Japan 2 Teikyo Sci. Univ., Uenohara, Japan 3 Grad. Sch. of Inf. Technol. Sci., The Univ. of Tokyo, Tokyo, Japan 4 Dept. of Arts and Sci., The Univ. of Tokyo, Tokyo, Japan

Structuring basis of the cytoskeletons is morphologically similar between C6 glioma cells and L6/C2C12 myoblast cells: view from tubulin/microtubule molecular chaperone αB-crystallin function

s / Neuroscience Research 68S (2010) e55–e108 e89 O2-4-1-2 Structuring basis of the cytoskeletons is morphologically similar between C6 glioma cells and L6/C2C12 myoblast cells: view from tubulin/microtubule molecular chaperone B-crystallin function Yoriko Atomi 1 , Eri Fujita 2, Miho Shimizu 2 1 Cell to Body Dynamics Laboratory, Radioisotope Center, The University of Tokyo 2 Grad Sch Inf Sci Tech, The Univ of Tokyo B-crystallin, is highly expressed in oxidative muscle cells compared with glycolytic muscle cells. It is also expressed in glia cells as well as heart cells, where oxidative metabolism is high. In skeletal muscle, structure and function are closely related, thus biochemical properties may be responsible for morphological character. We have previously reported that B-crystallin works as molecular chaperone for tubulin (Ohto et al., 2007) and microtubule (Fujita et al., 2004). When B-crystallin is expressed high quantity in both myoblast C2C12 cells and C6 glioma cells, increased resistance of microtubule against nocodazole treatment and calcium ions are seen. We have analyzed the relationships among three cytoskeleton (e.g. actin filaments, microtubules, and intermediate filaments) in both cell types, and found the morphological similarity between them. Here we show molecular chaperone B-crystallin colocalizes at both microtubule and intermediate filament networks, and also focal adhesion areas of the plus end of actin filaments. Thus B-crystallin seems to be essential for structure-related protein turn-over as well as metabolism in both neuronal and muscle cells keeping metabolism high. References: Eri Ohto-Fujita, Yoshinobu Fujita, and Yoriko Atomi. Cell Stress Chaperones, 12: 163-171 (2007); Yoshinobu Fujita, Eri Ohto, Eisaku Katayama, and Yoriko Atomi. Journal of Cell Science 117, 1719-1726 (2004). doi:10.1016/j.neures.2010.07.157 O2-4-1-3 Brain specific RasGEF, very-KIND in dendrite arborization Kanehiro Hayashi 1 , Asako Furuya 1, Jinhong Huang 1, Manabu Nakayama 2, Teiichi Furuichi 1 1 Lab for Molecular Neurogenesis, RIKEN BSI, Saitama 2 Department of Human Genome Technology, Kazusa DNA Research Institute, Chiba The regulation of cytoskeleton is important for neuronal formation and brain development. One of the regulator, very-KIND is a Ras guanine nucleotide exchange factor (Ras GEF) which contains two kinase non-catalytic C-lobe domains (KINDs) and Ras GEF domain. very-KIND is mainly expressed in cerebellar granule cells and hippocampal neurons and localized on soma and dendrites. However little is known about function of very-KIND in brain. We have found very-KIND plays an essential role on the control of dendrite growth and branching by binding to MAP2 via its KIND2 domain and regulating MAP2 stability through the activation of Ras small GTPase. Loss of very-KIND or introduction of its KIND2 domain induces more complex dendrite arborization whereas overexpression of this protein attenuates dendrite branching in primary hippocampal neurons. We also determine the functional modules on the very-KIND-MAP2 interaction and its function. Furthermore very-KIND KO mouse shows abnormal behavior phenotype and complex dendrite formation in cerebellar granule cells. These findings reveal an important role for very-KIND on dendrite arborization in neuronal development and maintenance. doi:10.1016/j.neures.2010.07.158 O2-4-1-4 Kinesin-1/Hsc70-dependent switching mechanism between slow and fast axonal transport and its relation to optic axonopathy Sumio Terada 1,2 , Masataka Kinjo 3, Makoto Aihara 4, Yosuke Takei 5, Nobutaka Hirokawa 5 1 Sect Neuroanat/Cell Neurobiol & CBIR, Tokyo Med Dent Univ, Tokyo, Japan 2 PRESTO, JST, Kawaguchi, Saitama, Japan 3 Lab Mol Cell Dynamics, Hokkaido Univ, Sapporo, Hokkaido, Japan 4 Dept Ophthal, Univ Tokyo, Tokyo, Japan 5 Dept Cell Biol/Anat, Univ Tokyo, Tokyo, Japan Cytoplasmic protein transport in axons (slow axonal transport) is essential for neuronal homeostasis, and involves Kinesin-1, the same motor for membranous organelle transport (fast axonal transport). However, both molecular mechanisms of slow axonal transport and difference in usage of Kinesin-1 between slow and fast axonal transport have been elusive. Here, we show that slow axonal transport depends on the interaction between the DnaJ-like domain of the kinesin light chain in the Kinesin-1 motor complex and Hsc70, scaffolding between cytoplasmic proteins and Kinesin-1. The domain is within the tetratricopeptide repeat, which can bind to membranous organelles, and competitive perturbation of the domain in squid giant axons disrupted cytoplasmic protein transport and reinforced membranous organelle transport, indicating that this domain might have a function as a switchover system between slow and fast transport by Hsc70. Transgenic mice overexpressing a dominant-negative form of the domain showed delayed slow transport, accelerated fast transport and optic axonopathy. These findings provide a basis for the regulatory mechanism of intracellular transport and its intriguing implication in neuronal dysfunction. doi:10.1016/j.neures.2010.07.159 O2-4-2-1 On the role of SUMOylation in the developing murine forebrain Mikako Sakurai 1 , Ai Yamamoto 2, Ottavio Arancio 1 1 Taub Institute, Columbia University, USA 2 Department of Neurology, Pathology and Cell Biology, Columbia University, USA Post translational modifications control a diverse array of cellular and developmental processes. One such modification, SUMOylation, can impact the half life, the activity and the cellular localization of many proteins; nonetheless, little is understood about the functional significance of SUMOylation in vivo. In this study, we examined the role of SUMOylation by SUMO-1 and SUMO2/3 during embryonic neurogenesis in mice. Western blot analyses between embryonic day (E11) and E16 revealed that levels of high molecular weight (HMW) SUMO-2/3 SUMOylated proteins were dramatically decreased at E16, the peak of neurogenesis. In contrast, little change was seen for SUMO-1 SUMOylated proteins during the same period. qRT-PCR and western blot analyses also revealed that monomeric levels of SUMO-1, SUMO-2 and SUMO-3 do not change, but the SUMO proteases SENP5 and SENP7 increase, suggesting that the decrease of HMW SUMO-2/3 may be due to increased de-SUMOylation of SUMO-2/3-modified proteins. To further understand the relationship between SUMO-2/3 SUMOylation and neurogenesis, we are using LC/MS/MS to identify the HMW proteins whose levels decrease. Moreover, we are performing cell fractionation and other biochemical analyses of embryonic brain to determine the significance of the SUMO-2/3 modification. We hypothesize that identifying the SUMO-2/3 targets and determining how SUMO-2/3 modification impacts their function will better our understanding of how SUMOylation by SUMO-2/3 impacts neurogenesis and proper brain development. doi:10.1016/j.neures.2010.07.160 O2-4-2-2 A growth factor signaling involving neurogenesis in the developing zebrafish tectum Tomomi Sato , Kazuya Sakaguchi, Ryoma Tanigome, Fuminori Sato, Tomohiro Kurisaki, Atsuko Sehara Department of Growth Regulation, Institute for Frontier Medical Sciences, Kyoto University In the developing brain, neurons are derived form neuroepithelial cells located in the ventricular zone (VZ). These cells self-renew and differentiate to generate neural progenitors that give rise to neurons, and then, produce glial cells. However, precise molecular mechanisms to regulate self-renewal and differentiation of those neural progenitors remain to be elucidated. Here we show a growth factor signaling that is involved in differentiation but not in self-renewal of neural progenitors in the VZ of the zebrafish tectum. Treatment of Tg(brn3a-hsp70:GFP) embryos with an kinase inhibitor of the growth factor receptors ErbBs showed an enlarged ventricle and absence of GFP-expressing neurons in the tectum without any abnormalities in gross morphology. This suggests that the growth factor signaling via ErbB receptors is involved in the generation of postmitotic neurons in the tectum. Photoconversion of Kaede expressed in Tg(huc:kaede) embryos showed suppression of the generation of neurons in the tectum of the treated embryos. Immunohistochemistry with an anti-phospho histone H3 antibody showed that proliferation occurred in the VZ of the treated tectum, suggesting that self-renewal of neural progenitors is unaffected. Expression of tis21, a marker for generating neurons, was absent from the treated tectum, suggesting a defect in differentiation of neural progenitors. By the analysis of expression profiles and the effects of antisense morpholinos against erbBs and their lig-