Disrupted Membrane Structure and Intracellular Ca2+ Signaling in Adult Skeletal Muscle with Acute Knockdown of Bin1

Efficient intracellular Ca2+ ([Ca2+]i) homeostasis in skeletal muscle requires intact triad junctional complexes comprised of t-tubule invaginations of plasma membrane and terminal cisternae of sarcoplasmic reticulum. Bin1 consists of a specialized BAR domain that is associated with t-tubule development in skeletal muscle and involved in tethering the dihydropyridine receptors (DHPR) to the t-tubule. Here, we show that Bin1 is important for Ca2+ homeostasis in adult skeletal muscle. Since systemic ablation of Bin1 in mice results in postnatal lethality, in vivo electroporation mediated transfection method was used to deliver RFP-tagged plasmid that produced short –hairpin (sh)RNA targeting Bin1 (shRNA-Bin1) to study the effect of Bin1 knockdown in adult mouse FDB skeletal muscle. Upon confirming the reduction of endogenous Bin1 expression, we showed that shRNA-Bin1 muscle displayed swollen t-tubule structures, indicating that Bin1 is required for the maintenance of intact membrane structure in adult skeletal muscle. Reduced Bin1 expression led to disruption of t-tubule structure that was linked with alterations to intracellular Ca2+ release. Voltage-induced Ca2+ released in isolated single muscle fibers of shRNA-Bin1 showed that both the mean amplitude of Ca2+ current and SR Ca2+ transient were reduced when compared to the shRNA-control, indicating compromised coupling between DHPR and ryanodine receptor 1. The mean frequency of osmotic stress induced Ca2+ sparks was reduced in shRNA-Bin1, indicating compromised DHPR activation. ShRNA-Bin1 fibers also displayed reduced Ca2+ sparks' amplitude that was attributed to decreased total Ca2+ stores in the shRNA-Bin1 fibers. Human mutation of Bin1 is associated with centronuclear myopathy and SH3 domain of Bin1 is important for sarcomeric protein organization in skeletal muscle. Our study showing the importance of Bin1 in the maintenance of intact t-tubule structure and ([Ca2+]i) homeostasis in adult skeletal muscle could provide mechanistic insight on the potential role of Bin1 in skeletal muscle contractility and pathology of myopathy

Une nouvelle maladie virale sur Danae racemosa causée par le virus de la mosaïque du chénopode

Depuis plusieurs années, on observe l'extension importante d'une maladie sur Danae racemosa, liliacée cultivée dans le sud-est de la France pour son feuillage d'ornement. Les symptômes observés sont une marbrure et un jaunissement caractéristiques du feuillage rendant ce dernier impropre à la commercialisation. L'utilisation de techniques virologiques classiques — inoculation d'une gamme d'hôtes, rétro-inoculation systématique, purification, microscopie électronique — ont permis de mettre en évidence la présence régulière d'un virus isométrique (26 nm) à partir des plantes malades de différentes provenances géographiques. À partir de ces premiers résultats, une étude sérologique — immunodiffusion et immunoélectrophorèse en milieu gélosé, technique immunoenzymatique, couplée à une analyse biochimique (détermination des poids moléculaires de la sous-unité capsidiale et de l'acide ribonucléique) — a permis de démontrer qu'il s'agit du virus de la mosaïque du chénopode (Sowbane mosaic virus). L'inoculation du virus sur D racemosa sain a permis de reproduire les symptômes typiques de la maladie et on a réisolé le virus. Le virus de la mosaïque du chénopode est donc le seul agent causal de la marbrure et du jaunissement du feuillage de D racemosa et il est, à notre connaissance, le premier virus signalé sur cette liliacée. Dans la plante malade, le virus est présent à des concentrations très faibles car non détectables par la méthode immunoenzymatique DAS-ELISA et il est nécessaire, pour le mettre en évidence, d'avoir recours à des rétro-inoculations systématiques sur plante hôte sensible (Chenopodium quinoa Wild). La transmission du virus par pucerons a été confirmée (mode non-persistant). Elle doit contribuer, avec la multiplication végétative des plants, à la propagation de la maladie.A new virus disease inducing a mottle and a yellowing of ornamental Danae racemosa foliage caused by a Sowbane Mosaic Virus strain. Since the beginning of the 1980s, a disease has been observed on Danae racemosa Moench (Alexander Laurel), a plant of horticultural interest. In the south of France, as in Italy, D racemosa is commonly cultivated for its ornamental foliage, like Asparagus plumosus L. This monocot from the Liliaceae family shows typical damage on its foliage: first a mottle appears on the young phylloclades which turn yellow with time. This disease is rarely widespread in a plantation. Only single plant or small areas are usually affected but the regular extension of the disease is responsible for important economic consequences. In the present paper we show that these symptoms are due to the propagation of a virus in the plant. The use of general virological tests, host range inoculation and retroinoculation, purification and EM, demonstrated the systematic presence of isometric virus particles (26 nm) in the diseased D racemosa collected from various geographic areas. The study of the serological properties of the virus (immunodiffusion, immunoelectrophoresis and DAS-ELISA) with the MW determination of the capsid-subunit and the RNA, led to its identification as a Sowbane mosaic virus strain. When healthy D racemosa were inoculated with the virus, the typical symptoms of the disease could only be identified after a long delay (9 months after the inoculation) on the young neoformed boughs, from which the virus was isolated. We therefore consider the Sowbane mosaic virus as the single pathogen inducing the mottle and the yellowing of the D racemosa foliage. In the diseased tissues, the virus was present in very low amounts and could not be detected by classical immunoenzymatic assay (DAS-ELISA). Retroinoculation and rare direct inoculations from crude extracts, on a sensitive host (ie Chenopodium quinoa) were necessary to propagate the virus. Disease diagnostic is discussed, especially as the virus seemed to be latent with no external symptoms during a certain growing period of the plant under special conditions (shadowing, high nitrogen fertilization). A rapid and efficient method for detection of the virus is absolutely necessary to carry out a phytosanitary programme and to obtain virus-free plants for vegetative propagation. Evidence was found that the virus could be transmitted by several aphids; this is an important parameter for the control of the disease

A gene coding for a monomeric form of cucumber mosaic virus satellite RNA confers tolerance to CMV

A gene whose transcript bears a monomeric form of cucumber mosaic virus (CMV) satellite RNA was introduced into tobacco (Nicotiana tabacum ‘Xanthi’ nc) plants by using an Ri plasmid-based vector system. On CMV infection, the transcript of the satellite RNA gene was used as a template to yield unit-length satellite RNA, which was efficiently amplified by the virus. Plants bearing the satellite RNA gene displayed long-term tolerance to CMV infection and were also tolerant to CMV infection by aphids, the natural vector of CMV. Implications of these results concerning the mechanism of satellite RNA replication are discussed

Voltage-gated and calcium-gated calcium release during depolarization of skeletal muscle fibers.

The role of elevated intracellular calcium concentration ([Ca2+]) in activating calcium release from the sarcoplasmic reticulum (SR) was studied in skeletal muscle fibers microinjected with strong calcium buffers. After the injection of 3.8 +/- 0.5 mM (mean +/- S.E. of mean, n = 16) BAPTA (1,2-bis[o-aminophenoxy]ethane- N,N,N',N'-tetraacetic acid) or 2.2-2.8 mM fura-2 the normal increase in [Ca2+] during a depolarizing pulse was virtually eliminated. Even though calcium was released from the SR the kinetics of this release were markedly altered: the extensive buffering selectively eliminated the early peak component of SR calcium release with no effect on the maintained steady level. Microinjections of similar volumes but with low concentrations of fura-2 had no significant effect on the release waveform. The calcium released by voltage-dependent activation during depolarization may thus be involved in activating further calcium release, that is, in a calcium-induced calcium release mechanism