Microtubules and mitochondria nanotunnels

Mitochondrial function strictly depends on mitochondrial structure, location and dynamics. Mitochondria and cytoskeleton interaction is crucial for normal mitochondrial morphology distribution and motility. In cardiac muscle, microtubules sustain muscle contraction and mitochondria disposition; the latter is crucial for localized uptake of calcium (Ca2+) and consequentially energy production needed for cardiac function. Few studies have been conducted on understanding mitochondria and microtubules interaction in cardiomyocytes. In cardiac muscle, recent studies have shown that cardiac mitochondria do not need to migrate to communicate with each other, but they can extend protrusions called nanotunnels to reach and communicate with other mitochondria in long-distance. This process does not involve mitochondria movement, but possibly involves a pulling action along microtubules. The association of mitochondria nanotunnels and microtubules have been identified using 2D and 3D electron microscopy [1]. These new findings highlight the need to investigate the action of microtubules on cardiac mitochondria and its effect on mitochondria dynamics and cardiac function.Sociedad Argentina de Fisiologí

A proposed role for non-junctional transverse tubules in skeletal muscle as flexible segments allowing expansion of the transverse network

Using a variety of technical approaches, we have detected the presence of continuous triads that cover the entire length of T tubules in the main white body muscles of several small fish. This is in contrast to the discontinuous association of SR with T tubules in the red muscles from the same fish as well as in all other previously described muscles in a large variety of skeletal muscles. We suggest that continuous triads are permissible only in muscle fibers that are not normally subject to significant changes in sarcomere length during normal in vivo activity, as is the case for white muscles in the trunk of fish

Endosulfan effects on Rana dalmatina tadpoles: Quantitative developmental and behavioural analysis

Endosulfan is an organochlorine pesticide that was recently labeled as a persistent organic pollutant, but it is still widely employed, particularly in developing countries. The goal of this study is to evaluate the acute (LC50) and chronic effects (developmental and behavioural traits) of this insecticide on Rana dalmatina tadpoles after exposure to ecologically relevant concentrations (0.005, 0.01, and 0.05 mg/L) by applying video-tracking techniques to evaluate the quantitative effect of endosulfan on amphibian behavioural patterns. The 96 h LC50 value was 0.074 mg endosulfan/L. Tadpoles chronically exposed to 0.01 and 0.05 mg endosulfan/L underwent high mortality rate, decreased larval growth, delayed development, and increased incidence of malformations, and they did not reach metamorphosis by the end of the experiment. Moreover, tadpoles exposed to these concentrations exhibited several abnormalities in swimming patterns, such as shorter distance moved, swirling, resting, and unusual use of space. The exposure to 0.005 mg endosulfan/L did not cause any significant effects on behaviour, larval growth, or development, but we observed a significant decrease in both survival and time to metamorphosis. We showed that developmental abnormalities are dose-dependent and that the pesticide effects could differ depending on the endosulfan concentration and the species tested. We also validated the hypothesis that behavioural analysis, along with the use of new analytical methods, could be a useful tool in amphibian ecotoxicological studies

De novo reconstitution reveals the proteins required for skeletal muscle voltage-induced Ca 2+

Skeletal muscle contraction is triggered by Ca2+ release from the sarcoplasmic reticulum (SR) in response to plasma membrane (PM) excitation. In vertebrates, this depends on activation of the RyR1 Ca2+ pore in the SR, under control of conformational changes of CaV1.1, located ∼12 nm away in the PM. Over the last ∼30 y, gene knockouts have revealed that CaV1.1/RyR1 coupling requires additional proteins, but leave open the possibility that currently untested proteins are also necessary. Here, we demonstrate the reconstitution of conformational coupling in tsA201 cells by expression of CaV1.1, β1a, Stac3, RyR1, and junctophilin2. As in muscle, depolarization evokes Ca2+ transients independent of external Ca2+ entry and having amplitude with a saturating dependence on voltage. Moreover, freeze-fracture electron microscopy indicates that the five identified proteins are sufficient to establish physical links between CaV1.1 and RyR1. Thus, these proteins constitute the key elements essential for excitation-contraction coupling in skeletal muscle

Effetti dell’endosulfan sugli Anfibi: uno studio morfologico e comportamentale

Dottorato di Ricerca in Biologia Animale, XIV Ciclo a.a. 2010-2011The declining amphibian population is a global phenomenon in conservation biology that has attracted the attention of many scientists (Russell et al., 1995; Stallard, 2001; Sparling e Fellers, 2009). As for the majority of recent reductions in Earth biodiversity, the amphibian decline can also be attributed to direct human impact on the environment such as the use of pesticides and other chemical pollutants. Therefore, understanding the impacts of agrochemicals on amphibians is a challenging proposition. Amphibians are sensitive to the action of pollutants and they are considered excellent bioindicators of the environmental quality due to their biological, ecological and anatomical characteristics. For these reasons, amphibians are broadly used as typical targets in evaluating the effects of chemicals on aquatic and agricultural ecosystems (Schuytema e Nebeker, 1996; Pollet e Bendell-Young, 2000; Venturino et al., 2003; Bernabò et al., 2008). Decreased species richness, reduced populations, and high deformity incidences have been reported in agroecosystems, which might be linked to the extensive use of pesticides (Berrill et al., 1994, 1997; Bonin et al., 1997; Knutson et al., 2002; Davidson, 2004; Bridges et al., 2004; Relyea, 2005). As outlined by Boone et al. (2005) there is a lack of data for many commonly used pesticides with regard to their effects on amphibians. We chose endosulfan, an organochlorine globally used on a wide variety of crops and other sectors. The environmental persistence and toxicity of endosulfan forced several national authorities to ban it and to propose its inclusion as a persistent organic pollutant in the Stockholm convention (Kelly et al., 2007; EPA, 2010). Endosulfan reaches aquatic systems through direct application, as well as spray drift and runoff from agricultural areas (Leonard et al., 1999, 2000, 2001; Broomhall, 2002; Jergentz et al., 2004) and levels in nature pose a risk to aquatic ecosystems. The last EPA (2010) report on endosulfan, highlights the need for further work to consider amphibians as model species. Previous studies on the effects of endosulfan on amphibians showed detrimental effects on survival, growth, development and metamorphosis, and even caused deformity, severe changes in gill morphology and behavioural alterations (Bernabò et al., 2008; Brunelli et al., 2009, 2010; Jones et al., 2009; Shenoy et al., 2009). Therefore, we proposed to assess the effects of chronic exposure to ecologically relevant concentrations of endosulfan on two anuran species during larval development. In particular, we focused attention on the morphological endpoint evaluating the effects on Bufo bufo skin, to validate the hypothesis of our research group using a morphological approach in a toxicological study. Recently we demonstrated that environmentally relevant endosulfan concentrations (0.1, 0.05 and 0.01 mg/L) may affect Bufo bufo gills morphology and ultrastructure (Brunelli et al., 2010). On this basis we proposed completing the study on endosulfan toxicity in Bufo bufo tadpoles evaluating the effects of the same sublethal concentrations on skin. In fact, the thin and permeable skin of amphibians directly interacts with all environmental constituents, including toxicants. Moreover there is a gap in literature on endosulfan effects on this target organ. We also analyzed the effects of endosulfan on Rana dalmatina tadpoles. Firstly, we examined the short-term toxicity of endosulfan determining the LC50 value and after this preliminary analysis we conducted a classical toxicological study evaluating the effects of sublethal concentrations of endosulfan (0.005, 0.01 and 0.05 mg/L) on survival, growth, development, metamorphosis, deformities presence in Rana dalmatina tadpoles and, in collaboration with the University of Liege we applied new tools for evaluating behavioural patterns. For the first time we used video-tracking analyses to test the quantitative effect of endosulfan on the locomotion. We exposed Bufo bufo tadpoles from Gosner stage 25 until stage 42 to a chronic static renewal system according to the experimental design and conditions of our previous studies (according to Brunelli et al., 2010). and the epidermis was removed after 96 h, 14 and 20 days and analyzed by Trasmission Electron Microscopy (TEM). Exposure to endosulfan caused a general defensive response by mucous secretion and the appearance of large secretory vescicles in Bufo bufo larval epidermis. The exposure to all concentrations, already after 96 h, caused an increase of intercellular spaces, alteration of the cell-cells interactions, cellular degeneration, in particular of the typical larval bufonidae cells (Riesenzellens). The epidermal-dermal junctions and the figures of Eberth in the basal layer, were completely lost at the end of exposure. Our results were successful in showing that environmentally relevant concentrations of endosulfan negatively affected the epidermis of Bufo bufo tadpoles after long-term exposure, resulting in ultrastructure modification in the treated animals compared to controls and that epidermal damage was strongly correlated with exposure time. These results are consistent with our previous findings (Bernabò et al., 2008; Brunelli et al., 2010) concerning the morphological alteration pattern of the gill epithelium during long term exposure. We confirmed that the morphological approach is an useful tool in evaluating the effects of environmental contamination at sublethal levels in sensitive organisms like amphibians, and we supported the role of amphibian skin as a good biomarker following pesticide exposure. Rana dalmatina tadpoles were exposed to several nominal concentrations of endosulfan to assess the sensitivity of the species by estimating the LC50 value at 96 h in a static exposure system. We found that the nominal 96 h LC50 value was 0.074 mg/L. After this preliminary analysis, Rana dalmatina tadpoles were exposed to sublethal concentrations of endosulfan (0.005, 0.01, 0.05 mg/L) in a chronic static renewal system from Gosner stage 25 for the entire course of larval development. Mortality, larval growth (mass), body length, development, time to metamorphosis and deformities presence were monitored regularly over the entire course of larval development. The behaviour (swimming activity) was recorded after 7, 14, 21 and 28 days of exposure and analyzed by a video-tracking software Ehovision 7 XT (Noldus Information Technology, The Netherlands). We analyzed behavioural traits such as: distance moved, velocity, immobility and space use. Our results showed that long-term exposure to environmentally relavant concentrations of endosulfan impaired survival, larval growth, development, metamorphosis, swimming activity and caused severe morphological alteration in tadpoles exposed to 0.01 and 0.05 mg endosulfan/L (medium and high concentrations). With regards to the inhibition of metamorphosis in the highest concentrations, this has a profound implication in the light of amphibian decline. Instead, the lowest concentration of endosulfan (0.005 mg/L) did not cause any significant effects on growth, development and on swimming activity, although we have observed a slightly significant reduction in survival without decreasing the success of metamorphosis. In natural environments, increased incidences of deformities, a reduced larval growth could increase susceptibility to predation (Rohr et al., 2003), delay metamorphosis or result in metamorphosis at a smaller size having negative effects on future fitness, growth and overwinter survival (Brodie and Formanowicz, 1983; Semlitsch et al., 1988; Smith, 1987; Boone and Semlitsch, 2002; Altwegg and Reyer, 2003; Boone and James, 2003). We used video-tracking analyses for the first time to test the quantitative effects of a pesticide on the behaviour of amphibians. And we observed that behavioural effects occurred from 7 days of exposure and tadpoles exposed to the medium and high concentrations of endosulfan exhibited several anomalies in swimming activity such as: shorter distance moved, slower speed, swirling, resting and different space use. Our findings are in agreement with the previous studies of our research group on endosulfan effects on Bufo bufo (Brunelli et al., 2009) and, with the use of video-tracking analysis, we had advantages over standard visual methods. In fact we precisely quantified swimming activity anomalies. We demonstrated that quantitative ecotoxicology is thus a valuable tool to assess conservation concerns when other techniques cannot detect detrimental effects. Our study thus confirms and extends the results of previous studies on the toxicity of this organochlorine pesticide in showing its possible role in amphibian decline.Università della Calabri