A pre-embedding immunogold approach for detection of synaptic endocytic proteins in situ

During the past decade, many molecular components of clathrin-mediated endocytosis have been identified and proposed to play various hypothetical roles in the process [Nat. Rev. Neurosci. 1 (2000) 161; Nature 422 (2003) 37]. One limitation to the evaluation of these hypotheses is the efficiency and resolution of immunolocalization protocols currently in use. In order to facilitate the evaluation of these hypotheses and to understand more fully the molecular mechanisms of clathrin-mediated endocytosis, we have developed a protocol allowing enhanced and reliable subcellular immunolocalization of proteins in synaptic endocytic zones in situ. Synapses established by giant reticulospinal axons in lamprey are used as a model system for these experiments. These axons are unbranched and reach up to 80-100 microm in diameter. Synaptic active zones and surrounding endocytic zones are established on the surface of the axonal cylinder. To provide access for antibodies to the sites of synaptic vesicle recycling, axons are lightly fixed and cut along their longitudinal axis. To preserve the ultrastructure of the synaptic endocytic zone, antibodies are applied without the addition of detergents. Opened axons are incubated with primary antibodies, which are detected with secondary antibodies conjugated to gold particles. Specimens are then post-fixed and processed for electron microscopy. This approach allows preservation of the ultrastructure of the endocytic sites during immunolabeling procedures, while simultaneously achieving reliable immunogold detection of proteins on endocytic intermediates. To explore the utility of this approach, we have investigated the localization of a GTPase, dynamin, on clathrin-coated intermediates in the endocytic zone of the lamprey giant synapse. Using the present immunogold protocol, we confirm the presence of dynamin on late stage coated pits [Nature 422 (2003) 37] and also demonstrate that dynamin is recruited to the coat of endocytic intermediates from the very early stages of the clathrin coat formation. Thus, our experiments show that the current pre-embedding immunogold method is a useful experimental tool to study the molecular mechanisms of synaptic vesicle recycling.

The Risk Implications of Globalisation: An Exploratory Analysis of 105 Major Industrial Incidents (1971–2010)

This paper revisits work on the socio-political amplification of risk, which predicts that those living in developing countries are exposed to greater risk than residents of developed nations. This prediction contrasts with the neoliberal expectation that market driven improvements in working conditions within industrialising/developing nations will lead to global convergence of hazard exposure levels. It also contradicts the assumption of risk society theorists that there will be an ubiquitous increase in risk exposure across the globe, which will primarily affect technically more advanced countries. Reviewing qualitative evidence on the impact of structural adjustment reforms in industrialising countries, the export of waste and hazardous waste recycling to these countries and new patterns of domestic industrialisation, the paper suggests that workers in industrialising countries continue to face far greater levels of hazard exposure than those of developed countries. This view is confirmed when a data set including 105 major multi-fatality industrial disasters from 1971 to 2000 is examined. The paper concludes that there is empirical support for the predictions of socio-political amplification of risk theory, which finds clear expression in the data in a consistent pattern of significantly greater fatality rates per industrial incident in industrialising/developing countries.