Photosynthesis dependent acidification of perialgal vacuoles in theParamedum bursaria/Chlorella symbiosis. Visualization by monensin

After treatment with the carboxylic ionophore monensin theChlorella containing perialgal vacuoles of the greenParamecium bursaria swell. TheParamecium cells remain motile at this concentration for at least one day. The swelling is only observed in illuminated cells and can be inhibited by DCMU. We assume that during photosynthesis the perialgal vacuoles are acidified and that monensin exchanges H+ ions against monovalent cations (here K+). In consequence the osmotic value of the vacuoles increases. The proton gradient is believed to drive the transport of maltose from the symbiont into the host. Another but light independent effect of the monensin treatment is the swelling of peripheral alveoles of the ciliates, likewise indicating that the alveolar membrane contains an active proton pump

A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress

Background: The secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER) stress in real-time based on the naturally secreted Gaussia luciferase (Gluc). Methodology/Principle Findings: An expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP), a well established assay for monitoring of protein processing and ER stress in mammalian cells. Conclusions/Significance: The Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced

Discovery and progress in our understanding of the regulated secretory pathway in neuroendocrine cells

In this review we start with a historical perspective beginning with the early morphological work done almost 50 years ago. The importance of these pioneering studies is underscored by our brief summary of the key questions addressed by subsequent research into the mechanism of secretion. We then highlight important advances in our understanding of the formation and maturation of neuroendocrine secretory granules, first using in vitro reconstitution systems, then most recently biochemical approaches, and finally genetic manipulations in vitro and in vivo