FRET-based voltage probes for confocal imaging: membrane potential oscillations throughout pancreatic islets

Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential (V_m) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Förster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from V_m –70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in V_m. When V_m was measured simultaneously with intracellular Ca²⁺, all active cells showed tight coupling of V_m to islet cell Ca²⁺ changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans. pancreatic -cell; optical electrophysiology; islet electrical coupling     

Dynamin regulates focal exocytosis in phagocytosing macrophages

Phagocytosis in macrophages is thought to involve insertion of cytoplasmic vesicles at sites of membrane expansion before particle ingestion ("focal" exocytosis). Capacitance (Cm) measurements of cell surface area were biphasic, with an initial rise indicative of exocytosis followed by a fall upon phagocytosis. Unlike other types of regulated exocytosis, the Cm rise was insensitive to intracellular Ca2+, but was inhibited by guanosine 5'-O-(2-thio)diphosphate. Particle uptake, but not Cm rise, was affected by phosphatidylinositol 3-kinase inhibitors. Inhibition of actin polymerization eliminated the Cm rise, suggesting possible coordination between actin polymerization and focal exocytosis. Introduction of anti-pan-dynamin IgG blocked Cm changes, suggesting that dynamin controls focal exocytosis and thereby phagocytosis. Similarly, recombinant glutathione S-transferase*amphiphysin-SH3 domain, but not a mutated form that cannot bind to dynamin, inhibited both focal exocytosis and phagocytosis. Immunochemical analysis of endogenous dynamin distribution in macrophages revealed a substantial particulate pool, some of which localized to a presumptive endosomal compartment. Expression of enhanced green fluorescent protein*dynamin-2 showed a motile dynamin pool, a fraction of which migrated toward and within the phagosomal cup. These results suggest that dynamin is involved in the production and/or movement of vesicles from an intracellular organelle to the cell surface to support membrane expansion around the engulfed particle.     

Visualizing superoxide production in normal and diabetic rat islets of Langerhans

Oxygen free radicals have been implicated in beta-cell dysfunction and apoptosis associated with type 1 and type 2 diabetes mellitus. The roles of free radicals in diabetes have thus far been defined indirectly by monitoring oxidative tissue damage and the effects of antioxidants, free radical scavengers, and overexpression of superoxide dismutase. We employed the superoxide-mediated oxidation of hydroethidine to ethidium to dynamically and directly assess the relative rates of mitochondrial superoxide anion generation in isolated islets in response to glucose stimulation. Superoxide content of isolated islets increased in response to glucose stimulation. We next compared the oxyradical levels in Zucker lean control and Zucker diabetic fatty rat islets by digital imaging microfluorometry. The superoxide content of Zucker diabetic fatty islets was significantly higher than Zucker lean control islets under resting conditions, relatively insensitive to elevated glucose concentrations, and correlated temporally with a decrease in glucose-induced hyperpolarization of the mitochondrial membrane. Importantly, superoxide levels were elevated in islets from young, pre-diabetic Zucker diabetic fatty animals. Overproduction of superoxide was associated with perturbed mitochondrial morphology and may contribute to abnormal glucose signaling found in the Zucker diabetic fatty model of type 2 diabetes mellitus.     

Ethanol-perturbed amyloidogenic self-assembly of insulin: looking for origins of amyloid strains

A model cosolvent, ethanol, has profound and diversified effects on the amyloidogenic self-assembly of insulin, yielding spectroscopically and morphologically distinguishable forms of beta-aggregates. The alcohol reduces hydrodynamic radii of insulin molecules, decreases enthalpic costs associated with aggregation-prone intermediate states, and accelerates the aggregation itself. Increasing the concentration of the cosolvent promotes curved, amorphous, and finally donut-shaped forms. According to FT-IR data, inter-beta-strand hydrogen bonding is stronger in fibrils formed in the presence of ethanol. Mechanisms underlying the polymorphism of insulin aggregates were investigated by spectroscopic (CD, FT-IR, and fluorescence anisotropy) and calorimetric (DSC and PPC) methods. The nonmonotonic character of the influence of ethanol on insulin aggregation suggests that both preferential exclusion (predominant at the low concentrations) and direct alcohol-protein interactions are involved. The perturbed hydration of aggregation nuclei appears to be a decisive factor in selection of a dominant mode of beta-strand alignment. It may override unfavorable structural consequences of an alternative strand-to-strand stacking, such as strained hydrogen bonding. A hypothetical mechanism of inducing different amyloid "strains" has been put forward. The cooperative character of fibril assembly creates enormous energy barriers for any interstrain transition, which renders the energy landscape comblike-shaped.     

Insulin forms amyloid in a strain-dependent manner: an FT-IR spectroscopic study

The presence of 20% (v/v) ethanol triggers growth of insulin amyloid with distinct infrared spectroscopic features, compared with the fibrils obtained under ambient conditions. Here we report that the two insulin amyloid types behave in the prion strain-like manner regarding seeding specificity and ability of the self-propagating conformational template to overrule unfavorable environmental factors and maintain the initial folding pattern. The type of the original seed has been shown to prevail over cosolvent effects and determines spectral position and width of the amide I' infrared band of the heterogeneously seeded amyloid. These findings imply that "strains" may be a common generic trait of amyloids.     

Phosphorylation Drives a Dynamic Switch in Serine/Arginine-Rich Proteins

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Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region

Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrP^Sc. One key operational parameter used to define differences between strains has been conformational stability of PrP^Sc as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrP^Sc, especially because large strain-specific differences in PrP^Sc stability are often observed despite a similar size of the PrP^Sc core region.     

Current state and perspectives in modeling and control of human pluripotent stem cell expansion processes in stirred‐tank bioreactors

Implementation of model‐based practices for process development, control, automation, standardization, and validation are important factors for therapeutic and industrial applications of human pluripotent stem cells. As robust cultivation strategies for pluripotent stem cell expansion and differentiation have yet to be determined, process development could be enhanced by application of mathematical models and advanced control systems to optimize growth conditions. Therefore, it is important to understand both the potential of possible applications and the apparent limitations of existing mathematical models to improve pluripotent stem cell cultivation technologies. In the present review, the authors focus on these issues as they apply to stem cell expansion processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:355–364, 2017     

The Granular Chloride Channel ClC-3 Is Permissive for Insulin Secretion

Insulin secretion from pancreatic β cells is dependent on maturation and acidification of the secretory granule, processes necessary for prohormone convertase cleavage of proinsulin. Previous studies in isolated β cells revealed that acidification may be dependent on the granule membrane chloride channel ClC-3, in a step permissive for a regulated secretory response. In this study, immuno-EM of β cells revealed colocalization of ClC-3 and insulin on secretory granules. Clcn3^−/− mice as well as isolated islets demonstrate impaired insulin secretion; Clcn3^−/− β cells are defective in regulated insulin exocytosis and granular acidification. Increased amounts of proinsulin were found in the majority of secretory granules in the Clcn3^−/− mice, while in Clcn3^+/+ cells, proinsulin was confined to the immature secretory granules. These results demonstrate that in pancreatic β cells, chloride channels, specifically ClC-3, are localized on insulin granules and play a role in insulin processing as well as insulin secretion through regulation of granular acidification.     

The genetic structure of red raspberry (<Emphasis Type="Italic">Rubus idaeus</Emphasis> L.) populations in Lithuania

The red raspberry (Rubus idaeus L.) is widely distributed in Lithuania and occupies a range of habitats. The presence of coadapted gene pools in local populations of R. idaeus is a question of interest not only to plant scientists, but also to plant breeders. In this study, we investigated the genetic structure of R. idaeus and the influence of local habitats on the genetic diversity within and among populations. Nineteen populations of R. idaeus were sampled from different habitats in various agroclimatic subregions of Lithuania, and analyzed using RAPD markers. 113 RAPD bands were identified among 315 individuals; 84.31% of these were polymorphic. The mean values of Shannon’s information index for different populations ranged from 0.341 to 0.455. Nei’s gene diversity established within populations averaged 0.266. An AMOVA revealed 74% of genetic variation among individuals within populations of R. idaeus, and 23% among populations. The remaining genetic variation was distributed among populations from different agroclimatic subregions (3%). The results of this study suggest that the genetic structure of R. idaeus populations in Lithuania may be influenced partially by isolation by distance as well as by local environmental conditions.