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Fluorescent proteins (FPs) have revolutionized cell biology by allowing genetic tagging of specific proteins inside living cells. In conjunction with Förster's resonance energy transfer (FRET) measurements, FP-tagged proteins can be used to study protein-protein interactions and estimate distances between tagged proteins. FRET is mediated by weak Coulombic dipole-dipole coupling of donor and acceptor fluorophores that behave independently, with energy hopping discretely and incoherently between fluorophores. Stronger dipole-dipole coupling can mediate excitonic coupling in which excitation energy is distributed near instantaneously between coherently interacting excited states that behave as a single quantum entity. The interpretation of FP energy transfer measurements to estimate separation often assumes that donors and acceptors are very weakly coupled and therefore use a FRET mechanism. This assumption is considered reasonable as close fluorophore proximity, typically associated with strong excitonic coupling, is limited by the FP β-barrel structure. Furthermore, physiological temperatures promote rapid vibrational dephasing associated with a rapid decoherence of fluorophore-excited states. Recently, FP dephasing times that are 50 times slower than traditional organic fluorophores have been measured, raising the possibility that evolution has shaped FPs to allow stronger than expected coupling under physiological conditions. In this study, we test if excitonic coupling between FPs is possible at physiological temperatures. FRET and excitonic coupling can be distinguished by monitoring spectral changes associated with fluorophore dimerization. The weak coupling mediating FRET should not cause a change in fluorophore absorption, whereas strong excitonic coupling causes Davydov splitting. Circular dichroism spectroscopy revealed Davydov splitting when the yellow FP VenusA206 dimerizes, and a novel approach combining photon antibunching and fluorescence correlation spectroscopy was used to confirm that the two fluorophores in a VenusA206 homodimer behave as a single-photon emitter. We conclude that excitonic coupling between VenusA206 fluorophores is possible at physiological temperatures.

Cholesterol-specific imeractions that affect membrane fusion were tested for using insect cells; cells that have namrally low cholesterol levels (

The role of the time dependence of calcium delivery in defining the differences in the calcium sensitivity of sea urchin egg secretory vesicles in vitro was studied. The calcium sensitivity of individual secretory vesicles was found to be invariant over a range of calcium delivery rates from faster than micromolar per millisecond to slower than micromolar per second. Any specific calcium concentration above threshold triggers subpopulations of vesicles to fuse, and the size of these subpopulations is independent of the time needed to attain that calcium concentration.

The role of calcium-dependent inactivation in defining the calcium sensitivity of sea urchin secretory vesicle exocytosis in vitro was studied. The cessation of fusion in the continued presence of calcium was not due to calcium-dependent inactivation. Instead, the calcium sensitivity of individual vesicles within a population of exocytotic vesicles is heterogeneous. Any specific calcium concentration above threshold triggered subpopulations of vesicles to fuse and the size of the subpopulations was dependent on the magnitude of the calcium stimulus.

Abstract Background Egress of Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca2+ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca2+ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca2+ in infected erythrocytes during the multistep egress programme of malaria parasites. Methods Live-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca2+ homeostasis on the egress programme. Results A steady increase in cytoplasmic free Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER) Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell. Conclusions The parasite egress programme requires intracellular free Ca2+ for egress initiation, vacuole swelling, and host cell cytoskeleton digestion. The evidence that parasitophorous vacuole swelling, a stage of unaffected egress, is dependent upon a rise in intracellular Ca2+ suggests a mechanism for ionophore-inducible egress and a new target for Ca2+ in the programme liberating parasites from the host cell. A regulatory pathway for egress that depends upon increases in intracellular free Ca2+ is proposed.

Although influenza kills about a half million people each year, even after excluding pandemics, there is only one set of antiviral drugs: neuraminidase inhibitors. By using a new approach utilizing giant unilamellar vesicles and infectious X-31 influenza virus, and testing for the newly identified pore intermediate of membrane fusion, we observed ~30-87% poration, depending upon lipid composition. Testing the hypothesis that spontaneous curvature (SC) of the lipid monolayer controls membrane poration, our Poisson model and Boltzmann energetic considerations suggest a transition from a leaky to a non-leaky fusion pathway depending on the SC of the target membrane. When the target membrane SC is below approximately -0.20 nm-1 fusion between influenza virus and target membrane is predominantly non-leaky while above that fusion is predominantly leaky, suggesting that influenza hemagglutinin (HA)-catalyzed topological conversion of target membranes during fusion is associated with a loss of membrane integrity. [ABSTRACT FROM AUTHOR]

Although the relationship between exocytosis and calcium is fundamental both to synaptic and nonneuronal secretory function, analysis is problematic because of the temporal and spatial properties of calcium, and the fact that vesicle transport, priming, retrieval, and recycling are coupled. By analyzing the kinetics of sea urchin egg secretory vesicle exocytosis in vitro, the final steps of exocytosis are resolved. These steps are modeled as a three-state system: activated, committed, and fused, where interstate transitions are given by the probabilities that an active fusion complex commits ([Alpha]) and that a committed fusion complex results in fusion, p. The number of committed complexes per vesicle docking site is Poisson distributed with mean [bar]n. Experimentally, p and [bar]n increase with increasing calcium, whereas a and the p/[bar]n ratio remain constant, reducing the kinetic description to only one calcium-dependent, controlling variable, [bar]n. On average, the calcium dependence of the maximum rate (R.sub.max) and the time to reach [R.sub.max] ([T.sub.peak]) are described by the calcium dependence of [bar]n. Thus, the nonlinear relationship between the free calcium concentration and the rate of exocytosis can be explained solely by the calcium dependence of the distribution of fusion complexes at vesicle docking sites. KEY WORDS: cytoplasmic vesicles * membrane fusion * sea urchins * secretion * neurosecretion