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In this work, the release of giant liposome (∼100 μm in diameter) content was imaged by shadow electrochemiluminescence (ECL) microscopy. Giant unilamellar liposomes were pre-loaded with a sucrose solution and allowed to sediment at an ITO electrode surface immersed in a solution containing a luminophore ([Ru(bpy)3]2+) and a sacrificial co-reactant (tri-n-propylamine). Upon polarization, the electrode exhibited illumination over its entire surface thanks to the oxidation of ECL reagents. However, as soon as liposomes reached the electrode surface, dark spots appeared and then spread over time on the surface. This observation reflected a blockage of the electrode surface at the contact point between the liposome and the electrode surface, followed by the dilution of ECL reagents after the rupture of the liposome membrane and release of its internal ECL-inactive solution. Interestingly, ECL reappeared in areas where it initially faded, indicating back-diffusion of ECL reagents towards the previously diluted area and thus confirming liposome permeabilization. The whole process was analyzed qualitatively and quantitatively within the defined region of interest. Two mass transport regimes were identified: a gravity-driven spreading process when the liposome releases its content leading to ECL vanishing and a diffusive regime when ECL recovers. The reported shadow ECL microscopy should find promising applications for the imaging of transient events such as molecular species released by artificial or biological vesicles. [ABSTRACT FROM AUTHOR]

Vesicular exocytosis is an essential biological mechanism used by cellular organisms to release bioactive molecules (hormones, neurotransmitters…) in their environment. For instance, this is the pathway by which chromaffin cells deliver catecholamines (adrenaline, nor-adrenaline, dopamine…) in blood. During this process, secretory vesicles that initially stored the (bio)chemical messengers dock to the cell membrane. The subsequent fusion of vesicle and cell membranes induces the formation of a fusion pore that initiates the first exchanges between the intravesicular and extracellular media. Its following expansion thus favours a larger release of the vesicular content into the external medium. Several analytical methods have been developed in order to study exocytosis at the single living cell level in real time. Among those techniques, mostly based on electric or optic measurements, amperometry with a carbon-fiber ultramicroelectrode [1], used in the first part of this report, and total internal reflection fluorescence microscopy (TIRFM) appear as the most powerful [2] Practically, physico-chemical properties of ultramicroelectrodes induce a high detection sensitivity and temporal resolution, thus being particularly well adapted to monitor exocytosis of electroactive molecules in real time.

The role of condensed counterions in transport properties, such as electrical conductivity and viscosity, has been investigated with solutions of a flexible polyelectrolyte. Comparisons with existing theories are proposed. Viscosity is strongly affected by confinement in thin films, depending whether polyelectrolyte chains are adsorbed or not at the film surfaces. The role of counterion mobility is however difficult to assess because the measurements are not accurate enough. It is proposed that this role could be tested by electrical conductivity measurements. [ABSTRACT FROM AUTHOR]

Intracellular trafficking between organelles is achieved by coat protein complexes, coat protomers, that bud vesicles from bilayer membranes. Lipid droplets are protected by a monolayer and thus seem unsuitable targets for coatomers. Unexpectedly, coat protein complex I (COPI) is required for lipid droplet targeting of some proteins, suggesting a possible direct interaction between COPI and lipid droplets. Here, we find that COPI coat components can bud 60-nm triacylglycerol nanodroplets from artificial lipid droplet (LD) interfaces. This budding decreases phospholipid packing of the monolayer decorating the mother LD. As a result, hydrophobic triacylglycerol molecules become more exposed to the aqueous environment, increasing LD surface tension. In vivo, this surface tension increase may prime lipid droplets for reactions with neighboring proteins or membranes. It provides a mechanism fundamentally different from transport vesicle formation by COPI, likely responsible for the diverse lipid droplet phenotypes associated with depletion of COPI subunits. [ABSTRACT FROM AUTHOR]

We present a large range of experimental data concerningthe influenceof surfactants on the well-known Landau-Levich–Derjaguin experimentwhere a liquid film is generated by pulling a plate out of a bath.The thickness hof the film was measured as a functionof the pulling velocity Vfor different kinds ofsurfactants (C12E6, which is a nonionic surfactant,and DeTAB and DTAB, which are ionic) and at various concentrationsnear and above the critical micellar concentration (cmc). We reportthe thickening factor α = h/hLLD, where hLLDis the filmthickness obtained without a surfactant effect, i.e., as for a purefluid but with the same viscosity and surface tension as the surfactantsolution, over a wide range of capillary numbers (Ca = ηV/γ, with η being the surfactant solution viscosityand γ its surface tension) and identify three regimes: (i) atsmall Ca α is large due to confinement and surface elasticity(or Marangoni) effects, (ii) for increasing Ca there is an intermediateregime where α decreases as Ca increases, and (iii) at larger(but still small) Ca α is slightly higher than unity due tosurface viscosity effects. In the case of nonionic surfactants, thesecond regime begins at a fixed Ca, independent of the surfactantconcentration, while for ionic surfactants the transition dependson the concentration, which we suggest is probably due to the existenceof an electrostatic barrier to surface adsorption. Control of thephysical chemistry at the interface allowed us to elucidate the natureof the three regimes in terms of surface rheological properties. [ABSTRACT FROM AUTHOR]

Strategies to harness photosynthesis from living organisms to generate electrical power have long been considered, yet efficiency remains low. Here, we aimed to reroute photosynthetic electron flow in photosynthetic organisms without compromising their phototrophic properties. We show that 2,6-dimethyl-p-benzoquinone (DMBQ) can be used as an electron mediator to assess the efficiency of mutations designed to engineer a novel electron donation pathway downstream of the primary electron acceptor QA of Photosystem (PS) II in the green alga Chlamydomonas reinhardtii. Through the use of structural prediction studies and a screen of site-directed PSII mutants we show that modifying the environment of the QA site increases the reduction rate of DMBQ. Truncating the C-terminus of the PsbT subunit protruding in the stroma provides evidence that shortening the distance between QA and DMBQ leads to sustained electron transfer to DMBQ, as confirmed by chronoamperometry, consistent with a bypass of the natural QA°− to QB pathway. [ABSTRACT FROM AUTHOR]

Electrochemistry and confocal fluorescence microscopy were successfully combined to selectively bleach and monitor the fluorescence of NBD (7-nitrobenz-2-oxa-1,3-diazole)-labeled phospholipids of giant liposomes. Three types of giant unilamellar vesicles have been investigated, the fluorescent phospholipids being localized either mainly on their outer-, inner-, or both inner/outer leaflets. We established that only the fluorescent lipids incorporated in the outer leaflet of the vesicles underwent electrochemical bleaching upon reduction. The relative fluorescence intensity decay was quantified all along the electrochemical extinction through an original fluorescence loss in electrobleaching (FLIE) assay. As expected, the reorganization of the fluorescent phospholipids followed diffusion-driven dynamics. This was also evidenced by comparison with fluorescence loss in photobleaching (FLIP) and the corresponding numerical model. The value of the lateral diffusion coefficient of phospholipids was found to be similar to that obtained by other methods reported in the literature. This versatile and selective bleaching procedure appears reliable to explore important biological and pharmacological issues., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Strategies to harness photosynthesis from living organisms to generate electrical power have long been considered, yet efficiency remains low. Here, we aimed to reroute photosynthetic electron flow in photosynthetic organisms without compromising their phototrophic properties. We show that 2,6-dimethyl-p-benzoquinone (DMBQ) can be used as an electron mediator to assess the efficiency of mutations designed to engineer a novel electron donation pathway downstream of the primary electron acceptor Q A of Photosystem (PS) II in the green alga Chlamydomonas reinhardtii. Through the use of structural prediction studies and a screen of site-directed PSII mutants we show that modifying the environment of the Q A site increases the reduction rate of DMBQ. Truncating the C-terminus of the PsbT subunit protruding in the stroma provides evidence that shortening the distance between Q A and DMBQ leads to sustained electron transfer to DMBQ, as confirmed by chronoamperometry, consistent with a bypass of the natural Q A ° - to Q B pathway.

In this work, Fluorescent False Neurotransmitter 102 (FFN102), a synthesized analogue of biogenic neurotransmitters, was demonstrated to show both pH-dependent fluorescence and electroactivity. To study secretory behaviors at the single-vesicle level, FFN102 was employed as a new fluorescent/electroactive dual probe in a coupled technique (amperometry and total internal reflection fluorescence microscopy (TIRFM)). We used N13 cells, a stable clone of BON cells, to specifically accumulate FFN102 into their secretory vesicles, and then optical and electrochemical measurements of vesicular exocytosis were experimentally achieved by using indium tin oxide (ITO) transparent electrodes. Upon stimulation, FFN102 started to diffuse out from the acidic intravesicular microenvironment to the neutral extracellular space, leading to fluorescent emissions and to the electrochemical oxidation signals that were simultaneously collected from the ITO electrode surface. The correlation of fluorescence and amperometric signals resulting from the FFN102 probe allows real-time monitoring of single exocytotic events with both high spatial and temporal resolution. This work opens new possibilities in the investigation of exocytotic mechanisms., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)