Manon Guille

Regulation of Exocytosis in Chromaffin Cells by Trans-Insertion of Lysophosphatidylcholine and Arachidonic Acid into the Outer Leaflet of the Cell Membrane

Vesicular exocytosis is an important complex process in the communication between cells in organisms. It controls the release of chemical and biochemical messengers stored in an emitting cell. In this report, exocytosis is studied amperometrically (at carbon fiber ultramicroelectrodes) at adrenal chromaffin cells, which release catecholamines after appropriate stimulation, while testing the effects due to trans‐insertion of two exogenous compounds (lysophosphatidylcholine (LPC) and arachidonic acid (AA)) on the kinetics of exocytotic events. Amperometric analyses showed that, under the present conditions (short incubation times and micromolar LPC or AA solutions), LPC favors catecholamine release (rate, event frequency, charge released) while AA disfavors the exocytotic processes. The observed kinetic features are rationalized quantitatively by considering a stalk model, for the fusion pore formation, and the physical constraints applied to the cell membrane by the presence of small fractions of LPC and AA diluted in its external leaflet (trans‐insertion). We also observed that the detected amount of neurotransmitters in the presence of LPC was larger than under control conditions, while the opposite trend is observed with AA.

Coupling Amperometry and Total Internal Reflection Fluorescence Microscopy at ITO Surfaces for Monitoring Exocytosis of Single Vesicles

Water-soluble hormones and neurotransmitters are packaged in secretory vesicles and secreted into the extracellular medium by exocytosis, a process involving the fusion of the vesicle membrane with the cell membrane. Transport of the secretory vesicles to the cell s periphery, the maturation stages they undergo there to acquire fusion competence, and the factors controlling the fusion process itself (including the dynamics of the fusion pore) are important biological questions that are not fully understood. To elucidate secretory mechanisms at the single-vesicle level, currently only a few analytical methods exist, which can be grouped into electrical or optical recordings. The great advantage of electrical recordings (patch–clamp membrane capacitance and electrochemical amperometry) is their excellent time resolution (ca. tens of microseconds), which allows studies of the dynamics of the fusion pore itself. However, a major disadvantage is the fact that signals appear only after fusion has commenced; that is, the dynamics of the secretory vesicle itself or any labeled regulatory protein prior to the fusion event cannot be detected. In contrast, optical recordings allow secretory vesicles or regulatory proteins to be visualized and tracked prior to their fusion, yet generally they lack the time resolution required to follow the dynamics of the fusion pore (typical time resolution is ca. 100 ms). In addition, depending on the technique, secretion may be probed from different areas of a cell (top or bottom), which makes comparison of the results obtained by different approaches difficult. Because of their complementary nature, it would be a great advance if electrical and optical measurements could be made simultaneously from the same side of a cell at the singlevesicle level. This will enable a comprehensive and precise analysis of the whole exocytotic event, from predocking through fusion steps up to the dynamics of vesicular release. Herein, we report a device based on transparent indium tin oxide (ITO) electrodes, which allows simultaneous total internal reflection fluorescence microscopy (TIRFM) and amperometric measurements (Figure 1). As a proof of concept, the ability of our device in the coupled optical and electrochemical detections of exocytotic events is demonstrated using enterochromaffin BON cells. Amperometry is based on detection at a microelectrode surface positioned near the emitting cell of electroactive vesicular contents that are released into the extracellular medium. With very high temporal resolution and sensitivity, the flux of the vesicular content (released through an initial fusion pore that is only a few nanometers wide) corresponding to an exocytotic event appears as a current spike, which features (frequency, time length, area, magnitude) the dynamics of release from single vesicles. Generally, amperometry involves placing a large collecting electrode near the investigated cell. The whole cell active surface area is covered so the spatial localization of a particular exocytotic event cannot be achieved. Nevertheless, a few studies involving smaller microelectrodes or microelectrode arrays allowed amperometric signals from different releasing sites to be identified, but with a random positioning for the small microelectrode and a spatial resolution necessarily limited by the array dimensions, respectively. Coupling of amperometric and optical recordings would allow precise localization of exocytosis events in space and time. The most widely used optical approach to study exocytosis, TIRFM, is based on the total internal reflection of a laser beam at the glass/water interface, which creates an evanescent field in the aqueous medium whose characteristic decay length (ca. 100 nm) provides a high signal-to-noise ratio and an axial resolution of about 10 nm. When a vesicle fuses with the plasma membrane, its labeled contents are released toward the glass/water interface where the excitation [*] A. Meunier, Dr. R. Fulcrand, Dr. S. Arbault, Dr. M. Guille, Dr. F. Lema tre, Prof. C. Amatore D partement de Chimie, Ecole Normale Sup rieure UMR 8640 (CNRS-ENS-UPMC Univ Paris 06) 24 rue Lhomond, 75005 Paris (France) Fax: (+33)1-4432-3863 E-mail: Christian.Amatore@ens.fr

Invariance of exocytotic events detected by amperometry as a function of the carbon fiber microelectrode diameter.

Etched carbon fiber microelectrodes of different radii have been used for amperometric measurements of single exocytotic events occurring at adrenal chromaffin cells. Frequency, kinetic, and quantitative information on exocytosis provided by amperometric spikes were analyzed as a function of the surface area of the microelectrodes. Interestingly, the percentage of spikes with foot (as well as their own characteristics), a category revealing the existence of sufficient long-lasting fusion pores, was found to be constant whatever the microelectrode diameter was, whereas the probability of overlapping spikes decreased with the electrode size. This confirmed that the prespike foot could not feature accidental superimposition of separated events occurring at different places. Moreover, the features of amperometric spikes investigated here (charge, intensity and kinetics) were found constant for all microelectrode diameters. This demonstrated that the electrochemical measurement does not introduce significant bias onto the kinetics and thermodynamics of release during individual exocytotic events. All in all, this work evidences that information on exocytosis amperometrically recorded with the usual 7 microm diameter carbon fiber electrodes is biologically relevant, although the frequent overlap between spikes requires a censorship of the data during the analytical treatment.

Prediction of Local pH Variations during Amperometric Monitoring of Vesicular Exocytotic Events at Chromaffin Cells

Electrochemical monitoring of the exocytosis process is generally performed through amperometric oxidation of the electroactive messengers released by single living cells. Herein, we consider the vesicular release of catecholamines by chromaffin cells. Each exocytotic event is thus detected as a current spike whose morphology (intensity, duration, area, etc.) features the efficiency of the secretion process. However, the electrochemical oxidation of catechols produces quinone derivatives and protons. As a consequence, unless specific mechanisms may be adopted by a cell to regulate the pH near its membrane, the local pH between the cell membrane and the electrode necessarily drops within the electrode-cell cleft. Though this consequence of amperometric detection is generally ignored, it has been investigated in this work through simulation of the local pH drop created during the amperometric recording of a sequence of exocytotic events. This was performed based on frequencies and magnitudes of release detected at chromaffin cells. The corresponding acidification was shown to severely depend on the microelectrode radius. For usual 10 μm diameter carbon fiber electrodes, pH values below six were predicted to be reached within the electrode-cell cleft after monitoring a few current spikes.