A Dual Functional Electroactive and Fluorescent Probe for Coupled Measurements of Vesicular Exocytosis with High Spatial and Temporal Resolution

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A Dual Functional Electroactive and Fluorescent Probe for Coupled Measurements of Vesicular Exocytosis with High Spatial and Temporal Resolution, Angew. Chem. Int. Ed. 2017, 56, 1 – 6

 

Vesicular exocytosis is a ubiquitous process for intercellular communication in living systems. During exocytosis, an intracellular vesicle fuses with the cell membrane and releases its contents to the extracellular space within a brief period of time (milliseconds to seconds), resulting in various physiological responses, such as insertion of receptors at defined areas of the plasma membrane, release of messengers, and removal of waste products from the cell. Owing to its involvement in many normal and pathological events in living cells, exocytosis has attracted increasing research attention.

 

 

In summary, the unique FFN102 probe was demonstrated to show pH-dependent fluorescence and electrochemical properties. Using transparent ITO microelectrodes, exocytotic events of N13 cells stained by FFN 102 were simultaneously observed as fluorescence variations and oxidation current spikes with the TIRFM/amperometry coupled technique. Considering the excellent spatial and temporal resolution of the coupled method, analysis of the amperometric signals as a function of the zone of release will help to investigate the kinetic properties of the release sites and, in particular, the specificities of “hot spots”. Further studies will also examine the specific roles of regulatory proteins involved in the exocytotic process (for example, SNARE proteins and actin). Finally and most importantly, this work offers an innovative idea to develop other probes capable of acting as optical/electrochemical dual reporters in a coupled method, especially those with longer excitation/emission wavelengths, which will greatly contribute to an understanding of various regulating factors of exocytotic secretion.

 

Consultez le communiqué de presse associé à cet article : Suivre la communication entre les cellules !

 

 

Résumé: 

Angew. Chem. Int. Ed. 2017, 56, 1 – 6

 

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 realtime monitoring of single exocytotic events with both high spatial and temporal resolution. This work opens new possibilities in the investigation of exocytotic mechanisms.

 

 

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A Dual Functional Electroactive and Fluorescent Probe for Coupled Measurements of Vesicular Exocytosis with High Spatial and Temporal Resolution

Xiaoqing Liu, Alexandra Savy, Sylvie Maurin, Laurence Grimaud, François Darchen, Damien Quinton, Eric Labbé, Olivier Buriez, Jérôme Delacotte, Frédéric Lemaître, and Manon Guille-Collignon

Angew. Chem. Int. Ed. 2017, 56, 1 – 6

DOI: 10.1002/anie.201611145