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Concentration-Dependent Staining of Lactotroph Vesicles by FM 4-64

Matja Stenovec ^*, Igor Poberaj , Marko Kreft ^*  and Robert Zorec ^* 

^* Celica Biomedical Sciences Center, 1000 Ljubljana, Slovenia; Laboratory of Neuroendocrinology-Molecular Cell Physiology, Medical School, University of Ljubljana, 1000 Ljubljana, Slovenia; and Department of Physics, Faculty of Mathematics and Physics, University of Ljubljana, 1001 Ljubljana, Slovenia

Correspondence: Address reprint requests to Robert Zorec, E-mail: robert.zorec{at}mf.uni-lj.si.

Hormones are released from neuroendocrine cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. An elegant way to study this process at the single-vesicle level is to use styryl dyes, which stain not only the membrane, but also the matrix of individual vesicles in some neuroendocrine cells. However, the mechanism by which the vesicle matrix is stained is not completely clear. One possibility is that molecules of the styryl dye in the bath solution dissolve first in the plasma membrane and are then transported into the vesicle by lateral diffusion in the plane of the membrane, and finally the vesicle matrix is stained from the vesicle membrane. On the other hand, these molecules may enter the vesicle lumen and reach the vesicle matrix by permeation through an open aqueous fusion pore. To address these questions, we exposed pituitary lactotrophs to different concentrations of FM 4-64 to monitor the fluorescence increase of single vesicles by confocal microscopy after the stimulation of cells by high K⁺. The results show that the membrane and the vesicle matrix exhibit different concentration-dependent properties: the plasma membrane staining by FM 4-64 has a higher affinity in comparison to the vesicle matrix. Moreover, the kinetics of vesicle loading by FM 4-64 exhibited a concentration-dependent process, which indicates that FM 4-64 molecules stain the vesicle matrix by aqueous permeation through an open fusion pore.

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