Compounds that induce exocytosis in

Compounds that induce exocytosis in cultured BAMB-4 include Ca2+-dependent [14] or Ca2+-independent [15] mechanisms. Ca2+-dependent exocytosis releases soluble enzyme content from lysosomes [16] operating by increasing the cytosolic concentration of Ca2+[13] by recruiting extracellular Ca2+[14], liberating Ca2+ from organelle stores [17] or by increasing cytosolic cAMP concentration [18], [19]. Ca2+-independent exocytosis is up-regulated in cultured cells in response to the induction of endocytosis [20], which can be achieved by increasing protein uptake at the cell surface [20], [21], [22] or by altering the specific lipid composition of the cell surface plasma membrane [15], [23]. Exposure to these compounds in culture is thought to lead to their incorporation into vesicular membranes, thereby changing membrane curvature to a more suitable configuration for vesicle-plasma membrane fusion [15], but may also alter membrane fluidity, ion channel permeability and membrane-bound enzyme activity [23]. The aim of this study was to determine the amount of exocytosis and glycogen release from Pompe skin fibroblasts treated with modulators of both Ca2+-dependent and Ca2+-independent exocytosis. The identification of compounds able to induce efficient glycogen release from Pompe cells may lend support to exocytic induction as an alternative treatment approach for Pompe disease.
Materials and methods
Results and discussion
Funding The authors acknowledge funding from the Masonic Foundation. CT was funded by the NHMRC Dora Lush Scholarship.
Acknowledgements
Introduction
TIRFM In a fluorescence microscope, we see only what is illuminated. Illuminated objects emit fluorescence, and emitted fluorescence is collected by objective lens. So if we want to improve the resolution of the fluorescence microscope, we either have to elaborate the ways of illuminations or collections. A stimulated emission depletion (STED) microscopy is an example of the latter (Willig et al., 2006), and a TIRFM is the former. TIRFM illuminates only a very thin layer by taking advantage of the total internal reflection mechanism. When a light travelling through a high refractive index medium (e.g. glass) encounters one of lower refractive index (e.g. water) with the shallow angle, a thin layer of light called evanescent field is formed at an interface between two mediums. When a beam of light faces the interface with the steep angle, light is refracted and propagates through the interface. But when the angle becomes shallow enough to exceeds a certain angle, the beam of light undergoes total internal reflection. Even under total internal reflection, a part of light penetrates the medium of lower refractive index as a thin layer of evanescent field. TIRFM uses this evanescent field to illuminates fluorescent molecules near the interface. The evanescent wave vanishes exponentially with distance from the interface, and the thickness of evanescent field could reach less than 100 nm. When the cell is adhered tightly to the glass and the light travels up through the glass side, evanescent field is formed at the glass-cell interface, thus TIRFM can excite fluorescent molecules only at the very thin layer beneath the plasma membrane and leaves more remote cytosolic molecules in the dark. The 100 nm thickness of illumination area is about two synaptic vesicles diameters and is comparable to the thickness of ultrathin sections used for electron microscopy. In depth discrimination, this feature gives TIRFM five-to-ten-fold better resolution than confocal microscopy. Besides this depth resolution, minimum exposure of out-of-focus fluorophore to illumination light, and unnecessary of scanning provide TIRFM a less photodamage and a higher time resolution. The another advantage of TIRFM is that it is easy to combine with electrophysiological technique (Zenisek et al., 2000). Through-the-lens TIRFM, which is the widely used configuration, allows free pipette access to the cell from the top, which is out of evanescent field and doesn’t distort the TIRFM imaging. On the other hand, to apply TIRFM the measuring object needs to be located immediately adjacent to the glass-water interface. Therefore, for the live cell imaging using TIRFM, the cells have to be adhered tightly to the coverslip, and the targets of observations are limited to the fluorophores immediately below the plasma membrane. For measurements of the fusion and pre-fusion activities of secretory vesicles, if one can adhere the fusion sites to the coverslip, one can fully utilize the positive sides of the TIRFM.