Abstract
It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2+ sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; however, the precise subcellular localization of Syt VII is still a matter of controversy (plasma membrane versus secretory granules). In this study we established a PC12 cell line "stably expressing" the Syt VII-GFP molecule and demonstrated by immunocytochemical and immunoelectron microscopic analyses that the Syt VII-GFP protein is localized on dense-core vesicles as well as in other intracellular membranous structures, such as the trans-Golgi network and lysosomes. Syt VII-GFP forms a complex with endogenous Syts I and IX, but not with Syt IV, and it colocalize well with Syts I and IX in the cellular processes (where dense-core vesicles are accumulated) in the PC12 cell line. We further demonstrated by an N-terminal antibody-uptake experiment that Syt VII-GFP-containing dense-core vesicles undergo Ca2+ -dependent exocytosis, the same as endogenous Syt IX-containing vesicles. Moreover, silencing of Syt VII-GFP with specific small interfering RNA dramatically reduced high KCl-dependent neuropeptide Y secretion from the stable PC12 cell line (approximately 60% of the control cells), whereas the same small interfering RNA had little effect on neuropeptide Y secretion from the wild-type PC12 cells (approximately 85-90% of the control cells), indicating that the level of endogenous expression of Syt VII molecules must be low. Our results indicate that the targeting of Syt VII-GFP molecules to specific membrane compartment(s) is affected by the transfection method (transient expression versus stable expression) and suggested that Syt VII molecule on dense-core vesicles functions as a vesicular Ca2+ sensor for exocytosis in endocrine cells.
Highlights
It has recently been proposed that synaptotagmin (Syt) VII functions as a plasma membrane Ca2؉ sensor for dense-core vesicle exocytosis in PC12 cells based on the results of transient overexpression studies using green fluorescent protein (GFP)-tagged Syt VII; the precise subcellular localization of Syt VII is still a matter of controversy
Synaptotagmin VII-GFP in PC12 cells Is Predominantly Localized on the Dense-core Vesicles— several independent groups have recently proposed that Syt VII regulates Ca2ϩdependent vesicle exocytosis, its precise subcellular localization is a matter of controversy [32, 36, 38, 40, 41, 43]
Because phospholipid binding to the C2 domains of Syt VII is activated by lower concentrations of Ca2ϩ (EC50 value ϭ 1–2 M Ca2ϩ) than is required to activate Syt I, and the recombinant Syt VII transiently overexpressed in PC12 cells is mainly localized at the plasma membrane, it has been proposed that Syt VII functions as a plasma membrane Ca2ϩ sensor for exocytosis [38, 39, 41]
Summary
Syt(s), synaptotagmin(s); GFP, green fluorescence protein; GST, glutathione S-transferase; NPY, neuropeptide Y; RNAi, double-stranded RNA-mediated interference; siRNA, small interfering RNA; TGN, trans-Golgi network; SNARE, soluble NSF attachment protein receptors. Sudhof and colleagues [38, 43] have reported the finding that transient overexpressed Syt VII molecules with a C-terminal green fluorescence protein (GFP) tag were targeted to the plasma membrane in PC12 cells (or non-neuronal cells) and not endocytosed from the membrane, unlike endogenous Syt I. They showed that Syt VII is abundantly and endogenously expressed in PC12 cells [43]. We propose that the Syt VII molecule is mainly targeted to dense-core vesicles and functions as a “vesicular Ca2ϩ sensor” in endocrine cells
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