ABSTRACT
The subunit B of cholera toxin (CTB), which specifically binds with ganglioside GM1 enriched in membrane lipid rafts, is known to interfere with multiple cell functions. However, the specific, stable and spatially defined membrane signaling induced by CTB binding is often difficult to investigate by applying CTB molecules in bulk solution due to quick internalization, elicited intracellular reactions, and homogeneous interaction with the entire cell membrane. Here, we interfaced the neuroendocrine PC12 cells with surface immobilized and patterned CTB molecules, and interrogated the effects of CTB binding on vesicular exocytosis using integrative single-cell study methods. It was discovered that CTB binding facilitates vesicle trafficking, docking and exocytosis in a cholesterol dependent manner. And these effects are probably attributable to the increased membrane GM1 and cholesterol, and enhanced Ca(2+) signaling.
Subject(s)
Cholera Toxin/metabolism , Animals , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cholesterol/metabolism , Exocytosis/physiology , G(M1) Ganglioside/metabolism , Microscopy, Fluorescence , PC12 Cells , Rats , Signal TransductionABSTRACT
Nanocarbon materials, including single-walled carbon nanotubes (SWCNTs) and graphene, promise various novel biomedical applications (e.g., nanoelectronic biosensing). In this Letter, we study the ability of SWCNT networks and reduced graphene oxide (rGO) films in interfacing several types of cells, such as neuroendocrine PC12 cells, oligodendroglia cells, and osteoblasts. It was found that rGO is biocompatible with all these cell types, whereas the SWCNT network is inhibitory to the proliferation, viability, and neuritegenesis of PC12 cells, and the proliferation of osteoblasts. These observations could be attributed to the distinct nanotopographic features of these two kinds of nanocarbon substrates.
Subject(s)
Biocompatible Materials/chemistry , Nanotubes, Carbon/chemistry , Oligodendroglia/cytology , Osteoblasts/cytology , Animals , Biocompatible Materials/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Drug Evaluation, Preclinical , Graphite/chemistry , Humans , Materials Testing , Membranes, Artificial , Neurites/drug effects , PC12 Cells , Particle Size , Rats , Surface PropertiesABSTRACT
Exocytosis of neurotransmitter or hormone-filled vesicles is a highly dynamic process regulated by various proteins and lipids. As mainly revealed indirectly by the electrophysiological methods, exocytosis is believed to involve multiple kinetic steps in which vesicles transit from one state to another. Using total internal reflection fluorescence microscopy which enables direct visualization of individual vesicles, we developed an analytical framework to track and analyze vesicle dynamics. We demonstrated that all subplasmalemmal vesicles generally undergo constant and caged Brownian motion. And they can be classified into three populations that differ in their motion characteristics and fusion competence. Furthermore, we showed that these vesicle pools are differentially modulated by phorbol-12-myristate-13-acetate, a phorbol ester analog to endogenous diacylglycerol, through both protein-kinase-C-dependent and -independent pathways.
