Abstract
The number of neurotransmitter receptors on the plasma membrane is regulated by the traffic of Âintracellular vesicles. Golgi-derived vesicles provide newly synthesized receptors to the cell surface, whereas clathrin-coated vesicles are the initial vehicles for sequestration of surface receptors, which are ultimately degraded or recycled. Here we use confocal laser scanning microscopy and multiple immunofluorescence analysis to study constitutive GABAB receptor internalization and intracellular trafficking in the single-celled organism Paramecium primaurelia. GABAB receptors display a dotted vesicular pattern dispersed on the cell surface and throughout the cytoplasm and are internalized via clathrin-dependent and -independent endocytosis. Indeed, GABAB receptors colocalize with the adaptin complex AP2, which is implicated in the selective recruitment of integral membrane proteins to clathrin-coated vesicles, and with caveolin 1, which is associated with uncoated membrane invaginations. After internalization, receptors are targeted to the early endosomes, characterized by the molecular markers EEA1 and rab5. Some of these receptors, addressed to recycling back to the plasma membrane, move from the early endosomes to the endosomal recycling compartment that is characterized by the presence of rab4 immunoreactivity. Receptors that are addressed to degradation exit the endosomal pathway at the early endosomes and move to the late endosome–lysosome pathway. In fact, some of the GABAB-positive compartments were identified as lysosomal structures by double staining with the lysosomal marker LAMP1. GABAB vesicle structures also colocalize with TGN38- and rab11-immunoreactivity. TGN38 and rab11 proteins are associated with post-Golgi and recycling endosomes, respectively.
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Ramoino, P., Bianchini, P., Diaspro, A., Usai, C. (2012). Visualizing GABAB Receptor Internalization and Intracellular Trafficking. In: Badoer, E. (eds) Visualization Techniques. Neuromethods, vol 70. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-897-9_4
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DOI: https://doi.org/10.1007/978-1-61779-897-9_4
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