Abstract
Transmembrane proteins are continuously shuttled from the endosomal compartment to the neuronal plasma membrane by highly regulated and complex trafficking steps. These events are involved in many homeostatic and physiological processes such as neuronal growth, signaling, learning and memory among others. We have previously shown that endosomal exocytosis of the B2 adrenergic receptor (B2AR) and the GluR1-containing AMPA receptor to the neuronal plasma membrane is mediated by two different types of vesicular fusion. A rapid type of exocytosis in which receptors are delivered to the plasma membrane in a single kinetic step, and a persistent mode in which receptors remain clustered at the insertion site for a variable period of time before delivery to the cell surface. Here, by comparing the exocytosis of multiple receptors in dissociated hippocampal and striatal cultures, we show that persistent events are a general mechanism of vesicular delivery. Persistent events were only observed after 10 days in vitro, and their frequency increased with use of the calcium ionophore A23187 and with depolarization induced by KCl. Finally, we determined that vesicles producing persistent events remain at the plasma membrane, closing and reopening their fusion pore for a consecutive release of cargo in a mechanism reminiscent of synaptic kiss-and-run. These results indicate that the delivery of transmembrane receptors to the cell surface can be dynamically regulated by kiss-and-run exocytosis.
Highlights
Cellular sensitivity to external stimuli is tightly controlled by the number and location of transmembrane receptors at the plasma membrane
We used live-cell total internal reflection fluorescence (TIRF) microscopy to investigate the dynamics of receptor exocytosis in neuronal cultures
Hippocampal cultures transfected with transferrin receptor (TfR)-SEP were imaged by TIRF microscopy at 10 Hz in a controlled environment
Summary
Cellular sensitivity to external stimuli is tightly controlled by the number and location of transmembrane receptors at the plasma membrane. Receptors are removed and delivered to the cell surface by endocytosis and exocytosis (Collingridge et al, 2004; von Zastrow and Williams, 2012). Calcium-evoked somatic and dendritic exocytosis (CEDE) was initially described in the 1990’s using time-lapse microscopy and capacitance measurements (Huang and Neher, 1996; MaleticSavatic and Malinow, 1998; Maletic-Savatic et al, 1998). In those studies, release of FM dyes from intracellular stores and changes in capacitance were used to investigate the fundamental mechanism underlying non-synaptic exocytosis from neurons. This work suggested that receptors could be delivered by exocytosis, direct visualization was not described until much later (Kopec et al, 2006; Yudowski et al, 2006; Kennedy and Ehlers, 2011)
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