AP180 Maintains the Distribution of Synaptic and Vesicle Proteins in the Nerve Terminal and Indirectly Regulates the Efficacy of Ca²⁺-Triggered Exocytosis

Abstract

. AP180 maintains the distribution of synaptic and vesicle proteins in the nerve terminal and indirectly regulates the efficacy of Ca 2ϩ -triggered exocytosis. J Neurophysiol 94: 1888 -1903, 2005. First published May 11, 2005 doi:10.1152/jn.00080.2005. AP180 plays an important role in clathrin-mediated endocytosis of synaptic vesicles (SVs) and has also been implicated in retrieving SV proteins. In Drosophila, deletion of its homologue, Like-AP180 (LAP), has been shown to increase the size of SVs but decrease the number of SVs and transmitter release. However, it remains elusive whether a reduction in the total vesicle pool directly affects transmitter release. Further, it is unknown whether the lap mutation also affects vesicle protein retrieval and synaptic protein localization and, if so, how it might affect exocytosis. Using a combination of electrophysiology, optical imaging, electron microscopy, and immunocytochemistry, we have further characterized the lap mutant and hereby show that LAP plays additional roles in maintaining both normal synaptic transmission and protein distribution at synapses. While increasing the rate of spontaneous vesicle fusion, the lap mutation dramatically reduces impulse-evoked transmitter release at steps downstream of calcium entry and vesicle docking. Notably, lap mutations disrupt calcium coupling to exocytosis and reduce calcium cooperativity. These results suggest a primary defect in calcium sensors on the vesicles or on the release machinery. Consistent with this hypothesis, three vesicle proteins critical for calcium-mediated exocytosis, synaptotagmin I, cysteinestring protein, and neuronal synaptobrevin, are all mislocalized to the extrasynaptic axonal regions along with Dap160, an active zone marker (nc82), and glutamate receptors in the mutant. These results suggest that AP180 is required for either recycling vesicle proteins and/or maintaining the distribution of both vesicle and synaptic proteins in the nerve terminal.

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

“…The carboxy-terminal half of the molecules contains a clathrin-binding segment and motifs that bind the AP2 appendages. Many lower organisms have a single AP180/ CALM ortholog, and deletion of these in Drosophila and Caenorhabditis elegans leads to an increase in the size of synaptic vesicles but to a decrease in their number and in transmitter release, causing unregulated movement (Nonet et al 1999;Bao et al 2005). Although overexpression of CALM inhibits clathrin-mediated endocytosis (Tebar et al 1999), knockdown by RNAi in HeLa cells leads to formation of aberrant coated structures: very large "coated pits" with some attached tubular structures, rather than the 1000 Å budding vesicles seen in control cells (Meyerholz et al 2005).…”

Loss of Drosophila melanogaster p21-activated kinase 3 Suppresses Defects in Synapse Structure and Function Caused by spastin Mutations

“…The standard third instar larval body-wall muscle preparation first developed by Jan and Jan (1976) was used for electrophysiological recordings (Zhang et al 1998;Bao et al 2005). Wandering third instar larvae were submerged in ice-cold HL-3 saline (0.8 mM calcium; Stewart et al 1994), incised with a sharp razor blade along the dorsal midline and held open on a magnetic dish with metal pins.…”

The atypical cadherin flamingo regulates synaptogenesis and helps prevent axonal and synaptic degeneration in Drosophila

“…The larva was bathed in HL-3 solution (Stewart et al, 1994) containing 1 mM calcium (Ca 2+ ) for measuring evoked synaptic potentials and 0.1 mM Ca 2+ and 1 ÎM tetrodotoxin for recording spontaneous synaptic potentials (Zhang et al, 1998;Bao et al, 2005). To preserve dorsal muscles for intracellular recordings, the dissection method was slightly modified.…”