Abnormal Synaptic Vesicle Biogenesis inDrosophila SynaptogyrinMutants

Abstract

Sustained neuronal communication relies on the coordinated activity of multiple proteins that regulate synaptic vesicle biogenesis and cycling within the presynaptic terminal. Synaptogyrin and synaptophysin are conserved MARVEL domain-containing transmembrane proteins that are among the most abundant synaptic vesicle constituents, although their role in the synaptic vesicle cycle has remained elusive. To further investigate the function of these proteins, we generated and characterized a synaptogyrin (gyr) null mutant in Drosophila, whose genome encodes a single synaptogyrin isoform and lacks a synaptophysin homolog. We demonstrate that Drosophila synaptogyrin plays a modulatory role in synaptic vesicle biogenesis at larval neuromuscular junctions. Drosophila lacking synaptogyrin are viable and fertile and have no overt deficits in motor function. However, ultrastructural analysis of gyr larvae revealed increased synaptic vesicle diameter and enhanced variability in the size of synaptic vesicles. In addition, the resolution of endocytic cisternae into synaptic vesicles in response to strong stimulation is defective in gyr mutants. Electrophysiological analysis demonstrated an increase in quantal size and a concomitant decrease in quantal content, suggesting functional consequences for transmission caused by the loss of synaptogyrin. Furthermore, high-frequency stimulation resulted in increased facilitation and a delay in recovery from synaptic depression, indicating that synaptic vesicle exo-endocytosis is abnormally regulated during intense stimulation conditions. These results suggest that synaptogyrin modulates the synaptic vesicle exo-endocytic cycle and is required for the proper biogenesis of synaptic vesicles at nerve terminals.

N-glycosylation requirements in neuromuscular synaptogenesis

“…Preparations were then either processed with detergent [phosphatebuffered saline (PBS) + 1% bovine serum albumin (BSA) + 0.2% Triton X-100] for cell-permeabilized studies, or detergent-free (PBS with 1% BSA) conditions for non-permeabilized studies. Primary antibodies used included: rabbit anti-HRP (1:200, Sigma); mouse anti-Fasciclin 2 [FAS2, 1:10, 1D4, Developmental Studies Hybridoma Bank (DSHB), University of Iowa]; rabbit anti-DG (1:1000, gift from Wu Deng, Department of Biological Sciences, The Florida State University); mouse anti-glutamate receptor IIA [GLURIIA, 1:100, 8B4D2(MH2B), DSHB], rabbit anti-GLURIIB (1:1000) (Marrus et al, 2004), and rabbit anti-GLURIIC (1:500) (Marrus et al, 2004); mouse anti-BRP (1:100, NC82, DSHB); rabbit anti-vesicular glutamate transporter (VGLUT, 1:10,000) (Daniels et al, 2004); rabbit anti-SYB (1:500) (Littleton et al, 1993); mouse anti-Cysteine string protein (CSP, 1:250) (Zinsmaier et al, 1990); rabbit anti-Synaptogyrin (GYR, 1:500) (Stevens et al, 2012); mouse anti-Wingless (WG, 1:2, 4D4, DSHB); rabbit anti-GBB (1:100) , guinea pig anti-JEB (1:2000) (Lee et al, 2003); mouse anti-DLG (1:200, DLG1, DSHB); rabbit anti-L(2)GL (1:300) (Ohshiro et al, 2000). .…”

Drosophila Neuroligin3 Regulates Neuromuscular Junction Development and Synaptic Differentiation

“…Impaired Synaptic Endocytosis in dnlg3 Mutants-Enlarged synaptic vesicles and more cisternaes have been reported in mutants with impaired endocytosis, such as AP180/lap, cyfip, synaptotagmin, synaptogyrin, dap160, tweek, and eps15 mutants (41)(42)(43)(44)(45)(46)(47), suggesting that DNlg3 may regulate endocytosis and/or vesicle recycling. To explore whether endocytosis and/or vesicle recycling is altered in dnlg3 mutants, we turned to the styryl dye FM1-43, which can be taken up in a synaptic vesicle by endocytosis and released by exocytosis.…”

Transmission, Development, and Plasticity of Synapses

“…Changes in Ca 2+ influx following exocytosis may play important trigger roles in helping to initiate the endocytotic process (Yao et al 2009;Haucke et al 2011;Xue et al 2012), which then can proceed through several distinct routes. Loss of the sole Drosophila synaptogyrin/synaptophysin homolog results in a defect in the ability to recycle vesicles from bulk cisternae that form following strong stimulation (Stevens et al 2012). Bulk membrane retrieval is also triggered after acute disruption of dynamin and clathrin (Heerssen et al 2008;Kasprowicz et al 2014).…”