Synaptic Vesicle Size and Number Are Regulated by a Clathrin Adaptor Protein Required for Endocytosis

Abstract

Clathrin-mediated endocytosis is thought to involve the activity of the clathrin adaptor protein AP180. However, the role of this protein in endocytosis in vivo remains unknown. Here, we show that a mutation that eliminates an AP180 homolog (LAP) in Drosophila severely impairs the efficiency of synaptic vesicle endocytosis and alters the normal localization of clathrin in nerve terminals. Most importantly, the size of both synaptic vesicles and quanta is significantly increased in lap mutants. These results provide novel insights into the molecular mechanism of endocytosis and reveal a role for AP180 in regulating vesicle size through a clathrin-dependent reassembly process.

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

“…In w control larvae, the segmental nerve was usually devoid of accumulation of synaptic proteins, such as synaptic vesicle proteins (CSP, Zinsmaier et al, 1994;n-Syb, Wu et al, 1999;and Syt I), the endocytotic and trafficking proteins (clathrin heavy chain, LAP, Zhang et al, 1998;and DAP160;Roos and Kelly, 1999), the active zone (nc82), and the peri-active zone protein Highwire (Hiw; Wan et al, 2000). Examples of axons in the control larvae stained for the vesicle protein Syt I in 1 st instar larvae and the endocytotic protein DAP160 in 3 rd instar larvae are shown in Figure S3B and Figure 6A, respectively.…”

“…Following one hour incubation with a blocker solution (2-5% bovine serum albumin, 0.2% Tween-20 in PBS, PBT), the preparation was stained overnight at 4 ÂC using primary antisera, followed by one hour washes with PBT and 1-2 hours incubation with secondary antibodies at room temperature, and visualized using a Nikon fluorescence microscope. The following primary antibodies were used: rat anti-Syb (1:300, Wu et al, 1999), mAb anti-CSP (ab49, 1:20, Zinsmaier et al, 1994), rabbit anti-Syt (1:300, Mackler et al, 2002), mAb anti-Futsch (22C10, 1:100, Developmental Studies Hybridoma Bank), mAb anti-Hiw (6H4, 1:3, Wan et al, 2000), rabbit anti-DAP 160 (1:300, Roos and Kelly, 1999), mAb nc82 (1:20; Wagh et al, 2005), a polyclonal antibody to clathrin heavy chain (1:50) and to LAP (1:200, Zhang et al, 1998), a polyclonal antibody to glutamate receptor III (Marrus et al, 2004), a polyclonal antibody to the Drosophila vesicular glutamate transporter (VGluT, 1:500, Daniels et al, 2004), and a polyclonal antibody to HRP (Jan and Jan, 1982). Fluorescence-conjugated secondary antibodies were used 1:100 (Jackson Immunology Labs).…”

“…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.…”

“…The Mini Analysis program (Synaptosoft Inc.) was used to measure the amplitude of individual miniature EJPs (mEJPs or minis). mEJPs with a slow rise time arising from neighboring electrically coupled muscle cells were excluded from analysis (Zhang et al, 1998). The unpaired Student's test was used for the data statistics using Origin software (OriginLab).…”

Properties of GST-CALM expressed in E. coli

“…Dynamin is a cytosolic guanosine triphosphatase (GTPase), has a role of pinching off the clathrin coated pit. Amphiphysin has a SH3 domain which binds the GTPase dynamin, the inositol-5-phophatase, synaptojanin-1 and tetrameric clathrin assembly protein AP2 (Zhang et al, 1998;Cestra et al, 1999).…”