Article Figures & Data
Figures
- Fig. 1.
A, The protein product (arrow) of the 11–2 cDNA binds to GAP-43 in a solid phase assay. The 30 kDa radiolabeled 11–2 polypeptide (arrow) is precipitated by GST-GAP-43 (lane 2) but not by GST alone (lane 1).B, The 11–2 antibody immunodetects a 115 kDa protein in human fetal brain homogenates (lane 1). Lanes 2 and 3, 11–2 antibody preabsorbed with GST-11–2 or with an unrelated GST fusion protein, respectively;lane 4, preimmune serum. C, GAP-43 (arrow) co-precipitates with rabaptin-5 from rat primary cortical culture homogenates in the presence of calcium (lanes 1–6) but not in the absence of calcium (lanes 7–9). Lanes 1–3, Cytosolic fraction (+1.2 mm CaCl2); lanes 4–6, membrane fraction (+1.2 mm CaCl2);lanes 7–9, membrane fraction (no CaCl2). Lanes 1, 4, and 7, Immunoprecipitation with the 11–2 antibody;lanes 2, 5, and 8, primary antibody omitted from immunoprecipitation; lanes 3,6, and 9, immunoprecipitation with an irrelevant antibody (directed against glutathioneS-transferase). The blot was probed with an anti-GAP-43 antibody. The GAP-43 protein band is indicated with anarrow. The bands above it that are present in all lanes represent nonspecific immunoglobulins. D, Rabaptin-5 mRNA is expressed at highest levels in the brain. The 11–2 cDNA was used as a probe.
- Fig. 2.
A, Biochemical fractionation studies suggest that GAP-43 and rabaptin-5 coexist in certain membranous cellular compartments. See Materials and Methods for description of each fraction. Note that an antibody to the integral synaptic vesicle membrane protein SV2 detects high levels of antigen in the synaptosome fraction (P2-0.8 m), indicating the presence of synaptic vesicles in this fraction. The two bands immunodetected with the antibody to GAP-43 represent human GAP-43 (arrowhead) expressed from the HSV vector and endogenous rat GAP-43 (arrowhead). Because the cultures were infected at a low moi, the human GAP-43 increases the overall expression of GAP-43 by <50%. Primary cultures infected with HSV/Lac vectors alone showed a distribution of endogenous GAP-43 and rabaptin-5 identical to that seen in the cultures infected with HSV/GAP-43 vectors (data not shown). B, Subfraction of rat brain synaptosomes reveals that GAP-43 and rabaptin-5 are present in a nonsynaptic vesicle membrane fraction of the synaptosome. See Materials and Methods for description of each fraction. Note that antibodies to these proteins fail to immunodetect bands in subfraction D, which is enriched for synaptic vesicle proteins as shown by SV2 immunostaining.
- Fig. 3.
GAP-43 and rabaptin-5 immunoreactivity are detected most readily in the neuropil. A, GAP-43 (arrowheads, 10 nm gold particles) and rabaptin-5 (arrows, 20 nm gold particles) are both seen most frequently on endosome-like structures, most often at what appears to be the junctions of these structures with the plasma membrane.B, GAP-43 and rabaptin-5 are also present at the plasma membrane. C, GAP-43 and rabaptin-5 are observed occasionally in association with clusters of synaptic vesicles in axons. Scale bars, 200 nm.
- Fig. 4.
Immunofluorescent co-localization of GAP-43 and rabaptin-5 in noninfected primary neuronal cultures. GAP-43 is represented by red fluorescence in A–E. Rabaptin-5 is represented by green fluorescence inA–D, and Rab5 is represented by greenfluorescence in E. Cells in A,B, and E were pretreated with saponin to enhance visualization of endosomes. A, B, Dual labeling of endosomes with antibodies to GAP-43 and rabaptin-5 was revealed with confocal microscopy. C, Fluorescence intensity profile measured along white line in B. Areas of overlapping as well as nonoverlapping fluorescence are evident.D, Cells not treated with saponin, to preserve neuronal morphology, displayed intense staining of axons with GAP-43 (arrows) and scattered areas of co-localization within cell bodies (arrowheads). E, Saponin pretreatment reveals that GAP-43 co-localizes with Rab5, an endosomal marker. Scale bar: A, B, D, 10 mm; E, 5 mm.
- Fig. 5.
A–C, GAP-43 immunofluorescence is present in soma of noninfected cells, where it is partially co-localized with the Golgi apparatus marker mannosidase II.A, GAP-43 immunofluorescence; B, mannosidase immunofluorescence; C, dual labeling showing superimposition of the images in A and B. The arrow in B points to perinuclear Golgi body staining, also seen as an area of strong overlap inC. D–F, GAP-43 and rabaptin-5 co-localize in neuronal growth cones. GAP-43 is represented bygreen fluorescence and rabaptin-5 by redfluorescence. Arrows indicate heaviest rabaptin-5 immunoreactivity in body of growth cones, proximal to leading edge and filopodia. Scale bar: A–E, 10 μm; F, 5 μm.
- Fig. 6.
Histogram showing that the number of gold particles/ endosome in HSV/GAP-43-infected neurons is greater than it is in HSV/Lac-infected neurons. The difference is significant:p < 0.035 by the one-tailed ttest.
- Fig. 7.
GAP-43 and its mutants exert differential effects on synaptic vesicle recycling as measured with FM1–43. HSV recombinants for each infection are listed on the left. Each column represents a different stimulation paradigm: low K+ followed by low K+, high K+ followed by low K+, and high K+ followed by high K+. Note that each stimulation paradigm for a given recombinant uses a different coverslip of neurons. The CAM mutation deletes the calmodulin binding domain, the G19 mutation changes Ser 41 to a Gly, and the Asp mutation changes Ser 41 to an Asp.
Tables
Mutation Functional consequences G19 Ser 41 → Gly Cannot be phosphorylated by PKC (equivalent to Ser 41 → Ala) Binds calmodulin constitutively Enlarged endosomes Deficient in depolarized-induced FM1-43 fluorescence CAM Delete calmodulin Cannot bind calmodulin binding domain Smaller endosomes Enhancement of depolarization-induced FM1-43 fluorescence Asp Ser 41 → Asp Mimics phosphorylation of Ser 41 by PKC Cannot bind calmodulin Enhancement of depolarization-induced FM1-43 fluorescence Greater depletion of FM1-43 fluorescence after second high-K+treatment
