Article Figures & Data
Figures
- Fig. 1.
Saturation binding kinetics of GAP-43 to actin filaments. Actin (15 μm) was incubated in polymerizing buffer for 20 min with (left toright) 1000, 850, 650, 500, 400, 300, 200, and 100 nm GAP-43 before centrifugation. The pellet containing f-actin and bound GAP-43 was quantitated by immunoblotting with either 7B10 (A) or 2G12 (B) mAb followed by 125I secondary antibody, autoradiography, and densitometry. C, Equilibrium binding of total (phosphorylated and unphosphorylated) GAP-43 (▪) and phosphorylated GAP-43 (▴) to actin, calculated from data including that presented inA and B (n = 4 independent experiments). The binding of unphosphorylated GAP-43 to actin (•) was calculated from the difference between total GAP-43 and the phosphorylated form (see Results). D, Scatchard analysis of the binding of phosphorylated (▴) and unphosphorylated (•) GAP-43 to actin. The arrow indicates the break point in the Scatchard plot, indicating two independent binding sites of GAP-43 for actin.
- Fig. 2.
A, Effect of GAP-43 on the critical concentration for actin polymerization. The actin critical concentration was evaluated from the decrease in fluorescence of polymerized pyrenyl-actin (5 μm, 5% labeled) induced by serial dilutions of the samples in polymerization buffer containing 2 mm MgCl2, 100 mm KCl, 15 mm NaCl, and the appropriate concentrations of GAP-43. The decay of f-actin fluorescence as a function of the monomer concentration was fit by least squares linear regression analysis. The intersections between the regression lines and the fluorescence baseline (critical concentration) were: 5 μm actin alone (▪), 231 ± 10 (SD) nm (n = 5); 5 μm actin plus 300 nm phospho-GAP-43 (▴), 212 ± 20 (SD) nm (n = 3); and 5 μm actin plus 300 nm dephospho-GAP-43 (•), 435 ± 6 (SD) nm (n = 3).B, Actin polymer self-assembly in the presence of GAP-43. The self-assembly of pyrenyl-g-actin (5 μm, 5% labeled) was analyzed by measuring the increase in fluorescence related to the g-actin to f-actin transition. Polymerization was triggered at time 0 by the addition of KCl and MgCl2 in the absence (▪) or presence of 0.5 μm phosphorylated GAP-43 (▴) or 0.5 μm dephosphorylated GAP-43 (•).C, Polymerization of actin from f-actin seeds in the presence of GAP-43. The polymerization of pyrenyl-g-actin (1 μm, 5% labeled) in the presence of 0.1 μmf-actin seeds was measured by an increase in fluorescence as before. Polymerization occurred in the absence (▪) or presence of 0.1 μm phosphorylated GAP-43 (▴) or 0.1 μmdephosphorylated GAP-43 (•).
- Fig. 3.
Electron microscopy of negatively stained specimens. Actin filaments were polymerized and then negatively stained on electron microscope grids. A, Actin (10 μm); note the smooth contours of the filament bundles. B, Actin (10 μm) at higher power. C, D, Actin (10 μm) polymerized in the presence of 30 μmphosphorylated GAP-43. Note the long filaments commonly seen in this condition but never when unphosphorylated GAP-43 was present. E, F, Actin (10 μm) polymerized in the presence of 30 μmunphosphorylated GAP-43. Note the short filaments andlarge aggregates seen commonly under this condition but only very rarely when actin is polymerized alone or with phosphorylated GAP-43. A, C, E, Actins were from the same experiment. B, D,F, Actins were from independent experiments. Scale bars, 200 nm (A), 100 nm (C), and 500 nm (D–F).
- Fig. 4.
Length distribution of actin filaments in the presence of GAP-43 and CaM. The lengths of actin filaments polymerized in the presence of GAP-43 and negatively stained as above were measured directly from EM grids. A total of at least 400 individual filaments from three independent experiments were measured for each condition. A, The percent ratios of filaments >100 nm were calculated for actin (10 μm; white column), actin polymerized in the presence of phosphorylated GAP-43 at molar ratios of 10:1 and 1:3, respectively (black columns), or dephosphorylated GAP-43 at molar ratios of 10:1 and 1:3, respectively (hatched columns). The mean ± SD is plotted. B, GAP-43 was preincubated with CaM before copolymerization (see Materials and Methods), and the ratios were calculated as before for actin (10 μm; white column), actin polymerized in the presence CaM (gray column), actin polymerized with dephosphorylated GAP-43 at a molar ratio of 1:3 (hatched column), and actin polymerized in the presence of GAP-43 that had been preincubated with CaM at a molar ratio of 1:2 (black column). Two hundred filaments from each condition from a single experiment are depicted.
- Fig. 5.
Cosedimentation of f-actin and GAP-43 in the presence of CaM. a, Western blots of pellet formed by cosedimentation of F-actin (5 μm) and 1 μmdephosphorylated GAP-43 (lanes 1–3) or f-actin (5 μm) and 1 μm dephosphorylated GAP-43 that had been preincubated with 2 μm CaM (lanes 4–6). Blots were incubated with 7B10 anti-GAP-43 mAb and visualized with chemiluminescence. The upper band is intact GAP-43, whereas the lower band represents a proteolytic fragment. b, Amount of f-actin appearing in the pellet after cosedimentation in the presence of either GAP-43 or GAP-43 and actin as above. There was significantly less actin and less GAP-43 in the pellet when both GAP-43 and CaM were present.
- Fig. 6.
Immunocytochemistry of f-actin and GAP-43 in neuronal cultures. Dorsal root ganglion cultures were plated onto laminin substrates for 24 hr, fixed, and then stained for f-actin with phalloidin and GAP-43 immunoreactivity.A, B,D, E, Double labeling of cultures with the 2G12 antiphosphorylated GAP-43 mAb (A, D) and rhodamine phalloidin (B, E). Growth cones (arrowheads) were heavily reactive with 2G12 and phalloidin. However, the phalloidin staining in the growth cone was attenuated [compare the growth cones with the fibroblast labeled inB (arrow), as well as the parallel cultures in C and F that had been labeled with rhodaminated phalloidin alone]. G,H, High magnification of a growth cone from a DRG culture that had been double labeled with 2G12 (G) and a polyclonal anti-actin antibody (H). Note the colocalization of staining at the lamella (arrowheads). Scale bars, 10 μm (A–F) and 1 μm (G, H).
