Fig. 2.

G-actin incorporates into presynaptic puncta but not cortical actin. Reticulospinal axon impaled with a microelectrode containing Alexa Fluor 488 G-actin (1.1 mg/ml) and buffer solution (2 mM HEPES, 0.2 mM CaCl₂, 0.2 mM ATP, pH 8.0). This was pressure injected into the axon. Fluorescent actin structures formed within minutes of injection and increased in fluorescence over 80 min. A: 3D reconstruction of actin fluorescence close to the axon perimeter 5 min after injection. Hemi-axon is shown in transverse section (left) and from the ventral spinal surface (right). Red structures are bright actin labeling consistent with synaptic vesicle cluster colocalization; gray is the diffuse labeled G-actin within the axon. B: single optical section shown in linear grayscale taken from the dashed line in A. The graph is a profile plot of intensity taken from between the vertical white lines. There is no cortical actin signal. C: same views as A but after an hour of recording. D: mean intensity (black) and cross-sectional area (red) of all clusters in A plotted with time after injection. E: mean area and intensity (6 preparations) normalized to value 80 min after injection.