Figure 2. Localization and microtubule association of SMN granules in neuronal processes and growth cones.

A, Rat spinal cord neurons were cultured for 3 d and fixed for double label immunofluorescence staining with a monoclonal antibody to SMN (red) and a rabbit polyclonal antibody to tau (green), as an axonal marker. SMN formed granules that localized to the axonal process (arrows) and its growth cone (arrowhead).

B, Cultured chick forebrain neurons stained with the monoclonal antibody to SMN showed SMN granules (red) within minor neurites (arrows) and growth cones (arrowhead).

C, Transfection of EGFP-SMN in chick forebrain neurons similarly revealed granules within processes. Microtubules were detected with a monoclonal antibody and Cy3-labeled anti-mouse antibody (red). Z-series stacks were acquired with a cooled CCD camera, and images were deconvolved (Hazebuster; Vaytek). Higher magnification of two regions (a, b) suggest association of EGFP-SMN granules (yellow, arrows) and microtubules.

D, Immunogold detection of SMN (arrows) at the EM levels shows proximity to microtubules (arrowheads) within neuronal processes. E, Rat spinal cord neuron showed colocalization of SMN (green) with ribosomal RNA (red). Ribosomal RNA was detected by biotin-labeled oligonucleotide probes and Cy3-conjugated streptavidin. SMN was stained with a monoclonal antibody and Cy5-conjugated anti-mouse antibody. Higher magnification region (c) indicates SMN colocalization with ribosomal RNA (yellow, arrows).