Coatomer associates with components of the SMN complex in vivo. (A) Flag-α-COP co-precipitated endogenous SMN, Gemin2 and Gemin3 proteins from HEK293 lysate (Flag-bacterial alkaline phosphatase). (B) The C-terminal half (amino acids 751–1224) of α-COP complexes with endogenous SMN and Gemin2. (C) Endogenous α-COP co-immunoprecipitated SMN, Gemin2, Gemin3 and δ-COP from SH-SY5Y cells, whereas control IgG or anti-clathrin antibody (TD.1) did not. (D) Anti-SMN rabbit antibodies co-precipitated α-COP from SH-SY5Y cells. As expected, Gemin2 and Gemin3 were also present. (E) RNA immunoprecipitation using anti-α-COP antibody or control IgG followed by RT–PCR for β-actin mRNA.

α-COP is transported within the axon toward the growth cone in primary neurons. (A–G) Cultured primary mouse hippocampal neurons (HPN). Endogenous α-COP in the cell body (arrowhead in A and B) and the growth cone (arrow in B and C) as detected by affinity-purified anti-α-COP antibody. Similarly, ectopically expressed α-COP: mCherry localized in the cell body (arrowhead) and the growth cone (arrow) in HPN (D). Anti-γ2-COP antibody detected endogenous γ2-COP in the growth cone (green, arrow in E and F) and co-localized with α-COP (red in G). (H and I) Human SH-SY5Y cells differentiated with retinoic acid and BDNF. α-COP localized in the cell body (green, arrowhead in H) and the growth cone (arrow in I). (J–O) Chicken primary RGC. Anti-α-COP detected α-COP in the cell body (green, arrowhead in J) and distributed as granules along the axon (green in K). It was also abundant in the growth cone (red, arrow in L and M). (N and O) Live chicken RGC neurons ectopically expressing α-COP: mCit. α-COP: mCit localized in the same perinuclear Golgi (black arrowhead in N) and moved toward the growth cone (O, arrow indicates the stationary granule; also see Supplementary Material, Movie S1).

SMN and α-COP localization partially overlap in neurites and growth cones. (A–E) Primary murine motor neuron. Immunodetection of α-COP (red in C, green in E) and SMN (green in D, red in E). Co-localization of SMN with α-COP appears yellow in (B) and (E). (F–H) Nerve growth factor-differentiated PC12 cells expressing α-COP:mCherry and SMN: mCit. Abundant α-COP extends from the neurite into the growth cone. All SMN granules (green) are adjacent to and move with α-COP. SMN transiently dissociate and re-associate with α-COP as they move distally (Supplementary Material, Movie S2).

Depletion of α-COP alters SMN localization. (A) Doxycycline (dox)-induced α-COP shRNA expression in SH-SY5Y cells. Western blot revealed ∼80% reduction in the α-COP protein at 72 h, whereas SMN and Gemin2 levels were unchanged. (B) Top four images show a reduction in the fluorescence intensity of α-COP by immunofluorescence analysis of dox-induced α-COP shRNA cells. Note that controls cells (left) retained a spread lamellipodia with almost no filipodia, whereas cells expressing α-COP shRNA (right) showed a reduction in the lamellipodium area (arrows). The bottom four panels show that compared with uninduced control (left), cells that express α-COP shRNA (right) had prominent filipodia, visualized by differential interference contrast (DIC) microscopy, which were β-actin positive by immunofluorescence. (C) SMN and F-actin localized at the leading edge in control cells (top nine panels). α-COP knockdown induced a loss or reduction of SMN from leading edge, loss of lamellae and increased F-actin positive filipodia (bottom six panels).