Figure 1.
Colocalization of SMN with the Golgi apparatus.
(A–C) NSC34D cells were stained with SMN antibody (green) and antibodies against Gm130 (A), Tgn38 (B), and Golgin-97 (C) (red). Magnified regions are shown in boxes at right of the merged figures. Obvious colocalization between each protein is indicated with pink arrows. Scale bar: 20 µm; 2 µm in magnified box. (D) In the control group, antibodies against mouse (Ms) or rabbit (Rb) immunoglobulin G (IgG) were used in place of SMN antibody or the Golgi marker antibodies. (E) Quantitation of the colocalization ratio between SMN with Gm130, Tgn38, Golgin-97, and WGA in selected Golgi enriched region. Data represent the mean ± SEM from three independent experiments (over 50 cells were counted in each group). ***, P<0.001. ns: not significant. (F) EGFP-SMN and dsRed-β1,4-Gal-T or (I) EGFP-SMN and mCherry-FYVE are shown in green and red, respectively. Time series images were taken every 10 sec, including z-stacks. Images are shown in three dimensions with views from the front, underside, and side-back (F). Selected planes 36–43 (F) and 1–8 (I) are shown. Arrows indicate obvious colocalizations of SMN and the Golgi. (G, J) Transfection efficiencies of EGFP-SMN with dsRed-β1,4-Gal-T were 49.4% ±1.0% with 41.1% ±1.2%, respectively; efficiencies for EGFP-SMN with mCherry-FYVE were 47.5% ±2.3% with 43.8% ±2.2%, respectively. Data represent the mean ± SEM, n = 4 in each group. (H, K) Quantitation of the colocalization ratio in each time-point between SMN with organelle markers in a selected view from a neurite. Bar: 2 µm.
Figure 2.
Cytoplasmic mSmn exists in the Golgi-enriched fractions.
Protein lysates including Golgi-enriched fractions (lanes 1–3) and other fractions (lanes 4–5) were analyzed by Western blotting with Cop-α, Gm130, Calnexin, Syncrip, U2af65, Tgn38, SMN, Gemin2, Pabp-C1, and VAPB antibodies. The relative amounts of proteins in each lane are expressed in relative densitometric units (RDUs).
Figure 3.
Characterization of mSmn granules through immuno-gold labeling TEM.
(A–C) Electromicroscopic analysis of NSC34 cells revealed mSmn signals can be found in the Golgi stack (indicated by blue arrows) and mSmn aggregates at the trans-Golgi membrane (indicated by a yellow arrow). Magnified images from the boxed areas show the mSmn signal in the Golgi secreted granules (indicated by red arrows). Bar: 0.5 µm (left) and 0.2 µm (right). In the control group (C), mouse IgG was used in place of SMN antibody. Bar: 0.5 µm (A, left), 1 µm (B, C, left) and 0.2 µm (right). N, Nucleus; G, Golgi; M, Mitochondria; ER, Endoplasmic Reticulum. (D) Electromicroscopic analysis characterizes different mSmn granules (d′, blue arrows indicate mSmn granules; red arrows indicate non-granule type mSmn signals in the cytoplasm). Note that some mSmn granules were coated (d′, middle) and some were uncoated (d′, right). In the control group, mouse IgG instead of SMN antibody was used (d″). Bar: 100 nm. (E) Electromicroscopic analysis of sciatic axons revealed that the mSmn signal presents in granules and in the axoplasm. M, myelin sheath; A, axoplasm. The mSmn signals are indicated with red arrows. Figure E–e′ shows an mSmn granule (red arrow) that appears in a long sausage-shaped organelle. Figure E–e″ shows a small mSmn granule (red arrow) in an axon. Figure E–e′″ shows the control group, using the mouse IgG instead of SMN antibody. Bar: 0.5 µm. (F) Time series images (5 sec/plane) acquired from EGFP-SMN-transfected NSC34D cells show constant fusion and fission of SMN granules in the axon (indicated with an arrow). Bar: 20 µm.
Figure 4.
Global disruption of the trans-Golgi mediated granule secretion reduces mSmn levels in neurites.
(A) The underside of the polycarbonate membrane that allowed neurite outgrowth was stained by βIII-tubulin antibody (green). Bar: 200 µm. (B) Total lysates (T, lanes 1–2) and neurite lysates (N, lanes 3–8) (lanes 3–5: ethanol treated control; lanes 6–8: monensin treated cells) were analyzed for βIII-tubulin (internal control), mSmn, Gemin2, histone H1, and ChAT expression by Western blotting. Histone H1 served as a negative control for the neurite fraction. (C–E) Quantitative analysis of the blot shown in panel A using βIII-tubulin for normalization. mSmn levels in the total lysate (C) and neurite fraction (D), and Gemin2 level (E) in the neurite fraction in the two treatment groups. Results represent the mean ± SEM from at least three independent experiments performed in triplicate. ns: not significant; **, P < 0.01.
Figure 5.
Global disruption of the trans-Golgi mediated granule secretion mimicked SMN-deficient growth cone defects.
(A) Growth cones in NSC34D cells receiving different treatments were visualized by staining with SMN antibody (green), βIII-tubulin antibody (white), and Alexa Fluor 647-conjugated phalloidin (red). Bar: 10 µm. (B) Analysis of mSmn knockdown by three siRNAs specific against Smn and quantitation of the knockdown of Smn by siRNA3, the siRNA achieving the highest efficiency. Data represent the mean ± SEM from at least three independent experiments performed in triplicate. ***, P<0.001. (C) Growth cone area of NSC34D cells treated either with scrambled siRNA (n = 133) or mSmn siRNA-3 (n = 120) and ethanol (n = 188) or monensin (n = 207) were quantitatively analyzed. ***, P < 0.001.
Figure 6.
Cop-α is involved in mSmn granule secretion from the Golgi apparatus.
(A) Cell lysates treated with scrambled siRNA (lanes 1–3) or Cop-α siRNAs (lanes 4–6) were analyzed for α-tubulin (internal control) and Cop-α expression by Western blotting. Quantitation of the blot using α-tubulin for normalization of Cop-α and mSmn expression level in the total lysates. Results represent the mean ± SEM from at least two independent experiments performed in triplicate. ***, P < 0.001. ns: not significant. (B, C) Immunocytochemical analysis of the mSmn granules and Tgn38 colocalization in proximity to the Golgi in NSC34D cells treated with scrambled siRNA (B) or Cop-α siRNA (C). Cells were stained with antibodies against Cop-α (green), mSmn (red), and Tgn38 (blue). DAPI was used for nuclei staining (white, shown in the merged panel). Bar: 10 µm (B); 20 µm (C). The boxed areas (b′ and c′) in the merged panel show the trans-Golgi area (for quantitation of the colocalization) and are shown magnified at the right side. Yellow arrows in (B) and (C) indicate the colocalization of mSmn granules with the trans-Golgi area. (D) Quantitative analysis of the colocalization of mSmn granules with Tgn38 in NSC34D cells treated either with scrambled siRNAs or Cop-α siRNAs. Results represent the mean ± SEM from at least two independent experiments performed in triplicate. ***, P < 0.001.