Figure 3.

Axonal Nucleolin Interacts with the Importin β1 MAIL Motif

(A) Bovine axoplasm (10 mg/lane) was precipitated on immobilized MAIL or GMAIL RNA motifs, and eluted proteins were separated by 10% SDS-PAGE. The gel region containing the major differential band is shown here and the complete gel is shown in Figure S3A. Mass spectrometry analyses identified nucleolin as the major unique MAIL-bound component (Figures S3A–S3C).

(B) Western blot of nucleolin precipitated from rat sciatic nerve axoplasm with MAIL, GMAIL, or β-actin Zipcode RNA motifs. Precipitates were separated on 10% SDS-PAGE, blotted onto nitrocellulose, and probed with antibody against nucleolin.

(C) Immunoprecipitation of 200 μg rat sciatic nerve axoplasm samples with control IgG or anti-nucleolin antibodies followed by RT-PCR for importin β1 or β-actin mRNAs.

(D) Western blot of recombinant nucleolin precipitated with MAIL, IMAIL, or β-actin Zipcode RNA motifs. Input was 1 μg recombinant nucleolin per lane.

(E) Primary cultured rat sensory neurons immunostained with antibodies against nucleolin (red) and NFH (green), revealing nucleolin in both neuronal cell bodies and axons. Scale bar, 20 μm; right overlay panel scale bar, 10 μm.

(F) Sciatic nerve cross-sections immunostained with antibodies against nucleolin (red) and NFH (green), revealing nucleolin within sensory axons in vivo. Scale bar, 20 μm.

(G) Electron micrographs showing immunogold labeling for nucleolin in axons on ultrathin monolayer sections of cultured mouse DRG neurons (left) or of sciatic nerve (right). Nucleolin is present in axons in vitro and in vivo. Scale bars, 200 nm; gold particle diameter, 10 nm.

(H) Colocalization of nucleolin protein (immunostaining, green) and importin β1 mRNA (FISH, red) in sensory axons. Importin β1 mRNA colocalized with nucleolin protein (yellow) is shown in a single optical plane (scale bar, 5 μm). For cell body signal and scrambled probe control, see Figure S3D. Pearson’s correlation coefficient for importin β1 colocalization with nucleolin 0.37 ± 0.04 (n = 29) differs significantly from Pearson’s for β-actin or GAP43 (see below) (p value for importin β1 versus β-actin < 0.004, p value for importin β1 versus GAP43 < 0.0001; ANOVA with Bonferroni post hoc correction in both cases).

(I) Colocalization of nucleolin protein (immunostaining, green) and β-actin mRNA (FISH, red) in sensory axons. Colocalization is shown in yellow in a single optical plane (scale bar, 5 μm). For cell body signal and scrambled probe control, see Figure S3D. Pearson’s correlation coefficient for β-actin colocalization with nucleolin 0.19 ± 0.03 (n = 20).

(J) Colocalization of nucleolin protein (immunostaining, green) and GAP43 mRNA (FISH, red) in sensory axons. Colocalization is shown in yellow in a single optical plane (scale bar, 5 μm). For cell body signal and scrambled probe control, see Figure S3D. Pearson’s correlation coefficient for GAP43 colocalization with nucleolin 0.05 ± 0.01 (n = 48).

See also Figure S3.