Figure 6.

MT plus end-binding activity of CLASP1/2 supports regenerative axon growth. (A) Representative images of CLASP immunostaining in adult DRG neurons showing its enrichment in the growth cone (inset, arrow). MT plus end bindings (arrowheads) are enlarged in the top and bottom right panels. Bar in the inset, 1 μm. (B) Fluorescence intensity profiles of CLASP. CLASP signals numbered in A were quantified by intensity line scans to present the relative fluorescence intensity profiles. (C) Quantification of plus end-binding and lattice-binding activities of CLASP in nerve growth cones. Embryonic cortical and adult DRG neurons were immunostained side by side for CLASP and tubulin, and CLASP–MT association was quantified as described in the Materials and Methods. Numbers of growth cones analyzed are shown. (***) P < 0.001, χ² test. (D,E) Adult DRG neurons were transfected as indicated and replated at DIV4 to allow axon growth anew. Cells were fixed at 18–20 h after replating and stained for neuronal tubulin, TuJ1 (red). Transfected neurons (arrowheads) are shown in green. Representative images (D) and quantification of axon length (E) are shown. Bar, 100 μm. (F) Adult DRG neurons were transfected as indicated and replated at DIV4 to reinitiate axon growth. To deplete endogenous CLASP1 and CLASP2, the ON-TARGETplus siRNA pool against CLASP1 and siRNA oligos targeting the L domain of CLASP2 were used. Cells were fixed at 18–20 h after replating and axon length was measured. (**) P < 0.01; (***) P < 0.001. (G,H) Adult DRG neurons were transfected with either EGFP alone (control) or together with siCLASP1 and siCLASP2 (siCLASP1/2, both ON-TARGETplus siRNA pools). Neurons were subjected to Western blot analysis at DIV4 (G) or immunocytochemistry (H) with the indicated antibodies. For H, fluorescence intensities of acetylated and tyrosinated tubulin in the growth cone were measured and presented as a ratio (MT stability index). (I) Adult DRG neurons transfected with either EGFP alone (control) or together with siCLASP1/2 (both ON-TARGETplus siRNA pools) were replated at DIV4 and allowed to grow axons anew. Taxol or nocodazole was treated at the time of replating. Neurons were fixed at 18–20 h after replating and axon length was measured. (***) P < 0.001.