Figure 5.

Taxol treatment increases all microtubule PTMs and disrupts the polarized localization of CA-Kinesin-1 motors and JIP-1 cargoes in polarized stage 3 and unpolarized stage 2 neurons. (A–E) Effects of Taxol in polarized stage 3 cells. (A) stage 3 cortical neurons were treated for 3 h with DMSO, 10 or 100 nM Taxol, and then lysates were analyzed by Western blotting with antibodies to acetylated α-tubulin, detyrosinated α-tubulin, polyglutamylated α- and β-tubulin, or total β-tubulin. Polyglutamylated samples are from nonadjacent lanes of the same gel. (B) Stage 3 hippocampal neurons were treated for 2–4 h with DMSO, 10 or 100 nM Taxol. The cells were then transfected with CA-Kinesin-1-mCherry and soluble YFP, allowed to express the exogenous proteins under additional treatment for 4–5 h, and then fixed and imaged. (C) Quantification of the data in B. The data are expressed as the percentage of cells that have accumulated CA-Kinesin-1 mCherry motors in minor neurites. (D) Stage 3 hippocampal neurons were treated for 3 h with DMSO, 10 or 100 nM Taxol, and then fixed and stained with antibodies to JIP1 and acetylated α-tubulin. Arrows, tips of minor neurites. Arrowheads, tips of axons. (E) Quantification of the data in D. The data are expressed as the percentage of stage 3 minor neurites or axons that have accumulated JIP1 protein. (F–H) Effects of Taxol in unpolarized stage 2 cells. (F) Stage 2 hippocampal neurons expressing CA-Kinesin-1-YFP motors since the time of plating were untreated or treated with 10 nM Taxol for 20 min and then imaged. Bar, 10 μm. (G) Stage 2 hippocampal neurons were treated for 3 h with DMSO, 10 or 100 nM Taxol, and then fixed and stained with antibodies to JIP1 and acetylated α-tubulin. (H) Quantification of the data in F and G. The data are expressed as the percentage of stage 2 neurites that have accumulated CA-Kinesin-1 motors or JIP1 cargoes. Bars, 20 μm. Error bars, SEM. t test: *p < 0.01 compared with control DMSO-treated cells.