Figure 2

Repolarization of the axis of the microtubule organizing center (MTOC) and nuclear centrosomal (NC) axis depends on the microglial activation state. Rat microglia were stimulated for 24 hr with 10 ng/ml lipopolysaccharide (LPS) or 20 ng/ml rat recombinant IL4. A) Confocal images of microglia labeled for α-tubulin (red) to visualize microtubules and the MTOC, and DAPI (blue) to label nuclei. F-actin was labeled with Alexa Fluor 488-conjugated phalloidin (green) to reveal the cell shape. In unipolar microglia, the MTOC was in one of three peri-nuclear orientations (and see cartoon in panel B): toward the leading edge (arrows), at the side (open arrow) or toward the uropod (arrowheads). Higher magnification images (bottom panel) show the anterior and posterior positions, and the lack of microtubule polarization in LPS-treated cells. Scale bars, 20 μm. B) Quantification of MTOC-nuclear orientation. Only unipolar (migrating) microglia were analyzed: that is, control and IL4-treated cells. For each slide, ten random images were acquired at 10× magnification on the confocal microscope. MTOC orientation was scored based on its peri-nuclear position (cartoon inset): anterior (MTOC toward leading edge), posterior (MTOC toward uropod), lateral (MTOC at side). For each treatment replicate, ≥90 unipolar microglia were scored. Results are expressed as percent of total cells counted (mean ± SEM, n = 7 individual cultures). Two-way ANOVA with Bonferroni correction revealed significant intergroup differences: ***control differs from IL4-treated; ^###MTOC position differs from anterior position of control cells. Three symbols indicate P <0.001.