TIG3 alters microtubule distribution.

<p><b>A</b> SCC-13 cells were infected with 10 MOI tAd5-EV or tAd5-TIG3 and at 24 h post-infection cells were fixed and stained with anti-TIG3 (green stain) and anti-β-tubulin (red stain). TIG3 accumulates at the expected perinuclear location (black arrow). β-tubulin accumulates in an atypical ring at the cell periphery (red arrows). The normal β-tubulin network in the TIG3-negative cell is indicated by a white arrow pointing to the centrosome. Nuclei were Hoechst stained (blue). The bottom panel is identical to the top, except that only the β-tubulin (red) signal is indicated. Bars = 10 µM. The graph shows the number of tAd-EV and tAd5-TIG3 infected cells with centrosome-originated microtubule arrays. Cells were counted in twenty independent microscope fields and a minimum of one-hundred cells were counted per treatment group. The values are mean ± SEM. Paired Student's t-test analysis reveals that the means are significantly different (p<0.001). To assess the impact of TIG3 on tubulin solubility, cells were infected with tAd5-EV or tAd5-TIG3 and after 24 h total extract, soluble and pellet fractions were prepared and electrophoresed followed by immunostaining to detect β-tubulin and β-actin. The presence of the majority of β-actin in the soluble fraction indicates that the fractionation was successful. <b>B</b> TIG3 expression causes actin filament collapse around the nucleus. SCC-13 cells were infected with adenovirus as above and after 24 h stained with anti-β-actin (red stain) and anti-TIG3 (green stain). Nuclei were Hoechst stained (blue). For the TIG3-positive cells, the left panel shows the TIG3 and β-actin signals (red and green), while the right panel shows only the β-actin (red) channel. The black arrow indicates TIG3 accumulation at the centrosome and the red arrow indicates the β-actin microfilament nuclear ring. Bars = 10 µm. The plot shows the number of tAd-EV and tAd5-TIG3 infected cells displaying actin microfilament rings surrounding the nucleus. Cells were counted in eighteen independent microscope fields and a minimum of one-hundred cells were counted per treatment group. The values are mean ± SEM. Paired Student's t-test analysis reveals that the means are significantly different (p<0.001).