Dynactin binding to tyrosinated microtubules promotes centrosome centration in C. elegans by enhancing dynein-mediated organelle transport
Fig 5
The C-terminal tyrosine of α-tubulin is required for centration/rotation of the nucleus-centrosome complex but not for MT tip tracking of dynactin.
(A) Cartoon depicting the C-terminal tail of TBA-1 and TBA-2, the major α-tubulin isotypes in early embryogenesis. The C-terminal tyrosine residues were mutated to alanine using CRISPR-Cas9-based genome editing. (B) Immunoblot of adult worms showing decreased levels of tubulin tyrosination in α-tubulin YA mutants. The same membrane was probed sequentially with an antibody against tyrosinated α-tubulin (antibody YL1/2) and against total α-tubulin (antibody B512). GAPDH served as a loading control. Molecular mass is indicated in kilodaltons. (C) Immunofluorescence images showing lack of tubulin tyrosination in tba-1/2(YA/YA) embryos at the one-cell stage (anaphase). Embryos were co-stained with antibodies against tyrosinated α-tubulin and total α-tubulin. Image with adjusted intensity levels shows unspecific background signal in the α-tubulin tyrosine mutant. Scale bars, 5 μm. (D)—(H) Still images from time-lapse sequences (D) and analysis of male pronuclear migration (E), centrosome positioning (F), NCC/spindle orientation (G), and anaphase spindle oscillations (H) during the first embryonic division for controls and α-tubulin tyrosine mutants. Data is displayed as described for Fig 3. Statistical significance was determined by one-way ANOVA followed by Bonferroni's multiple comparison test. ****P < 0.0001; **P < 0.01; ns = not significant, P > 0.05. Scale bar, 5 μm. (I) (left) Cortical confocal section of a control and tba-1/2(YA/YA) one-cell embryo in metaphase expressing GFP::p50DNC-2. Images correspond to maximum intensity projections over time (12 images acquired every 5 s). Scale bar, 5 μm. (right) Quantification of dynactin levels at MT plus ends using fluorescence intensity measurements of GFP::p50DNC-2 at the cortex. Error bars represent the SEM with a 95% confidence interval, and n indicates the number of individual measurements from 8–9 different embryos per condition. The t-test was used to determine statistical significance. ns = not significant, P > 0.05.