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. 2022 May 5;11:e76189. doi: 10.7554/eLife.76189

Figure 3. TUBA1A-V409I/A microtubules have altered microtubule dynamics in neurons.

(A) Representative days in vitro 2 (DIV2) cortical neurons expressing plasmids with cytoplasmic GFP and 6X-His-tagged TUBA1A-WT, -V409I, and -V409A. Cells stained with tyrosinated tubulin and acetylated tubulin. Scale bar = 10 µm. (B) Quantification of acetylated tubulin fluorescence intensity or tyrosinated tubulin (C) binned in two, 10 µm segments at the distal end of the axon tip. Images were acquired in three independent experiments; n=61 cells for wild-type (WT), n=54 for V409I, n=59 for V409A. Each dot represents the quantification from one cell. Bars are mean ± 95% confidence interval. Statistical analysis between multiple groups was analyzed by two-way ANOVA and corrected for multiple comparisons post hoc by Tukey test. All statistics with p≤0.05 are indicated on graph. (D) Calculated ratio of acetylated tubulin over tyrosinated tubulin measurements displayed in (B) and (C). (E) Example image of DIV2 cortical neuron co-expressing TUBA1A-WT and GFP-MACF43. Scale bar = 10 µm. (F) Quantification of the number of microtubule polymerization events that occur in each cell over the course of 4 min. Images were obtained from three independent experiments; n=19 cells for WT, n=18 for V409I, n=19 for V409A. Each dot represents one cell. Bars represent mean ± 95% confidence interval. Statistical analysis for panels F–H was done using an unpaired t-test. All statistics with p≤0.05 are indicated on graph. (G) Quantification of microtubule polymerization rates measured from GFP-MACF43 comets. Each dot represents the rate of one microtubule polymerization rate. Larger dots outlined in black indicate the average of all polymerization rates from the three independent experiments where these images were obtained. (H) Quantification of GFP-MACF43 comet lifetime in seconds.

Figure 3—source data 1. Microtubule post-translational modifications fluorescence intensity.
Figure 3—source data 2. Quantification of microtubule dynamics in neurons by GFP-MACF43 comets.

Figure 3.

Figure 3—figure supplement 1. TUBA1A-V409I/A microtubules have increased microtubule acetylation but not tyrosination.

Figure 3—figure supplement 1.

(A–C) Example line scan from one TUBA1A-WT (A), -V409I (B), and -V409A (C) neuron measuring the fluorescence intensity along the distal 10 µm of the axon and the next proximal 10 µm. Acetylated tubulin values are in magenta and tyrosinated tubulin values are in aqua. (D) Quantification of acetylated tubulin fluorescence intensity or tyrosinated tubulin (E) binned in two, 5 µm segments at the distal end of a dendrite tip. Images were acquired in three independent experiments; n=61 for wild-type (WT), n=54 for V409I, n=59 for V409A. Each dot represents the quantification from one cell. Bars are mean ± 95% confidence interval. Statistical analysis between multiple groups was analyzed by two-way ANOVA and corrected for multiple comparisons post hoc by Tukey test. All statistics with p≤0.05 are indicated on graph. (F) Calculated ratio of acetylated tubulin over tyrosinated tubulin measurements displayed in (D) and (E).
Figure 3—video 1. Microtubule dynamics in neurons.
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Primary cortical neurons transfected with a pCIG2 plasmid co-expressing TUBA1A and GFP-MACF43 imaged at days in vitro 2 (DIV2). Z-series of 5 µm were collected at 2.2 s intervals for 4 min.