Figure 2. TUBA1A-V409I/A mutants alter neuron morphologies.

(A) Representative days in vitro 2 (DIV2) cortical neurons expressing cytoplasmic GFP and 6X-His-tagged TUBA1A-WT, -V409I, and -V409A. Scale bar = 10 µm. (B) Quantification of number of primary, secondary, and tertiary branches along axons and dendrites in each condition. Images were collected from three independent experiments; n=65 cells for wild-type (WT), n=56 for V409I, and n=59 for V409A. Each dot represents a single 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. (C) Example image of DIV2 cortical neuron expressing cytoplasmic GFP and TUBA1A-WT. Scale bar = 10 µm. Yellow box indicates a representative region measured every 30 s for 15 min at 37°C, 5% CO₂. Scale bar in insets = 5 µm. (D) Proportion of events classified as the neurite growing, paused, or shrinking, depending on if the length of the neurite grew, stayed the same, or shrunk, respectively. Images were collected from three independent experiments; n=10 cells for WT, n=13 for V409I, and n=13 for V409A. Bars are mean ± standard error of the mean. (E) Quantification of the amount of time a neurite was in a paused state, neither growing nor shrinking. Each dot represents a period of pause; cell numbers are the same as stated in (D). Bars are mean ± 95% confidence interval. Statistical analysis was done using an unpaired t-test. All statistics with p≤0.05 are indicated on graph. (F) Representative images of neurons expressing the above plasmids, exposed to 4°C for indicated time, and stained with TUBB2A/B. Scale bar = 10 µm. (G) Quantification of the number of neurites per cell every 2 hr over a 6 hr 4°C cold shock. Images were collected from three independent experiments; for WT n=36 cells at 0 hr, n=35 at 2 hr, n=41 at 4 hr, n=44 at 6 hr; for V409I n=32 at 0 hr, n=30 at 2 hr, n=27 at 4 hr, n=29 at 6 hr; for V409A n=37 at 0 hr, n=29 at 2 hr, n=27 at 4 hr, n=22 at 6 hr. Dots represent averages from the three separate experiments and error bars are ± 95% confidence interval. (H) The proportion of neurites that have retraction bulbs per cell measured every 2 hr over a 6 hr 4°C cold shock. Cell numbers and error bars are the same as stated in (G).

Figure 2—figure supplement 1. Effect of TUBA1A-V409I/A on neuronal morphologies.

(A) Representative days in vitro 1 (DIV1) cortical neurons expressing GFP and 6X-His-tagged TUBA1A-WT, -V409I, and -V409A. Scale bar = 10 µm. (B) Quantification of number of primary, secondary, and tertiary branches along axons and dendrites in each condition. Images were collected from three independent experiments; n=32 cells for wild-type (WT), n=41 for V409I, and n=36 for V409A. Each dot represents a single 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. (C) Individual values of the proportion of neurite events that classify as growing, paused, or shrinking that make up the average values in Figure 2D. (D) Representative merge images of neurons expressing the above plasmids, exposed to 4°C for indicated time, stained with TUBB2A/B in magenta, and expressing cytoplasmic GFP in green. Scale bar = 10 µm. (E) Individual values of the number of neurites per cell that make up the average values in Figure 2G. Cell numbers are the same as stated in Figure 2G. 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) Individual quantifications of the proportion of neurites per cell that have retraction bulbs measured every 2 hr over a 6 hr 4°C cold shock that make up the average values in Figure 2H. Each dot represents one cell. Cell numbers and error bars are the same as stated in (E). 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.

Figure 2—video 1. Neurite dynamics.

Download video file ^{(182.3KB, mp4)}

Primary cortical neurons transfected with a pCIG2 plasmid co-expressing TUBA1A-6X-His and cytoplasmic GFP imaged at days in vitro 2 (DIV2). Z-series of 6 µm were collected at 30 s intervals for 15 min.