Deletion of the Actin-Associated Tropomyosin Tpm3 Leads to Reduced Cell Complexity in Cultured Hippocampal Neurons—New Insights into the Role of the C-Terminal Region of Tpm3.1

Figures

• 

  Figure 1. Effects of Tpm3 knock-out on axonal morphology of Bl6Tpm3flox neurons. Representative fluorescent images of mouse primary hippocampal neurons expressing EGFP-C1 and Cre-IRES-GFP for 96 hours, fixed at 4DIV and stained with GFP, Tau-1 and β3-tubulin antibodies. Pseudo images are inverted black and white GFP fluorescence images with pseudocoloured axon and dendrite labelling for clearer visualizations of the compartments. Curves in pink—axonal shaft, burgundy—primary axonal branches, orange—secondary axonal branches, light blue—dendritic shaft, dark blue—primary dendritic branches, and purple—secondary dendritic branches (A). Quantitative analysis of axonal morphology at 4DIV after transfection with EGFP-C1 and Cre-IRES-GFP constructs. The analysed axonal morphological parameters are (B) length of axonal shaft, (C) number of primary axonal branches, (D) average length of primary axonal branches, and (E) total axonal length. In total, 55–60 neurons from three biological replicates (with at least 15 neurons per replicate) were analysed for each construct. Error bars represent standard error of the mean. Significance was determined by Mann–Whitney nonparametric test: **** p < 0.0001.…

• 

  Figure 2. Quantitative analysis of dendritic morphology of Bl6Tpm3flox neurons. Mouse primary hippocampal neurons expressed EGFP-C1 and Cre-IRES-GFP constructs for 96 h and were fixed at 4DIV. The analysed dendritic morphological parameters are: (A) average total length of dendritic trees, (B) average length of dendritic shaft, (C) number of primary dendritic branches, (D) average length of primary dendritic branches, and (E) Sholl analysis. In total, 55–60 neurons from three biological replicates (with at least 15 neurons per replicate) were analysed for each construct. Error bars represent standard error of the mean. Significance was determined by Mann–Whitney nonparametric test: * p < 0.05, and **** p < 0.0001. Significance of Sholl analysis was determined by nonparametric two-way ANOVA test with Sidak’s test for multiple comparisons: * p < 0.05, **** p < 0.0001.

• 

  Figure 3. Expression of C-terminal deletion plasmids in NIH3T3 cells. NIH3T3 cells were transfected with hTpm3.1, hTpm3.1Δ2AA and hTpm3.1Δ6AA plasmids and fixed after 24 h. (A) Alternatively, spliced exons 1b, 6a and 9d that give rise to the hTpm3.1 isoform are shown in yellow, green and purple, respectively. Amino acids deleted at the C-terminus are given in grey for hTpm3.1Δ2AA and hTpm3.1Δ6AA. (B) NIH3T3 cells were stained with γ-actin and LC-1 (the areas with prominent stress fibres are highlighted). (C) Expression of hTpm3.1, hTpm3.1Δ2AA and hTpm3.1Δ6AA on Western blot. (D) Association between actin and hTpm3.1 was quantified via Pearson’s correlation coefficient (PCC). Significance was determined by Kruskal–Wallis test with Dunn’s correction for multiple comparisons: **** p < 0.0001. Scale bars are 5 µm.

• 

  Figure 4. Effects of hTpm3.1 C-terminal truncation on morphology of C57/Bl6 hippocampal neurons. Representative fluorescent images of mouse primary hippocampal neurons expressing EGFP-C1, hTpm3.1, hTpm3.1Δ6AA and hTpm3.1Δ2AA for 48 h, fixed at 4DIV and stained with GFP, Tau-1 and β3-tubulin antibodies (EGFP-C1 transfected neurons) or LC-1, MAP2 and β3-tubulin (hTpm3.1, hTpm3.1Δ6AA, hTpm3.1Δ2AA transfected neurons). Pseudo images are inverted black and white GFP or LC-1 fluorescence images with pseudo-coloured axon and dendrite labelling for clearer visualizations of the compartments. Curves in pink—axonal shaft, burgundy—primary axonal branches, orange—secondary axonal branches, bright green—tertiary axonal branches, dark green—quaternary axonal branches, light blue—dendritic shaft, dark blue—primary dendritic branches, and purple—secondary dendritic branches. Scale bars = 50 µm.

• 

  Figure 5. Quantitative analysis of axonal morphology of C57/Bl6 hippocampal neurons. Mouse primary hippocampal neurons expressed EGFP-C1, hTpm3.1, hTpm3.1Δ6AA and hTpm3.1Δ2AA for 48 h and were fixed at 4DIV. The analysed axonal morphological parameters are (A) total axonal length, (B) length of axonal shaft, (C) total length of primary axonal branches, (D) average length of primary axonal branches, (E) number of primary axonal branches, and (F) number of secondary axonal branches. In total, 55–60 neurons from three biological replicates (with at least 15 neurons per replicate) were analysed for each construct. Error bars represent standard error of the mean. Significance was determined by Kruskal–Wallis test with Dunn’s correction for multiple comparisons: * p < 0.05, ** p < 0.01.

• 

  Figure 6. Quantitative analysis of dendritic morphology of C57/Bl6 hippocampal neurons. Mouse primary hippocampal neurons expressed EGFP-C1, hTpm3.1, hTpm3.1Δ6AA and hTpm3.1Δ2AA for 48 h and were fixed at 4DIV. The analysed dendritic morphological parameters are: (A) average length of dendritic shaft, (B) number of dendritic trees, (C) average number of primary branches per dendritic trees, (D) average length of primary dendritic branches, and (E) Sholl analysis. In total, 55–60 neurons from three biological replicates (with at least 15 neurons per replicate) were analysed for each construct. Error bars represent standard error of the mean. Significance was determined by Kruskal–Wallis test with Dunn’s correction for multiple comparisons: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Significance in Sholl analysis was determined by nonparametric two-way ANOVA test with Sidak’s test for multiple comparisons: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Significant difference between EGFP-C1 and other constructs is indicated by the following symbols: ^-hTpm3.1, + -hTpm3.1Δ6AA, and *-hTpm3.1Δ2AA. Significant difference between hTpm3.1 and other constructs is…

• 

  Figure 7. Effects of hTpm3.1 C-terminal truncation on segregation of hTpm3.1 to the tips of neurites. Mouse primary hippocampal neurons expressed EGFP-C1, hTpm3.1, hTpm3.1Δ6AA and hTpm3.1Δ2AA for 48 h and were fixed at 4DIV. Fluorescence intensity in the 488 nm channel (EGFP-C1 and LC-1) was measured as the average intensity along the 10% of the length of each neurite at either proximal or distal end. Difference of intensity at each end of neurite is displayed as a ratio of fluorescence intensity at distal end versus proximal end in (A) axons and (B) dendrites. 39 neurons from three biological replicates (with 13 neurons per replicate) were analysed for each construct. Error bars represent standard error of the mean. Significance was determined by Kruskal–Wallis test with Dunn’s correction for multiple comparisons: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

• 

  Figure 8. Effects of hTpm3.1 C-terminal truncation on the amount of F-actin pool in the growth cone of 3DIV old mouse primary hippocampal neurons. Representative fluorescent images of mouse primary hippocampal neurons expressing (A) EGFP-C1, (B) hTpm3.1, (C) hTpm3.1Δ6AA and (D) hTpm3.1Δ2AA for 24 h, fixed at 3DIV and stained with GFP, phalloidin and MAP2 antibodies (EGFP-C1 transfected neurons), or LC-1, phalloidin and MAP2 (hTpm3.1, hTpm3.1Δ6AA and hTpm3.1Δ2AA transfected neurons). Growth cones are highlighted and enlarged for each experimental group. The 488 nm channel was overexposed on purpose to obtain a better outline of the growth cone. Scale bars = 50 µm.

• 

  Figure 9. Effects of Tpm3.1 C-terminal truncation on the amount of F-actin at the growth cone. Actin intensity in the growth cone was measured as mean fluorescence intensity of the area of the growth cone in phalloidin channel (A). Absolute actin intensity in the growth cone was obtained by multiplying the value of actin intensity by the growth cone surface (B). Surface of the growth cone was measured by manually outlining the area of the growth cone and measuring the surface (C). 60–69 growth cones from four biological replicates (with at least 5 growth cones per replicate) were analysed for each construct. Error bars represent standard error of the mean. Significance was determined by Kruskal–Wallis test with Dunn’s correction for multiple comparisons: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.