Functional characterisation of filamentous actin probe expression in neuronal cells

<div><p>Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.</p></div

Summary of quantitative morphological analysis.

<p>Summary of quantitative morphological analysis.</p

Sholl analysis of dendritic complexity of mouse hippocampal neurons transfected with filamentous actin tracking probes.

<p><b>Dendritic complexity was</b> measured as number of intersections per shell as a function of distance from the soma. A Neurons transfected with Lifeact-GFP(1) displayno significant changes in dendritic compliexity compared to EGFP control. Lifeact-GFP(2) transfected neuron consistently had the lowest number of intersections per shell from 0–60 μm from the soma. Lifeact-EGFP transfected neurons showed an increase in dendritic complexity from 0–20 μm and a decrease from 20–60 μm compared to EGFP control. Lifeact-7-mEGFP transfected neurons showed an increase in dendritic complexity from 0–30 μm and a significant decrease from 30–40 μm. Utr261-EGFP also showed an increase in dendritic complexity from 0–30 μm. F-tractin-EGFP transfection resulted in increased dendritic complexity at 0–20 μm from the cell soma. Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by two-way ANOVA with Tukey's test for multiple corrections. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Significant difference between EGFP and each actin tracking probe is indicated by the following symbols: Lifeact-GFP(2) = #, Lifeact-EGFP = §, Lifeact-7-mEGFP = £, Utr-261-EGFP = ⁺, F-tractin-EGFP = ‡.</p

Effects of filamentous actin tracking probes on the morphology of primary mouse hippocampal neurons [EGFP control and Lifeact-GFP(1)].

<p>Representative images of neurons transfected with EGFP (A-D) or Lifeact-GFP(1) (E-H). (A, E) expressed probes; (B, F) axonal marker Tau1; (C, G) pan-neuronal β3-tubulin; (D, H) merged images. Scale bar = 50μm.</p

Quantitative analysis of dendritic morphology at DIV3 after transfection with EGFP, Lifeact-GFP(1), Lifeact-GFP(2), Lifeact-EGFP, Lifeact-7-mEGFP, Utr261-EGFP and F-tractin-EGFP expressing constructs.

<p>Compared to EGFP control, neurons transfected with Lifeact-GFP(2) showed significant decreases in the total length of dendritic trees (B), the mean length of primary dendritic shaft (C), the mean length of primary dendritic branches (E) and the mean length of dendritic trees (H). Neurons transfected with Utr261-EGFP showed an increase in the number of primary dendritic branches (D) and mean number of primary dendritic branches per dendritic tree per cell (G), compared to EGFP control. Neurons transfected with Lifeact-GFP(1), Lifeact-EGFP, Lifeact-7-mEGFP and F-tractin-EGFP did not show any significant changes in dendritic morphology when compared to EGFP control (A-H) Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by Kruskal-Wallis Test (non-parametric one-way ANOVA) and Dunn's multiple corrections test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Effects of filamentous actin tracking probes on the morphology of primary mouse hippocampal neurons [Lifeact-GFP(2) and Lifeact-EGFP].

<p>Representative images of neurons transfected with Lifeact-GFP(2) (A-D) or Lifeact-EGFP (E-H). (A, E) expressed probes; (B, F) axonal marker Tau1; (C, G) pan-neuronal β3-tubulin; (D, H) merged images. Scale bar = 50μm.</p

Quantitative analysis of axonal morphology at DIV3 after transfection with EGFP, Lifeact-GFP(1), Lifeact-GFP(2), Lifeact-EGFP, Lifeact-7-mEGFP, Utr261-EGFP and F-tractin-EGFP expressing constructs.

<p>Neurons transfected with Lifeact-GFP(1) show decreased total axon length (A), decreased primary axon total length (B) and decreased total length of primary axon branches (D), compared to EGFP control. Neurons transfected with Lifeact-EGFP and Utr261-EGFP show also show decreased primary axon total length (B). Neurons transfected with Lifeact-GFP(2), Lifeact-7-mEGFP and F-tractin-EGFP did not show any significant changes in axonal morphology when compared to EGFP control (A-F). Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by Kruskal-Wallis Test (non-parametric one-way ANOVA) and Dunn's multiple corrections test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p