Functional characterisation of filamentous actin probe expression in neuronal cells

Abstract

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.

Lifeact-TagGFP2 alters F-actin organization, cellular morphology and biophysical behaviour

“…Our findings are more strongly marked in undifferentiated human stem cells, which may be due to a higher capacity to uptake the adenovirally-delivered Lifeact plasmid. Nevertheless, we obtain similar dose-response trends in immortalized cell lines (NIH/3T3 and COS-7), thus expanding the range of cells types were Lifeact has been shown to induce aberrant morphologies 8,9,24 . It is worth stressing that, in our hands, Lifeact affects different cells types to a different degree, and some cytoskeletal features more strongly than others.…”

“…Nevertheless, we note that the dose-response trends are typically preserved between cell types, thus suggesting a common origin of the observed changes. The Lifeact plasmid we used included a CMV promoter, which has been shown by others to induce milder aberrations than pBABE and CAG 8 . Of note, our results using a recombinant Lifeact-TagGFP2 protein show that the effect of Lifeact is similar regardless of the way in which the DNA (or protein) is delivered and expressed into the cell.…”

“…Of note, our results using a recombinant Lifeact-TagGFP2 protein show that the effect of Lifeact is similar regardless of the way in which the DNA (or protein) is delivered and expressed into the cell. Similarly, the GFP tag used (TagGFP2) is a next-generation monomeric fluorescent protein, again being linked to milder aberrations than other dimerization-prone GFP tags 8 . Our results are thus obtained in conditions identified by others as conductive to fewest aberrant morphologies in terms of choice of promoter and fluorescence protein tag used.…”

“…Similarly, strong in vivo Lifeact expression causes sterility in fruit flies 7 , associated with severe actin defects and multiple nuclei in follicle cells. In addition, the detrimental effects of strong Lifeact expression in cells appear to be linked to the specific promoter and fluorescent protein tag used 8,9 . The aforementioned studies have focused on highlighting the abnormal morphologies, dynamics and overall behaviour of cells associated with strong Lifeact expression.…”

The functional architecture of axonal actin

“…In addition to submembrane actin assemblies, a few electron microscopy studies had reported the presence of deeper intra-axonal actin filaments interspersed among microtubules (Bearer and Reese, 1999;Fath and Lasek, 1988;Nagele et al, 1988). Using a GFP-tagged calponin-homology domain of utrophina non-perturbing, high fidelity probe for filamentous actin (Burkel, 2007;Melak et al, 2017;Patel et al, 2017) -Ganguly et al observed the dynamics of actin along axons (Ganguly et al, 2015). Beyond the random bidirectional movement previously described (Chetta et al, 2015), they identified two new actin structures: static clusters of actin every 3-4 μm along axon shafts that appear and disappear within minutes called actin hotspots, and filaments up to 10 μm in length that grow in both directions at~1 μm/ s, called actin trails (Ganguly et al, 2015) (Fig.…”