Comparative analysis of actin visualization by genetically encoded probes in cultured neurons

Abstract

Actin cytoskeleton predominantly regulates the formation and maintenance of synapses by controlling dendritic spine morphology and motility. To visualize actin dynamics, actin molecules can be labelled by genetically fusing fluorescent proteins to actin monomers or using fluorescently tagged actin-binding proteins or single-chain anti-actin antibodies. However, the effects of these labels on the morphology of neurons have not been quantitatively compared yet. In the present study, we analysed Actin-Chromobody-GFP, LifeAct-GFP and EGFP-actin with respect to their effects on actin-related features in mouse cultured hippocampal neurons. The actin-binding probes LifeAct and Actin-Chromobody showed similar affinity to F-actin, and along with EGFP-actin, were enriched in dendritic protrusions. In contrast to EGFP-actin, neither of these constructs was able to detect subtle changes in actin remodelling between mature mushroom shaped spine and less developed filopodia. None of the compared probes altered filopodial motility compared to control EGFP expression, however, within 24 hours expression, minor changes in dendritic spine morphology and density were visible. Furthermore, while EGFP-actin and LifeAct-GFP expression did not alter dendritic arborization, AC-GFP expressing neurons displayed a reduced dendritic arborization. We therefore conclude that careful consideration of cellular consequences is required before performing experiments with a particular actin labelling probe in primary neurons.