COMPARISON OF POPULAR FLUORESCENT ACTIN MARKERS TO MEASURE ACTIN DYNAMICS IN DENDRITIC SPINES
Synaptic plasticity changes the shape and size of dendritic spines via regulating the extension and turnover of the actin cytoskeleton. Impaired regulation of actin dynamics is often linked with learning deficits, therefore analysing actin dynamics is a widely used approach to reveal regulatory factors behind memory disorders. One of the best ways to investigate the dynamics of actin cytoskeleton in living neurons is by fluorescently labelling actin and measuring the fluorescence recovery after photobleaching (FRAP).To compare popular actin labelling methods, three different fluorescent actin markers were expressed in hippocampal cell cultures. Actin-EGFP is a covalently labelled monomer which incorporates into the F-actin network. Actin-Chromobody-GFP is a monomeric camelid antibody while LifeAct-GFP has an actin binding domain which can bind to filamentous and monomeric actin. Actin-FRAP experiments were performed in dendritic spines under control conditions and after F-actin stabilization by Jasplakinolide. In addition, we compared how the different labelling methods affect the motility and shape of dendritic protrusions. As expected, fluorescence recovery after bleaching the covalently labelled actin-EGFP was completely blocked by F-actin stabilization. On the other hand, both indirect actin labelling constructs recovered almost completely after bleaching, indicating that non-bound diffusable fusion proteins mask the detection of actual actin dynamics. Additionally, the motility of dendritic protrusions was increased when expressing LifeAct-GFP or Actin-Chromobody-GFP compared to actin-EGFP. Thus, only the covalently labelled actin-EGFP method is suitable for FRAP experiments. This work was completed by the National Brain Research Program (2017-1.2.1-NKP-2017-00002) and the VEKOP-2.3.3-15-2016-00007 grant provided by NRDIO, Hungary.