Assessment of cellular actin dynamics by measurement of fluorescence anisotropy

Abstract

To study cellular actin dynamics, a cell-free assay based on fluorescence anisotropy was developed. Using G-actin-Alexa as a probe, we found that anisotropy enhancement reflects F-actin elongation. Anisotropy enhancement varies with the concentration of magnesium and calcium cations and with ethylenediaminetetraacetate or well-known effectors of the polymerization. This assay gives the overall status of actin dynamics in cell extracts which are the closest conditions to in vivo, implying most of the regulating proteins that are missing in purified actin measurements. It can be used in a large-scale screening for chemical compounds which modulate actin polymerization.

Cell-penetrating Peptides with Intracellular Actin-remodeling Activity in Malignant Fibroblasts

“…In this context, we have screened some cell-penetrating peptide analogues (25) (penetratin, (R/L)16, (R/W)16, (R/W)9, and R9), which present basic amino acids or sequences of charged/ hydrophobic residues with the potential to form amphipathic helices, for their ability to influence actin dynamics in a cell-free assay based on fluorescence anisotropy (26). In this actin polymerization assay, only (R/W)16, (R/W)9, and R9 were found active.…”

The β-carboline Harmine Induces Actin Dynamic Remodeling and Abrogates the Malignant Phenotype in Tumorigenic Cells

“…Thus, it is possible that a drug mimicking the effects of zyxin could suppress the malignant phenotype in the absence of a direct cytotoxic effect. In order to isolate such molecules, we have developed an in vitro assay that allows the assessment of actin polymerization by fluorescence anisotropy in cellular extracts [28]. Using this screening assay linked to a secondary screening assay that evaluates cell-cell adhesion recovery, we were able to identify harmine, that increases actin polymerization in vitro and recovers actin filament network organization and cell-cell adhesion in transformed cells, as a potential drug candidate.…”

“…In order to identify modulators of cellular actin dynamics, we have developed a cell-free screening assay based on the measurement of the incorporation of actin-Alexa488 molecules in polymerizing actin filaments by fluorescence anisotropy [28]. Briefly, a crude cellular extract is incubated in the presence of actin-Alexa488 used as tracer and the variation of Alexa488 fluorescence anisotropy, which depends upon the state of actin polymerization, is measured.…”

“…In order to identify such molecules, firstly, we developed a cell-free high-throughput screening assay for compounds that increase the actin polymerization rate constant and the F-actin to G-actin ratio in the presence of cellular extracts [28]. Then, we assessed the potential of the hit compound to restore the actin cytoskeleton and reverse the malignant phenotype in the E/F cells, as compared to their non-transformed NIH-3T3 counterparts.…”

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

“…Time-resolved fluorescence anisotropy has been used on cells for single point measurements (Keating and Wensel, 1991;Spitz et al, 2007;Swaminathan et al, 1997;Tramier et al, 2000) and for mapping solvent interactions in microfluidic devices (Benninger et al, 2005a), as well as the viscosity in the cell cytoplasm (Clayton et al, 2002;Lidke et al, 2003;Suhling et al, 2004) and membrane (Botchway et al, 2011).…”