The difference in FRET efficiency fluctuation at each end of the actin filament

Abstract

Actin filaments are involved in various cell motility processes. Actin polymerization is primarily governed by monomer association and dissociation occurring at the rapid-growing end called the barbed end, which generates the force to push the plasma membrane forward. Individual actin filaments bind to one nucleotide and its hydrolysis energy is used to maintain the filamentous form by changing the characteristics of the subunits. The asymmetry of the individual actin filaments is also important for detecting the asymmetry of the cell. However, asymmetry at the subunit level including conformational and temporal changes in actin filaments has not been visualized yet. Here, we used "Forster (or fluorescence) resonance energy transfer (FRET)-actin filament" by copolymerizing an equal amount of donor and acceptor labelled actin. FRET efficiency change was measured along each actin filament under a light microscope. The FRET efficiency was lower near the end region than in the interior regions. Fluctuations in the FRET efficiency (fFRET) were used to monitor local flexibilities along each actin filament. The fFRET was larger near the end region than the interior region. Our quantified data showed that spatial change of fFRET along actin filaments was rapidly decayed from the barbed end from near the pointed end toward internal region, suggesting that the behaviour of actin subunits near ends is affected from each end. Our result revealed that actin filaments have different orientations locally. These orientations appear when actin forms filaments, which may contribute the cell motility.