Spatiotemporal fluctuations in fluorescence intensity of rhodamine phalloidin-labeled actin filaments.

Phalloidin is widely used for fluorescent labeling of actin filaments. We observed ADP-actin filaments labeled with rhodamine-phalloidin or Alexa488-phalloidin in vitro and discovered that the fluorescence intensities along the filaments showed a mottled pattern of bright and dark regions. Filaments labeled with sub-stoichiometric rhodamine-phalloidin exhibited more significant fluorescence inhomogeneities than those labeled with excess rhodamine-phalloidin. Because the quantum yield of Alexa488 fluorescence is hardly affected by the environment, we concluded that the inhomogeneities arise from non-uniform phalloidin binding density rather than locally inhomogeneous quantum yield of the fluorophores. Simulations assuming random rhodamine-phalloidin binding alone partially produced fluorescence inhomogeneities, but the degree of inhomogeneities was significantly smaller than the experimental results. Furthermore, filaments co-labeled with rhodamine-phalloidin and Alexa488-phalloidin showed a positive correlation in fluorescence intensities of rhodamine and Alexa488. Moreover, addition of Pi suppressed the fluorescence inhomogeneities and the correlation between the rhodamine and Alexa488 fluorescence intensities. These results indicated that two mechanisms contribute to the non-uniform binding density of phalloidin: (i) stochastic binding and (ii) local differences in phalloidin binding affinity caused by Pi-sensitive structural polymorphism of actin filaments. This structural polymorphism may also affect the binding of various actin-binding proteins, contributing to the functional differentiation of actin filaments in vivo. Moreover, those mottled fluorescence patterns dynamically fluctuated over time. These temporal fluorescence fluctuations required glucose and glucose oxidase but were suppressed by Trolox, likely reflecting photophysical properties of fluorophores influenced by oxygen scavengers and triplet-state quenchers. Taken together, we provide new insights into the structural polymorphism of actin filaments.