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Spatiotemporal fluctuations in fluorescence intensity of rhodamine phalloidin–labeled actin filaments

Summary

Researchers discovered that fluorescent labels attached to actin filaments (cell structures involved in movement and shape) don’t glow uniformly along their length. Instead, they create a mottled pattern of bright and dark regions. This happens because the filament structure varies along its length, affecting how many labels stick to different parts. Interestingly, when the cell’s energy molecule contains a phosphate group, the filament structure becomes more uniform and the pattern disappears.

Background

Phalloidin is widely used for fluorescent labeling of actin filaments in cellular imaging. This study investigated unexpected observations of nonuniform fluorescence intensity patterns along actin filaments labeled with rhodamine phalloidin, suggesting structural heterogeneity in actin organization.

Objective

To determine whether spatial and temporal fluorescence intensity fluctuations in phalloidin-labeled actin filaments arise from nonuniform phalloidin binding density or from variations in fluorophore quantum yield, and to elucidate the underlying mechanisms.

Results

Spatial inhomogeneities in fluorescence were attributed to nonuniform phalloidin binding density rather than quantum yield variations. Simulations showed random binding alone could not explain the observed degree of inhomogeneity, indicating structural polymorphism of actin filaments creates regions of varying phalloidin affinity. Inorganic phosphate suppressed these inhomogeneities by stabilizing a more homogeneous actin conformation. Temporal fluorescence fluctuations depended on glucose oxidase and were suppressed by Trolox, reflecting triplet-state photophysics rather than actin dynamics.

Conclusion

Actin filaments exhibit structural polymorphism creating spatially variable phalloidin binding affinities, particularly in the ADP state, which is suppressed by bound phosphate. These findings suggest actin’s nucleotide state modulates conformational fluctuations at the submicron scale, potentially influencing actin-binding protein interactions and functional differentiation in cells. Temporal fluctuations reflect photophysical properties rather than biological phenomena.
  • Published in:Journal of Biological Chemistry,
  • Study Type:Experimental Research,
  • Source: PMID: 40570959, DOI: 10.1016/j.jbc.2025.110417
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