Toshino Kenta, Yamazaki Yosuke, Ando Shunsuke, Kaneda Ryuichi, Ono Kazunori, Suzuki Takahiro, Kijima Saku T, Uyeda Taro Q P
Department of Pure and Applied Physics, Graduate School of Advanced Science and Engineering, Waseda University, Shinjuku, Tokyo, Japan.
Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Ibaraki, Japan.
J Biol Chem. 2025 Jun 24;301(8):110417. doi: 10.1016/j.jbc.2025.110417.
Phalloidin (Ph) is widely used for fluorescent labeling of actin filaments. We observed ADP-actin filaments labeled with rhodamine phalloidin (RhPh) or Alexa488-Ph in vitro and discovered that the fluorescence intensities along the filaments showed a mottled pattern of bright and dark regions. Filaments labeled with substoichiometric RhPh exhibited more significant fluorescence inhomogeneities than those labeled with excess RhPh. Because the quantum yield of Alexa488 fluorescence is hardly affected by the environment, we concluded that the inhomogeneities arise from nonuniform Ph binding density rather than locally inhomogeneous quantum yield of the fluorophores. Simulations assuming random RhPh binding alone partially produced fluorescence inhomogeneities, but the degree of inhomogeneities was significantly smaller than the experimental results. Furthermore, filaments colabeled with RhPh and Alexa488-Ph showed a positive correlation in fluorescence intensities of Rh and Alexa488. Moreover, addition of inorganic phosphate suppressed the fluorescence inhomogeneities and the correlation between the Rh and Alexa488 fluorescence intensities. These results indicated that two mechanisms contribute to the nonuniform binding density of Ph: (i) stochastic binding and (ii) local differences in Ph binding affinity caused by inorganic phosphate-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.
鬼笔环肽（Ph）被广泛用于肌动蛋白丝的荧光标记。我们在体外观察了用罗丹明鬼笔环肽（RhPh）或Alexa488-Ph标记的ADP-肌动蛋白丝，发现沿丝的荧光强度呈现出亮区和暗区相间的斑驳图案。用亚化学计量的RhPh标记的丝比用过量RhPh标记的丝表现出更显著的荧光不均匀性。由于Alexa488荧光的量子产率几乎不受环境影响，我们得出结论，这种不均匀性源于Ph结合密度的不均匀，而不是荧光团局部不均匀的量子产率。仅假设随机RhPh结合的模拟部分产生了荧光不均匀性，但不均匀程度明显小于实验结果。此外，用RhPh和Alexa488-Ph共标记的丝在Rh和Alexa488的荧光强度上呈现正相关。此外，添加无机磷酸盐可抑制荧光不均匀性以及Rh和Alexa488荧光强度之间的相关性。这些结果表明，有两种机制导致了Ph结合密度的不均匀：（i）随机结合和（ii）由肌动蛋白丝对无机磷酸盐敏感的结构多态性引起的Ph结合亲和力的局部差异。这种结构多态性也可能影响各种肌动蛋白结合蛋白的结合，有助于体内肌动蛋白丝的功能分化。此外，那些斑驳的荧光图案随时间动态波动。这些时间荧光波动需要葡萄糖和葡萄糖氧化酶，但被生育三烯酚抑制了，这可能反映了受氧清除剂和三重态猝灭剂影响的荧光团的光物理性质。综上所述，我们对肌动蛋白丝的结构多态性提供了新的见解。
