Microscopic and structural observations of actin filament capping and severing by cytochalasin D

• 成果类型: Article
• 署名作者: Mitani, Takahiro; Takeda, Shuichi; Oda, Toshiro; Narita, Akihiro; Maeda, Yuichiro; Honda, Hajime; Fujiwara, Ikuko
• 署名单位: Nagaoka University of Technology; Nagoya University; Tokai Gakuin University; Nagoya University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-14268
• DOI: 10.1073/pnas.2502164122
• 发表日期: 2025-07-22
• 关键词: barbed-end; crystal-structure; atp-actin; adp-actin; binding; mechanism; polymerization; gelsolin; refinement; elongation

摘要

Cytochalasin D (CytoD), a widely used actin inhibitor, is typically employed in cell studies as a simple barbed end capper. However, accumulating evidence suggests broader effects on actin dynamics. We addressed this by observing single actin filaments using total internal reflection fluorescence microscopy. Our depolymerization assay confirmed that, at nanomolar concentrations, CytoD tightly caps barbed ends. The K1/2 for inhibition was 4.1 nM, consistent with previous bulk measurements, and our approach revealed a capping duration of similar to 2 min. In polymerization assays, nanomolar CytoD completely suppressed barbed end elongation. Interestingly, at subnanomolar concentrations, CytoD caps barbed ends only transiently, rapidly associating and dissociating under both polymerizing and depolymerizing conditions. We interpreted this contradictory behavior as arising from differences in binding modes: capping one strand (fast dissociation) or both strands (slow dissociation). CytoD severs actin filaments at micromolar levels, a concentration range commonly used in cell biological studies. Although the severing rate is slower than cofilin, its higher frequency leads to filament fragmentation. Severing activity was suppressed by inorganic phosphate or cofilin. Our crystal structure of CytoD bound to filamentous conformation (F-form) actin showed that CytoD fits better in the hydrophobic cleft of F-form actin than monomeric conformation actin, explaining its preference for barbed end subunits. CytoD prevents barbed end depolymerization by stabilizing the terminal subunits in the F-form, which is supported by our MD simulations. These findings reveal the molecular mechanisms by which CytoD modulates actin dynamics and highlight the need for careful dosage control when treating cells with CytoD.