Fidgetin binds spastin to attenuate the microtubule-severing activity.

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Microtubule-severing enzymes such as spastin, katanin, and fidgetin, characterized by their AAA ATPase domains, are pivotal in modulating microtubule dynamics and behavior across various cellular processes. While spastin and katanin are recognized for their predominant and robust severing of stable microtubules, thereby enhancing microtubule turnover, fidgetin exhibits comparatively weaker severing activity and selectively targets labile microtubules. The interplay among these enzymes and their mutual regulatory mechanisms remains inadequately understood. In this study, we elucidate the functional interaction between spastin and fidgetin, focusing on their roles in microtubule severing and neurite outgrowth. Our findings demonstrate that fidgetin serves as a negative regulator of spastin's severing activity. Co-expression assays revealed that fidgetin significantly attenuates spastin's severing efficiency, as confirmed by fluorescence-based microtubule polymerization assays and quantitative imaging of microtubule dynamics. Co-immunoprecipitation and Förster Resonance Energy Transfer (FRET) analyses further established a direct interaction between fidgetin and spastin, suggesting that fidgetin modulates spastin's activity through direct binding, possibly contributing to forming the hetero-hexmeric ring for their severing activities. Functionally, spastin overexpression in neuronal cells enhances neurite outgrowth, an effect that is suppressed upon co-expression with fidgetin, indicating that fidgetin counterbalances spastin's activity to regulate neurite extension. Therefore, this study uncovers a previously unrecognized mechanism by which fidgetin modulates spastin's function, providing critical insights into the intricate regulation of microtubule severing. These findings have significant implications for therapeutic strategies targeting microtubule-severing activities, particularly in neurodevelopmental and neurodegenerative disorders where microtubule dysregulation is a hallmark.