Functional Interdependence Between the Septin and Actin Cytoskeletons

Research Article

Functional Interdependence Between the Septin and Actin Cytoskeletons

Noah Carter¹*, Sophie Reynolds² and Amelie Laurent³

1Department of Microbiology, University of Harvard University, Boston, USA
2Department of Virology, University of Cambridge, Cambridge, UK
3Department of Biochemistry, University of Lyon, France

Published: 07 May 2014

Abstract

Background: Accurate chromosome segregation during mitosis must be tightly coordinated with cytokinesis and exit from mitosis to ensure genomic stability. The anaphase spindle midzone, a structure composed of antiparallel interpolar microtubules formed between separating sister chromatids, serves as a crucial platform for orchestrating cytokinesis. Key regulatory proteins, including the centralspindlin complex, the Chromosomal Passenger Complex (CPC) component Aurora B kinase and Polo-like kinase 1 (Plk1), concentrate at the midzone and play essential roles in contractile ring formation and abscission. However, whether the midzone also actively participates in signaling pathways that regulate the timing of mitotic exit and progression into the next cell cycle phase remains less clear.

Objective: This study aimed to investigate the role of the anaphase spindle midzone and its associated key regulators, not only in cytokinesis execution but also in controlling the timing of mitotic exit events.

Methods: Human HeLa and U2OS cells were synchronized and analyzed using live-cell fluorescence microscopy and immunofluorescence. The recruitment kinetics and localization of key midzone proteins (MKLP1, Aurora B, Plk1) tagged with fluorescent proteins were monitored relative to anaphase onset and cytokinesis progression. The functional requirement for these proteins and midzone integrity was assessed using siRNA-mediated depletion and specific kinase inhibitors (ZM447439 for Aurora B, BI 2536 for Plk1). The timing of cytokinesis events (furrow ingression, midbody formation, abscission) and mitotic exit markers (Cyclin B1 degradation, Cdk1 inactivation via FRET biosensor) were quantified following perturbations. RhoA activation at the equatorial cortex was also assessed.

Results: Key regulators, including centralspindlin, Aurora B and Plk1, exhibited dynamic recruitment to the forming spindle midzone early in anaphase. Live-cell imaging revealed a strong correlation between the proper assembly and maturation of the midzone structure and the timely initiation of furrow ingression. Depletion of centralspindlin components (MKLP1) or inhibition of Aurora B or Plk1 activity not only severely impaired cytokinesis, leading to furrow regression or abscission failure, but also significantly delayed the degradation of Cyclin B1 and prolonged Cdk1 activity, indicative of a delayed mitotic exit. Perturbations that specifically disrupted midzone microtubule stability, without directly targeting kinases, also led to similar delays in mitotic exit markers, alongside cytokinesis defects. Inhibition of Aurora B or Plk1 reduced RhoA activation at the cell equator.

Conclusion: Our findings suggest that the anaphase spindle midzone functions as more than just a structural scaffold for cytokinesis. It acts as a critical signaling    hub where the assembly state and the activity of associated kinases like Aurora B and Plk1 are monitored. Proper midzone formation and function are required not only to drive cytokinesis but also to generate signals that contribute to the timely inactivation of Cdk1 and ensure coordinated exit from mitosis. This highlights a potential midzone-based checkpoint mechanism linking successful chromosome segregation and cytokinesis progression to cell cycle control.

Keywords: Anaphase; Cell Cycle Regulation; Cytokinesis; Spindle Midzone; Cyclin B1; Central spindling