Tracking Mitotic Spindle Dynamics and Protein Localization in Fission Yeast With <scp>FYSKA</scp> , the Fission Yeast Spindle Kymograph Analyzer

Quantitative analysis of mitotic spindle dynamics requires accurate tracking despite challenges such as cell drift, spindle rotation, and fluctuating fluorescence signals. We developed the Fission Yeast Spindle Kymograph Analyzer (FYSKA), an automated software tool that tracks the spindle and constructs kymographs of spindle-associated proteins in Schizosaccharomyces pombe. FYSKA uses fluorescent spindle pole markers to achieve sub-pixel precision, applies error correction for transient signal loss, and maintains robustness under rotation or drift. Compared to semi-automated approaches, it generates kymographs with more consistent intensity profiles and improved capture of the spindle axis. Using FYSKA, we quantified spindle length fluctuations and examined localization patterns of the kinesin-5 motor Cut7 including asymmetric spindle pole recruitment in Cut11-7. These examples show how FYSKA enables automated, reproducible analysis of mitotic spindle organization and protein dynamics.