Quantifying Actin Persistence Length in S. pombe and S. cerevisiae: The Impact of Data Size, Filament Length and...

Actin filaments exhibit isoform and species-specific mechanical properties that are commonly quantified via their persistence length, yet the reliability of such measurements depends not only on the analysis method but also on dataset characteristics. Here, we measure and compare the persistence lengths of actin filaments from Schizosaccharomyces pombe and Saccharomyces cerevisiae as two exemplars, focusing on statistical and methodological factors that influence their determination. We evaluate common cosine-correlation-based analysis strategies and systematically assess the impact of filament length and dataset size on the accuracy of extracted persistence lengths. Because the bending stiffness of actin filaments can scale non-linearly with filament numbers in bundles, even small errors in the persistence length can have disproportionately large impact on the estimation of mechanical behavior. We validate our findings through comparative analysis utilizing simulated thermal fluctuations of semi-flexible polymers. Our results reinforce the importance of filament length and dataset size for the reliability of persistence length estimation, suggesting that their influence may have been underestimated. We introduce a weighting strategy that reduces the influence of error-dominated correlation regimes and improves the robustness of the analysis. This work contributes a quantitative framework for designing experiments and analyzing actin filament mechanics, ensuring robust and reproducible persistence length measurements. Furthermore, applying this approach revealed a significant difference in the persistence lengths of S. pombe and S. cerevisiae actin filaments.