Simultaneous quantification of actin monomer and filament dynamics with modelling assisted analysis of photoactivation

Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three dimensional cellular environment. We use this method to determine differences in the dynamic behavior of beta and gamma actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that beta actin exists in a higher monomer-to-filament ratio. We also demonstrate that Cofilin equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the beta actin monomer pool. Finally, we used modelling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the user’s own experimental system, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments. Jo ur na l o f C el l S ci en ce • A dv an ce a rt ic le