Simultaneous quantification of actin monomer and filament dynamics with modeling-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 β- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that β-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the β-actin monomer pool. Finally, we used modeling-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 experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments.
Department of Cell Biology and Physiology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA mkapust@med.unc.edu evitriol@ufl.edu.
Maryna
M
Kapustina
Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA.
Tracy-Ann
TA
Read
Department of Anatomy and Cell Biology, University of Florida, Gainesville, FL 32610, USA mkapust@med.unc.edu evitriol@ufl.edu.
Eric A
0000-0003-3398-1450
EA
Vitriol
D008841:Actin Cytoskeleton / Q000378:metabolism* / Q000528:radiation effects; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001369:Axons / Q000378:metabolism / Q000528:radiation effects; D002454:Cell Differentiation / Q000528:radiation effects; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008027:Light; D008954:Models, Biological
D000199:Actins; D049452:Green Fluorescent Proteins
Actin; Axonal cytoskeleton; Cofilin; Modeling; Photoactivation