Computational tools for quantifying actin filament numbers, lengths, and bundling

Abstract

ABSTRACT The actin cytoskeleton is a dynamic filamentous network that assembles into specialized structures to enable cells to perform essential processes. Direct visualization of fluorescently-labeled cytoskeletal proteins has provided numerous insights into the dynamic processes that govern the assembly of actin-based structures. However, accurate analysis of these experiments is often complicated by the interdependent and kinetic natures of the reactions involved. It is often challenging to disentangle these processes to accurately track their evolution over time. Here, we describe two programs written in the MATLAB programming language that facilitate counting, length measurements, and quantification of bundling of actin filaments visualized in fluorescence micrographs. To demonstrate the usefulness of our programs, we describe their application to the analysis of two representative reactions: (1) a solution of pre-assembled filaments under equilibrium conditions, and (2) a reaction in which actin filaments are crosslinked together over time. We anticipate that these programs can be applied to extract equilibrium and kinetic information from a broad range of actin-based reactions, and that their usefulness can be expanded further to investigate the assembly of other biopolymers.