Background

Actin filaments (F-actin) are highly charged double-stranded semiflexible polyelectrolytes formed by the polymerization of G-actin subunits. Due to their hydrodynamic, mechanical, and electrostatic properties, these cytoskeleton filaments have the extraordinary ability to dynamically change conformations in response to alterations in G-actin concentration and type of crosslinker/binding proteins, as well as electrolyte concentration. These cytoskeleton properties are crucial for eukaryotic cells to achieve specific biological functions in different cellular compartments, which may vary depending on the cell type and location conditions. While a substantial amount of research has been done in the biochemistry and biophysics fields (Lanni and Ware, 1984; Janmey et al., 1986, 1994; Hou et al., 1990; Käs et al., 1996; Xu et al., 1998; Bonet et al., 2000; Niranjan et al., 2001; Y. H. Wang and Narayan, 2008; Crevenna et al., 2013; Del Rocio Cantero et al., 2020), the underlying principles and molecular mechanisms that support the hydrodynamic, electrostatic, and stability properties of these filaments remain elusive (Lanni and Ware, 1984; F. Wang et al., 1989; Hou et al., 1990; Käs et al., 1996; Bonet et al., 2000).

Modern dynamic light scattering (DLS) and electrophoretic light scattering (ELS) instruments are the most accurate, non-invasive, experimental techniques to characterize hydrodynamic and electrostatic properties of polydisperse charged biomolecules even at low concentrations and using small sample volumes. These instruments use advanced technology and multi-functional software to measure the hydrodynamic size, polydispersity, dispersion stability and aggregation, biomolecular charge, and zeta potential of particles and polymers immersed in aqueous biological environments. To assure high accuracy and reproducibility of the results, it is essential to use high-quality samples, accurate instruments, robust software, and optimized measurement protocols.

This article presents a complete, accurate, detailed polymerization protocol to prepare high-quality actin filament samples for light scattering experiments. Obtaining actin filament samples that are stable, dispersed, clean from aggregates and impurities, and homogenous could benefit many other scientific research groups in the field. We describe the polymerization procedure for three typical actin buffers in physiological conditions (Janmey et al., 1986, 1994; F. Wang et al., 1989). Nevertheless, this protocol can easily be adapted to prepare samples using other buffers and biological fluids. Additionally, we provide a unique methodology based on fundamental biostatistical tools (McDonald, 2009) and a measurement protocol with optimized configuration to perform DLS and ELS experiments on actin filament samples at critical G-actin concentrations. We measured actin filament’s translational diffusion coefficient and electrophoretic mobility using the same sample and experimental conditions for both DLS and ELS experiments to assure unicity and consistency in the results (Alva et al., 2022). A fitting approach was adequate to characterize other filaments’ essential properties, such as asymmetric length distribution, effective electric charge, hydrodynamic size, effective diameter, dispersion stability, aggregation, and semi-flexibility (Kroy and Frey, 1997; Tassieri et al., 2008; Alva et al., 2022). Additionally, the comparison of the light scattering results obtained for different polymerization buffers elucidated the impact of their chemical composition, reducing agents, pH values, and ionic strengths on the hydrodynamic, electrostatic, and stability properties of these actin structures. This kind of comparison and characterization provides a deeper understanding of how actin filaments behave in various cellular environments and conditions. Overall, suitable modifications of the proposed experimental method might allow other scientific groups to obtain accurate and reproducible light scattering results on other highly charged anionic polyelectrolyte filaments, commonly found in biological cells (e.g., microtubules, DNA, RNA, or filamentous viruses), having hydrodynamic, electrostatic, and stability properties resembling those characterizing actin filaments (Janmey et al., 2014).