Reversibility and Viscoelastic Properties of Micropillar Supported and Oriented Magnesium Bundled F-Actin

PLOS ONE, Dec 2019

Filamentous actin is one of the most important cytoskeletal elements. Not only is it responsible for the elastic properties of many cell types, but it also plays a vital role in cellular adhesion and motility. Understanding the bundling kinetics of actin filaments is important in the formation of various cytoskeletal structures, such as filopodia and stress fibers. Utilizing a unique pillar-structured microfluidic device, we investigated the time dependence of bundling kinetics of pillar supported free-standing actin filaments. Microparticles attached to the filaments allowed the measurement of thermal motion, and we found that bundling takes place at lower concentrations than previously found in 3-dimensional actin gels, i.e. actin filaments formed bundles in the presence of 5–12 mM of magnesium chloride in a time-dependent manner. The filaments also displayed long term stability for up to hours after removing the magnesium ions from the buffer, which suggests that there is an extensive hysteresis between cation induced crosslinking and decrosslinking.

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Reversibility and Viscoelastic Properties of Micropillar Supported and Oriented Magnesium Bundled F-Actin

August Reversibility and Viscoelastic Properties of Micropillar Supported and Oriented Magnesium Bundled F-Actin Timo Maier 0 1 2 Tamás Haraszti 0 1 2 0 1 Max Planck Institute for Intelligent Systems, Department of New Materials and Biosystems , Heisenberg str. 3, D-70569 Stuttgart, Germany , 2 University of Heidelberg, Institute of Physical Chemistry, Department of Biophysical Chemistry , Im Neuenheimer Feld 253, D-69120 Heidelberg , Germany 1 Funding: This work was financially supported by the Max Planck Society. Part of the research leading to these results has received funding from the ERC Advanced grant “SynAd” (No° 294852). This work is also part of the excellence cluster CellNetwork at the University of Heidelberg ( 2 Editor: Pontus Aspenstrom , Karolinska Institutet, SWEDEN Filamentous actin is one of the most important cytoskeletal elements. Not only is it responsible for the elastic properties of many cell types, but it also plays a vital role in cellular adhesion and motility. Understanding the bundling kinetics of actin filaments is important in the formation of various cytoskeletal structures, such as filopodia and stress fibers. Utilizing a unique pillar-structured microfluidic device, we investigated the time dependence of bundling kinetics of pillar supported free-standing actin filaments. Microparticles attached to the filaments allowed the measurement of thermal motion, and we found that bundling takes place at lower concentrations than previously found in 3-dimensional actin gels, i.e. actin filaments formed bundles in the presence of 5-12 mM of magnesium chloride in a time-dependent manner. The filaments also displayed long term stability for up to hours after removing the magnesium ions from the buffer, which suggests that there is an extensive hysteresis between cation induced crosslinking and decrosslinking. - Competing Interests: The authors have declared that no competing interests exist. Actin is one of the most abundant cytoskeletal proteins with crucial roles in maintaining the cellular shape, elasticity, adhesion and motility [1–3]. In its natural monomer form it is a globular protein (G-actin), but in cells and under proper buffer conditions these globular units dynamically assemble into filaments (F-actin). Numerous previous studies have analyzed the morphology and rheological properties of actin gels. In vitro, actin filaments can reach a length of *20–30 μm. These studies show that the actin filaments possess a negative net charge with a linear charge density of about 4 e−/nm, a helical structure with a twisting increment of *36 nm/turn and a diameter of *7–9 nm [2, 4–6]. Mechanically, actin filaments are semiflexible, with a persistence length in the order of 8–17 μm[7, 8], which is about 1/4–1/2 of their contour length, depending on the presence and type of the stabilization agent. The rheological properties of actin gels depend on the concentration of actin and the crosslinker, as well as the chemical composition of the latter [9–16] (for a summary see Ref. [17]). While chemistry can be precisely controlled in these in vitro gel experiments, determination of network formation dynamics is greatly hindered because polymerization and bundling (crosslinking) occur simultaneously. Nevertheless, several physical and chemical parameters of network formation have been identified in such actin gels. For example, using light scattering of 0.5 mg/ml (c.a. 12 μM) actin solutions, Tang and Janmey showed that actin is bundled by divalent cations at various concentrations, i.e. Co2+ at 5.5 mM, Mn2+ at 7 mM, Ca2+ at 20 mM and Mg2+ at 27 mM[18]. Later, experiments indicated that the elasticity of isotropic and nematic actin gels depend on the magnesium concentration at much lower values than in the previous study, but a detailed correlation was not investigated [19]. Furthermore, experiments using actin comets, protrusion systems formed by polymerizing actin bundles, also indicated an effect at low magnesium concentrations [20]. The aforementioned studies have described the rheological properties of actin gels by modeling the filaments as linear, charged, semiflexible polyelectrolytes interacting with the divalent cations according to the counterion condensation theory. However, emerging information suggests a deviation from this classical mechanism, indicating the ability of divalent cations to promote bundling of filaments at lower concentrations than the previously estimated critical values. Therefore, anchoring actin filaments on supporting pillars or microparticles allows for dynamic control of the chemical environment resulting in time dependent control of the morphology and mechanical properties of the network [4, 21–23]. While this anchoring helps separate the polymerization and bundling processes, it also imposes a specific mechanical constraint: the diffusion of the filaments is hindered, and the bundling evolves in a characteristic zippering manner. Qualitatively, this (...truncated)


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Timo Maier, Tamás Haraszti. Reversibility and Viscoelastic Properties of Micropillar Supported and Oriented Magnesium Bundled F-Actin, PLOS ONE, 2015, Volume 10, Issue 8, DOI: 10.1371/journal.pone.0136432