Journal of Molecular Biology
Volume 411, Issue 5, 2 September 2011, Pages 1062-1071
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Nonlinear Viscoelasticity of Actin Transiently Cross-linked with Mutant α-Actinin-4

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Abstract

Filamentous actin and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. They can also play a critical role in disease; for example, mutations in α-actinin-4 (Actn4), a dynamic actin cross-linking protein, cause proteinuric disease in humans and mice. Amino acid substitutions strongly affect the binding affinity and protein structure of Actn4. To study the physical impact of such substitutions on the underlying cytoskeletal network, we examine the bulk mechanical behavior of in vitro actin networks cross-linked with wild-type and mutant Actn4. These networks exhibit a complex viscoelastic response and are characterized by fluid-like behavior at the longest timescales, a feature that can be quantitatively accounted for through a model governed by dynamic cross-linking. The elastic behavior of the network is highly nonlinear, becoming much stiffer with applied stress. This nonlinear elastic response is also highly sensitive to the mutations of Actn4. In particular, we observe that actin networks cross-linked with Actn4 bearing the disease-causing K255E mutation are more brittle, with a lower breaking stress in comparison to networks cross-linked with wild-type Actn4. Furthermore, a mutation that ablates the first actin binding site (ABS1) in Actn4 abrogates the network's ability to stress-stiffen is standard nomenclature. These changes in the mechanical properties of actin networks cross-linked with mutant Actn4 may represent physical determinants of the underlying disease mechanism in inherited focal segmental glomerulosclerosis.

Introduction

The functional behavior of cells is strongly influenced by the mechanical properties of their cytoskeleton. Cytoskeletal elasticity is governed by a complex interplay of biopolymer networks and their associated cross-linking proteins.1, 2, 3 The response of such networks depends sensitively on the timescale on which they are probed and is best quantified by a measure of the frequency-dependent mechanical response or viscoelasticity of the network. The richness of the mechanical behavior of these networks in vivo has led to extensive in vitro studies of networks of individual biopolymers. Many studies have focused on reconstituted networks of filamentous actin (F-actin), which make an essential contribution to the mechanics of the cytoskeleton of many cells.4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 F-actin is composed of 42-kDa monomers that assemble into double-stranded semiflexible filaments. In the absence of cross-linkers, F-actin forms an entangled network that behaves like a weak viscoelastic solid. However, the addition of a wide range of cross-linking proteins such as scruin, filamin, fascin, and α-actinin-4 (Actn4) dramatically strengthens these networks.15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 Moreover, cross-linked F-actin networks often exhibit strain stiffening; their stiffness increases dramatically upon the application of an external load that induces network strain.

Physiologically, the elastic properties of the actin cytoskeleton are critical to the function of cells. Of particular interest here are podocytes-highly differentiated epithelial cells unique to the kidney. Podocytes possess multiple cellular extensions, known as foot processes, which interdigitate over capillaries, contributing to the kidney's filtration barrier and preventing deformation due to distensive forces related to blood flow. Each foot process contains an actin-rich contractile apparatus that is cross-linked, in part, by Actn4.30, 31 Multiple proteins localize to foot processes and cause proteinuric kidney disease when mutated in humans and mice.32, 33, 34, 35, 36

Mutations in the gene encoding Actn4 cause an inherited form of proteinuric kidney disease, focal segmental glomerulosclerosis.32, 33, 34, 35, 36 The associated actin-binding domain of these mutant Actn4 can be generated as glutathione S-transferase fusion proteins. Through the use of actin cosedimentation, the dissociation constants of the various forms of Actn4 can be measured, allowing the determination of their binding affinity for actin filaments. The wild-type (WT) dissociation constant is measured to be KdWT = 31.72 μM.37 By comparison, one particular disease-causing mutation of the lysine residue at position 255 to glutamate (K255E) increases actin affinity (KdK255E = 5.33 μM). This is mediated by the exposure of an additional actin binding site (ABS1) that is normally buried and relatively inactive in the WT Actn4 protein.37 Structural studies of the actin-binding domains of actinin family members and other related proteins suggest a picture in which the actin-binding domain of Actn4 undergoes a conformational change between an “open” configuration, in which ABS1 is exposed, and a “closed” configuration, in which ABS1 is largely buried.38, 39, 40 Consistent with this model, when ABS1 function is ablated with a double-amino-acid substitution of the glutamine at position 52 and the threonine residue at position 55 (QTAA), there is only a modest effect on Actn4 binding to actin filaments (KdQTAA = 63.31 μM). However, when the QTAA mutation is present in parallel with K255E (K255E/QTAA), the binding affinity of the double mutant Actn4 reverts back to a level similar to that of WT Actn4, with KdDM = 33.06 μM.37 These data support the picture that the disease-causing K255E mutation shifts the balance of the Actn4 actin-binding domain toward the open configuration.

While this picture based on structural studies provides insight, understanding the consequences of these disease-causing mutants on the mechanical properties of the F-actin networks is essential. For example, networks cross-linked with the K255E mutant exhibit structural relaxation below a relaxation frequency, which is an order of magnitude lower than those cross-linked with WT Actn4, providing support for the mechanical consequences of this picture.41 However, while the complex linear viscoelastic behavior demonstrates the richness of the network's mechanical properties, the functioning of these networks in cells depends not only on their linear properties but also, critically, on their nonlinear properties,3, 7, 9, 20 which remain largely unexplored.17 An understanding of the structure and viscoelasticity of networks cross-linked with WT and mutant Actn4 is essential to elucidate the full consequences of these mutations on the networks and on the mechanical origins of proteinuric kidney disease.

Here, we discuss the results of a detailed, systematic investigation of the linear and nonlinear viscoelastic properties of networks of F-actin cross-linked with Actn4. We utilize four different types of Actn4—WT, K255E, QTAA, and K255E/QTAA double mutant—to explore the effects of mutations on network viscoelasticity. We show that the networks form cross-linked elastic gels and can exhibit strong nonlinearity as applied stress is increased. Notably, the presence of the ABS1 binding site plays a crucial role in nonlinear strain-stiffening. In addition, we demonstrate that the K255E mutation results in the formation of more brittle networks, with a breaking stress nearly an order of magnitude lower than those of corresponding networks formed with WT Actn4. The frequency dependence of the viscoelastic response of both WT- and mutant-Actn4-cross-linked networks is quantitatively described by a network model involving transient cross-links,29, 42 further confirming that network dynamics can be understood within the framework of ABS1 activity.

Section snippets

Results and Discussion

To investigate the microstructure of actin networks cross-linked with WT or mutant Actn4, we polymerize purified monomeric actin in vitro in their presence. As actin filaments elongate, they are cross-linked by WT Actn4 to form nearly isotropic, finely reticulated three-dimensional networks, as shown in Fig. 1a.37, 29, 48 Crucially, such microscopy demonstrates that the reconstituted networks are free of large-scale inhomogeneities. By comparison, networks polymerized in the presence of K255E

Conclusion

The ABS1 actin binding site within Actn4 (and other α-actinins) is highly conserved through evolution, suggesting an important role for this domain in the protein's function. Mutations in Actn4 appear to disrupt the proper folding of the protein, leading to variations in the accessibility of ABS1 and, thus, in the affinity for actin filaments.37 Such variations in the linkers' binding kinetics are manifest in network relaxation rates and are consistent with the picture of ABS1-governed binding

Materials and network visualization

Purified rabbit muscle globular actin (G-actin) is a kind gift of Fumihiko Nakamura (Brigham and Women's Hospital, Boston, MA, USA). Full-length human recombinant Actn4 protein is expressed in and purified from baculovirus-infected Sf21 insect cells by ProteinOne (Bethesda, MD). To form fluorescently labeled actin–Actn4 networks in vitro, we diluted actin to 1 μM in polymerization buffer [100 mM NaCl, 10 mM Tris–HCl (pH 7.4), 1 mM MgCl2, 0.5 mM ethylene glycol bis(β-aminoethyl ether) N,N

Acknowledgements

The authors acknowledge the insights of Sirfyl Pincus and inspiration from Judy Bowers. This work was supported, in part, by the National Science Foundation (DMR-1006546), the Harvard Materials Research Science and Engineering Center (DMR-0820484), the National Institutes of Health (DK59588), the United States Department of Energy (FG02-97ER25308), the Foundation for Fundamental Research on Matter, and the Netherlands Organization for Scientific Research.

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