Intermediate filaments are common structural elements found in abundance in all metazoan cells, where they form networks that contribute to the elasticity. Here, we report measurements of the linear and nonlinear viscoelasticity of networks of two distinct intermediate filaments, vimentin and neurofilaments. Both exhibit predominantly elastic behavior with strong nonlinear strain stiffening. We demonstrate that divalent ions behave as effective cross-linkers for both networks, and that the elasticity of these networks is consistent with the theory for that of semiflexible polymers. DOI: 10.1103/PhysRevLett.104.058101 PACS numbers: 87.15.La, 83.60.Df, 83.80.Rs, 87.16.Ka The mechanical response of cells depends largely on the structure and elasticity of their cytoskeleton, consisting of a variety of biopolymer networks, including filamentous actin, microtubules, and intermediate filaments (IFs) [1]. While filamentous actin and microtubules have been extensively studied, much less is known about IFs, although some key parameters such as their persistence length have been measured. Compared to actin and microtubules, IFs are more varied and specialized. Their networks are nevertheless cytoskeletal components contributing to the elasticity of the cell: there are five families of IFs found in a variety of cell types, ranging from muscles to neurons. Intermediate filament networks exhibit pronounced nonlinear elasticity similar to that observed in actin networks that are cross-linked. However, there are myriad associated actin-binding proteins that lead to this cross-linking; by contrast, fewer cross-linking proteins have been identified for IFs. Thus, the origin of the nonlinear elasticity in IF networks has not been identified.Here, we investigate the elasticity of two different IF networks, vimentin and neurofilaments (NFs). The networks exhibit remarkably similar mechanical properties: they are weak elastic solids even at the lowest frequencies probed and they exhibit strong nonlinear strain stiffening over several decades in stress. This behavior requires cross-linking of the network and we show that divalent ions act as effective cross-linkers. By comparing the linear and nonlinear macroscopic behavior, we extract microscopic network parameters. These observations suggest a general design principle for regulating the elasticity of intermediate filament networks even in the absence of specific cross-linking proteins.To explore the generality of this behavior, we use NFs, found only in neurons, and vimentin, found in nearly all mesenchymal cells. The main difference between these IFs lies in the length of their negatively charged carboxy terminal sidearms; NF sidearms are much longer than those of vimentin. Furthermore, optimal assembly is obtained at different ionic conditions, with NFs favoring a pH of 6.2, where vimentin does not assemble properly [2]. Neurofilaments are purified from bovine spinal cords [3][4][5]: fresh tissue is homogenized, then centrifuged, after which the crude NF pellet is purified ove...

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Stomata play an important role in plant innate immunity by limiting pathogen entry into leaves but molecular mechanisms regulating stomatal closure upon pathogen perception are not well understood. Here we show that the Arabidopsis thaliana L-type lectin receptor kinase-V.5 (LecRK-V.5) negatively regulates stomatal immunity. Loss of LecRK-V.5 function increased resistance to surface inoculation with virulent bacteria Pseudomonas syringae pv tomato DC3000. Levels of resistance were not affected after infiltration-inoculation, suggesting that LecRK-V.5 functions at an early defense stage. By contrast, lines overexpressing LecRK-V.5 were more susceptible to Pst DC3000. Enhanced resistance in lecrk-V.5 mutants was correlated with constitutive stomatal closure, while increased susceptibility phenotypes in overexpression lines were associated with early stomatal reopening. Lines overexpressing LecRK-V.5 also demonstrated a defective stomatal closure after pathogen-associated molecular pattern (PAMP) treatments. LecRK-V.5 is rapidly expressed in stomatal guard cells after bacterial inoculation or treatment with the bacterial PAMP flagellin. In addition, lecrk-V.5 mutants guard cells exhibited constitutive accumulation of reactive oxygen species (ROS) and inhibition of ROS production opened stomata of lecrk-V.5. LecRK-V.5 is also shown to interfere with abscisic acid-mediated stomatal closure signaling upstream of ROS production. These results provide genetic evidences that LecRK-V.5 negatively regulates stomatal immunity upstream of ROS biosynthesis. Our data reveal that plants have evolved mechanisms to reverse bacteria-mediated stomatal closure to prevent long-term effect on CO2 uptake and photosynthesis.

Microtubules are filamentous protein biopolymers found in eukaryotic cells. They form networks that guide active intracellular transport and support the overall cell structure. Microtubules are very rigid polymers, with persistence lengths as large as a millimeter. As such, they constitute an example of rodlike polymers, whose mechanical and rheological properties are as yet poorly understood. We measure the linear and nonlinear viscoelastic properties of isotropic solutions of purified microtubules, as well as networks permanently cross-linked with biotin−NeutrAvidin. In the linear regime both solutions and networks are soft elastic materials with elastic moduli on the order of a few pascals. The elastic moduli show a power-law dependence on tubulin concentration, c T, with G‘ ∼ c T ν, where ν ≈ 1.4 for solutions and increases slightly to ν ≈ 1.6−1.8 for networks. At large deformations, we observe a concentration-dependent yield stress. The rheology of microtubule solutions cannot be explained by the Doi−Edwards model, which treats noninteracting rigid rods. Instead, they show behavior very similar to the permanently cross-linked networks, suggesting the presence of effective cross-linking even in pure microtubule solutions. We develop a simple model based on transient cross-linking interactions between microtubules to interpret the rheological response. We also calculate a lower bound estimate of the strength of this interaction. Our data provide a framework with which to understand the dynamics and mechanics of more physiological networks of microtubules with microtubule-associated cross-linking and motor proteins, and ultimately to understand the role of microtubules in cell mechanics.