Alex J. Levine

Oxidative Stress Increased Respiration and Generation of Reactive Oxygen Species, Resulting in ATP Depletion, Opening of Mitochondrial Permeability Transition, and Programmed Cell Death

Mitochondria constitute a major source of reactive oxygen species and have been proposed to integrate the cellular responses to stress. In animals, it was shown that mitochondria can trigger apoptosis from diverse stimuli through the opening of MTP, which allows the release of the apoptosis-inducing factor and translocation of cytochrome c into the cytosol. Here, we analyzed the role of the mitochondria in the generation of oxidative burst and induction of programmed cell death in response to brief or continuous oxidative stress in Arabidopsis cells. Oxidative stress increased mitochondrial electron transport, resulting in amplification of H2O2 production, depletion of ATP, and cell death. The increased generation of H2O2 also caused the opening of the MTP and the release of cytochromec from mitochondria. The release of cytochromec and cell death were prevented by a serine/cysteine protease inhibitor, Pefablock. However, addition of inhibitor only partially inhibited the H2O2 amplification and the MTP opening, suggesting that protease activation is a necessary step in the cell death pathway after mitochondrial damage.

Distinct regimes of elastic response and deformation modes of cross-linked cytoskeletal and semiflexible polymer networks

Semiflexible polymers such as filamentous actin (F-actin) play a vital role in the mechanical behavior of cells, yet the basic properties of cross-linked F-actin networks remain poorly understood. To address this issue, we have performed numerical studies of the linear response of homogeneous and isotropic two-dimensional networks subject to an applied strain at zero temperature. The elastic moduli are found to vanish for network densities at a rigidity percolation threshold. For higher densities, two regimes are observed: one in which the deformation is predominately affine and the filaments stretch and compress; and a second in which bending modes dominate. We identify a dimensionless scalar quantity, being a combination of the material length scales, that specifies to which regime a given network belongs. A scaling argument is presented that approximately agrees with this crossover variable. By a direct geometric measure, we also confirm that the degree of affinity under strain correlates with the distinct elastic regimes. We discuss the implications of our findings and suggest possible directions for future investigations.

Deformation of Cross-Linked Semiflexible Polymer Networks

Networks of filamentous proteins play a crucial role in cell mechanics. These cytoskeletal networks, together with various cross-linking and other associated proteins largely determine the (visco)elastic response of cells. In this Letter we study a model system of cross-linked, stiff filaments in order to explore the connection between the microstructure under strain and the macroscopic response of cytoskeletal networks. We find two distinct regimes as a function primarily of cross-link density and filament rigidity: one characterized by affine deformation and one by nonaffine deformation. We characterize the crossover between these two.

One- and two-particle microrheology

We study the dynamics of rigid spheres embedded in viscoelastic media and address two questions of importance to microrheology. First, we calculate the complete response to an external force of a single bead in a homogeneous elastic network viscously coupled to an incompressible fluid. From this response function we find the frequency range where the standard assumptions of microrheology are valid. Second, we study fluctuations when embedded spheres perturb the media around them and show that mutual fluctuations of two separated spheres provide a more accurate determination of the complex shear modulus than do the fluctuations of a single sphere.

Suppression of Arabidopsis vesicle-SNARE expression inhibited fusion of H2O2-containing vesicles with tonoplast and increased salt tolerance.

Intracellular vesicle trafficking performs essential functions in eukaryotic cells, such as membrane trafficking and delivery of molecules to their destinations. A major endocytotic route in plants is vesicle trafficking to the vacuole that plays an important role in plant salt tolerance. The final step in this pathway is mediated by the AtVAMP7C family of vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs) that carry out the vesicle fusion with the tonoplast. Exposure to high-salt conditions causes immediate ionic and osmotic stresses, followed by production of reactive oxygen species. Here, we show that the reactive oxygen species are produced intracellularly, in endosomes that were targeted to the central vacuole. Suppression of the AtVAMP7C genes expression by antisense AtVAMP711 gene or in mutants of this family inhibited fusion of H2O2-containing vesicles with the tonoplast, which resulted in formation of H2O2-containing megavesicles that remained in the cytoplasm. The antisense and mutant plants exhibited improved vacuolar functions, such as maintenance of ΔpH, reduced release of calcium from the vacuole, and greatly improved plant salt tolerance. The antisense plants exhibited increased calcium-dependent protein kinase activity upon salt stress. Improved vacuolar ATPase activity during oxidative stress also was observed in a yeast system, in a ΔVamp7 knockout strain. Interestingly, a microarray-based analysis of the AtVAMP7C genes showed a strong down-regulation of most genes in wild-type roots during salt stress, suggesting an evolutionary molecular adaptation of the vacuolar trafficking.

How Sandcastles Fall

Exxon Research and Engineering, Route 22 East, Annandale, N.J. 08801(February 1, 2008)Capillary forces significantly affect the stability of sandpiles. We analyze the stability of sandpileswith such forces, and find that the critical angle is unchanged in the limit of an infinitely largesystem; however, this angle is increased for finite-sized systems. The failure occurs in the bulk ofthe sandpile rather than at the surface. This is related to a standard result in soil mechanics. Theincrease in the critical angle is determined by the surface roughness of the particles, and exhibitsthree regimes as a function of the added-fluid volume. Our theory is in qualitative agreement withthe recent experimental results of Hornbaker et al., although not with the interpretation they makeof these results.81.05.Rm, 68.45.Gd, 91.50.Jc

Response function of a sphere in a viscoelastic two-fluid medium.

In order to address basic questions of importance to microrheology, we study the dynamics of a rigid sphere embedded in a model viscoelastic medium consisting of an elastic network permeated by a viscous fluid. We calculate the complete response of a single bead in this medium to an external force, and compare the result to the commonly-accepted, generalized Stokes-Einstein relation (GSER). We find that our response function is well approximated by the GSER only within a particular frequency range determined by the material parameters of both the bead and the network. We then discuss the relevance of this result to recent experiments. Finally we discuss the approximations made in our solution of the response function by comparing our results to the exact solution for the response function of a bead in a viscous (Newtonian) fluid.

Nonequilibrium mechanics and dynamics of motor-activated gels.

The mechanics of cells is strongly affected by molecular motors that generate forces in the cellular cytoskeleton. We develop a model for cytoskeletal networks driven out of equilibrium by molecular motors exerting transient contractile stresses. Using this model we show how motor activity can dramatically increase the networks bulk elastic moduli. We also show how motor binding kinetics naturally leads to enhanced low-frequency stress fluctuations that result in nonequilibrium diffusive motion within an elastic network, as seen in recent in vitro and in vivo experiments.

An Elicitor from Botrytis cinerea Induces the Hypersensitive Response in Arabidopsis thaliana and Other Plants and Promotes the Gray Mold Disease.

ABSTRACT Botrytis cinerea is a necrotrophic fungus that infects over 200 plant species. Previous studies showed that host cells collapse in advance of the hyphae, suggesting secretion of toxins or elicitors. We have partially characterized elicitor activity from intercellular fluid extracted from Arabidopsis thaliana leaves infected with B. cinerea. Treatment of intact leaves or cell cultures with either intercellular fluid from infected leaves or medium from inoculated A. thaliana cell culture induced generation of reactive oxygen species, resulting in reduced photosynthesis, electrolyte leakage, and necrotic lesions that resembled the hypersensitive response (HR). The necrosis was inhibited by diphenyleneiodonium, a specific inhibitor of NADPH oxidase, and by chelating free iron, suggesting the involvement of hydroxyl radicals. The necrosis was also suppressed in dnd1 mutants that are compromised in HR. In contrast, increased cell death was observed in acd2 mutants, indicating the involvement of the host defense signaling pathways. Treatment with the intercellular fluid from infected leaves also induced transcription of pathogenesis-related (PR) genes, such as PR-1, PR-5, HSR203J, and of senescence-associated gene SAG-13. Moreover, rapid transcription of the ethylene-dependent AtEBP gene was detected, indicating induction of ethylene production. The inter-cellular fluid from infected A. thaliana induced cell death in other plants, in line with the lack of B. cinerea specificity. In summary, the intercellular fluid mimicked a range of molecular and physiological host responses that are observed during infection with a live fungus. Moreover, it accelerated the B. cinerea infection, suggesting that the elicitor may act as a pathogenicity factor in the progression of gray mold disease.

Dynamics of viscoelastic membranes

We determine both the in-plane and out-of-plane dynamics of viscoelastic membranes separating two viscous fluids in order to understand microrheological studies of such membranes. We demonstrate the general viscoelastic signatures in the dynamics of shear, bending, and compression modes. We show that these modes remain independent in the presence of hydrodynamic interactions. The full response functions for motion both in-plane and out-of-plane are derived for the general case of viscoelastic films in contact with arbitrary viscous fluids. Specifically, we derive closed-form expressions for the in-plane longitudinal and transverse response functions for viscous membranes embedded in fluid media. We also find a screening of the otherwise two-dimensional character of the response to point forces due to the presence of the solvent.