The Pseudomonas aeruginosa Isohexenyl Glutaconyl Coenzyme A Hydratase (AtuE) Is Upregulated in Citronellate-Grown Cells and Belongs to the Crotonase Family.

Pseudomonas aeruginosa is one of only a few Pseudomonas species that are able to use acyclic monoterpenoids, such as citronellol and citronellate, as carbon and energy sources. This is achieved by the acyclic terpene utilization pathway (Atu), which includes at least six enzymes (AtuA, AtuB, AtuCF, AtuD, AtuE, AtuG) and is coupled to a functional leucine-isovalerate utilization (Liu) pathway. Here, quantitative proteome analysis was performed to elucidate the terpene metabolism of P. aeruginosa. The proteomics survey identified 187 proteins, including AtuA to AtuG and LiuA to LiuE, which were increased in abundance in the presence of citronellate. In particular, two hydratases, AtuE and the PA4330 gene product, out of more than a dozen predicted in the P. aeruginosa proteome showed an increased abundance in the presence of citronellate. AtuE (isohexenyl-glutaconyl coenzyme A [CoA] hydratase; EC 4.2.1.57) most likely catalyzes the hydration of the unsaturated distal double bond in the isohexenyl-glutaconyl-CoA thioester to yield 3-hydroxy-3-isohexenyl-glutaryl-CoA. Determination of the crystal structure of AtuE at a 2.13-Å resolution revealed a fold similar to that found in the hydratase (crotonase) superfamily and provided insights into the nature of the active site. The AtuE active-site architecture showed a significantly broader cavity than other crotonase superfamily members, in agreement with the need to accommodate the branched isoprenoid unit of terpenes. Glu139 was identified to be a potential catalytic residue, while the backbone NH groups of Gly116 and Gly68 likely form an oxyanion hole. The present work deepens the understanding of terpene metabolism in Pseudomonas and may serve as a basis to develop new strategies for the biotechnological production of terpenoids.

Direct assessment of living cell mechanical responses during deformation inside microchannel restrictions.

The deformation of suspended cells inside microchannel restrictions mimics passive cell transportation in the blood circulation system of the body. The cells traverse or get stuck in narrow vessels, as, e.g., during the metastasis of tumor cells. In this work, the mechanical responses of suspended pancreatic cancer cells as they move through and deform inside microchannel restrictions are assessed with a cantilever-based polydimethylsiloxane (PDMS) force sensor. Incorporated into a flow cell chip, the PDMS cantilever is integrated into the boundary wall of a narrow microrestriction. Upon being forced to enter the restriction by an applied flow, the cell exerts pressure on the cantilever, which then bends. By assuming a uniformly loaded cantilever, the total force and pressure on the cantilever can be calculated using elastic beam theory. This technique has the advantage of presenting an absolute and direct measure, which is independent of the applied flow and frictional processes at the channel-cell interface; in contrast to, e.g., measuring cell mechanics indirectly via cell sliding velocities. Furthermore, a high number of cells can be examined in a short time compared to other single cell mechanical testing devices.

Elastic moduli of living epithelial pancreatic cancer cells and their skeletonized keratin intermediate filament network.

In simple epithelia, such as living epithelial pancreatic cancer cells (Panc-1), unusual amounts of keratin filaments can be found, which makes these cells an ideal model system to study the role of keratin for cell mechanical properties. In this work, the elastic moduli of Panc-1 cells and their extracted in-situ subcellular keratin intermediate filament network are determined and compared with each other. For this, the living adherent cells and their extracted keratin network were probed with local quasistatic indentation testing during large deformations using the Atomic Force Microscope (AFM). We determined the elastic modulus of the skeletonized but structurally intact keratin network to be in the order of 10 Pa, while the living cell elastic modulus ranged from 100 to 500 Pa. By removing microfilaments, microtubules, membranes and soluble cytoplasmic components during keratin network extraction, we excluded effects caused by crosslinking with other filamentous fibers and from the viscosity of the cytoplasm. Thus, the determined elastic modulus equals the actual elastic modulus inherent to such a keratin filamentous network. In our assessment of the effective mechanical contribution of the architecturally intact, skeletonized keratin network to living cell mechanics, we come to the conclusion that it plays only a very limited role. Evidently, the quantitative dominance of keratin in these cells does not reflect a strong influence on determining the cell's elastic modulus. Instead, keratin like other filamentous structures in the cell's scaffolding, e.g., F-actin and microtubuli, is one part of a greater whole.

Intense laser alignment as a route to control of surface reactions.

We explore the possibility of controlling the orientation of adsorbates, and their adsorption site, through alignment of a beam of gas-phase molecules prior to the surface reaction. To that end, we carry out classical trajectory simulations using ab initio data for the specific example of the I(2)/Si(100) adsorption reaction. I(2) is found to adsorb with the molecular axis roughly parallel to the surface plane independently of the initial alignment. The orientation of the molecule in the surface plane and the adsorption site are controllable through alignment of the gas-phase projectiles. Our results are explained in terms of the surface properties and the reaction dynamics, and the extent to which and way in which they may be generalized is discussed.

Head-neck domain of Arabidopsis myosin XI, MYA2, fused with GFP produces F-actin patterns that coincide with fast organelle streaming in different plant cells.

BACKGROUND: The cytoskeletal mechanisms that underlie organelle transport in plants are intimately linked to acto-myosin function. This function is mediated by the attachment of myosin heads to F-actin and the binding of cargo to the tails. Acto-myosin also powers vigorous cytoplasmic streaming in plant cells. Class XI myosins exhibit strikingly fast velocities and may have extraordinary roles in cellular motility. Studies of the structural basis of organelle transport have focused on the cargo-binding tails of myosin XI, revealing a close relationship with the transport of peroxisomes, mitochondria, and Golgi-vesicles. Links between myosin heads and F-actin-based motility have been less investigated. To address this function, we performed localization studies using the head-neck domain of AtMYA2, a myosin XI from Arabidopsis. RESULTS: We expressed the GFP-fused head-neck domain of MYA2 in epidermal cells of various plant species and found that it associated with F-actin. By comparison to other markers such as fimbrin and talin, we revealed that the myosin-labeled F-actin was of a lower quality and absent from the fine microfilament arrays at the cell cortex. However, it colocalized with cytoplasmic (transvacuolar) F-actin in areas coinciding with the tracks of fast organelles. This observation correlates well with the proposed function of myosin XI in organelle trafficking. The fact that organelle streaming was reduced in cells expressing the GFP-MYA2-head6IQ indicated that the functionless motor protein inhibits endogenous myosins. Furthermore, co-expression of the GFP-MYA2-head6IQ with other F-actin markers disrupted its attachment to F-actin. In nuclei, the GFP-myosin associated with short bundles of F-actin. CONCLUSION: The localization of the head of MYA2 in living plant cells, as investigated here for the first time, suggests a close linkage between this myosin XI and cytoplasmic microfilaments that support the rapid streaming of organelles such as peroxisomes. Potential roles of MYA2 may also exist in the cell nucleus. Whether the low quality of the F-actin-labeling by MYA2-head6IQ compared to other F-actin-binding proteins (ABPs) signifies a weak association of the myosin with actin filaments remains to be proven by other means than in vivo. Clues for the mode of contact between the myosin molecules and F-actin so far cannot be drawn from sequence-related data.

Cellular unbinding forces of initial adhesion processes on nanopatterned surfaces probed with magnetic tweezers.

To study the dependence of unbinding forces on the distance of molecularly defined and integrin specific c(-RGDfK-) ligand patches in initial cellular adhesion processes, we developed a magnetic tweezers setup for applying vertical forces of up to 200 pN to rat embryonic fibroblasts. The ligand patch distance is controlled with a hexagonally close packed pattern of biofunctionalized gold nanoparticles prepared by block-copolymer micelle nanolithography. Each gold nanoparticle potentially activates up to one alpha(v)beta(3)-integrin. The distances between the gold nanoparticles determine the separation of individual integrins and thus the assembly of integrin clusters. The results show an increase in cellular unbinding forces from approximately 6 to more than 200 pN for a decreasing ligand distance of 145 to 58 nm after 5 min of cell adhesion. Furthermore, we observe a strong dependence on adhesion time during the first 10 min of cell surface contact suggesting an active, cooperative cell response that is controlled by the spacing between individually activated integrins.