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International audience; A method to extract proteins from natural rubber (NR) samples has been developed to characterize the protein composition of NR by SDS-PAGE electrophoresis. While the incubation of NR pieces in aqueous buffers did not provide satisfactory results (no protein bands detected by SDS-PAGE), the solubilization of NR pieces in an organic solvent, prior to centrifugation and protein extraction from the obtained pellet, was found efficient to highlight protein bands by SDS-PAGE. Pure cyclohexane was used to solubilize NR pieces and resulted in a low share of nitrogen in the pellet (29%). The addition of 10% (v/v) ethanol to cyclohexane allowed to significantly increase this share to 73% and to detect 13 protein bands by SDS-PAGE. The molecular weights of the four most intense bands suggest the presence in NR of the following proteins: rubber elongation factor, small rubber particle protein, hevamine, and β-1-3 glucanase.

This work is part of a larger project aiming at elucidating the mechanisms by which the biochemical components naturally occurring in Hevea brasiliensis latex influence the structuration of natural rubber (NR). To achieve this overall objective, our strategy consists of measuring the physical properties of NR samples made of reconstructed lattices which were obtained by mixing different proportions of C-serum and bottom fraction (lutoids) to rubber fraction made of small and large particles. This work represents the first step of the above-mentioned project where we carried out a systematic study to identify and locate the main biochemical components of latex that might drive NR structuration. Fresh latex from RRIM600 and PB235 clones was fractionated by centrifugation into 4 fractions: large rubber particles, small rubber particles, C-serum and bottom fraction (lutoids + Frey-Wyssling particles). The fractions were further analyzed to provide a comprehensive description of their biochemical composition (lipids, proteins, minerals) in comparison with the original whole latex. The results gathered in this work are essential for further study of the quantitative influence of each component on NR structuration and properties.

International audience; The latex collected from the Hevea brasiliensis tree is today the only commercial source of natural rubber (NR), the cis-1,4-polyisoprene polymer, a strategic raw material. The Hevea latex is a very complex material both in its structure and composition. In terms of structure, it is a colloidal dispersion where various micrometric objects, mainly rubber particles and lutoids, are dispersed in the cytoplasmic serum (C-serum). The rubber fraction is the most abundant, followed by the C-serum and the lutoids. In terms of composition, the fresh latex contains about 60% of water, 35% of cis-1,4-polyisoprene and 5% of non-isoprene molecules. These non-isoprenes are biochemical compounds mostly including proteins, lipids, carbohydrates and minerals, and their distribution in the fractions of latex is not homogeneous. Although the non-isoprenes represent a minor part of the latex, some of them are retained in NR after latex processing and recognized to play a crucial role on the NR properties. Actually, the non-isoprene molecules are likely behind the better mechanical properties of NR over its synthetic counterpart, but they are also responsible for the high variability of NR quality. This variability of NR quality is a major drawback in NR industry and is directly linked to the latex composition, which is influenced by various physical and physiological parameters. The biochemical composition of latex matters, and this chapter is thus an overview of the protein, lipid, carbohydrate and mineral contents in latex, as well as their distribution in the three main fractions of latex.

Improving the productivity of existing rubber plantations is necessary to cope with the growing demand for natural rubber (NR) while limiting the environmental and social impacts linked to the expansion of rubber cultivation at the expense of natural ecosystems and food crops. The effect of fertilization on NR yield is still unclear and is poorly documented particularly in Thailand, the world leader in NR production. Hence, the main objective of this study was to re-assess the possible effects of fertilization on the yield of intensively managed rubber plantations in Thailand. Our main hypothesis was that the effect of fertilization would be higher with intensive latex harvesting practices (high tapping frequency with ethylene stimulation). To test this hypothesis, we set-up a split-plot experiment with four fertilization doses (T1, no fertilization, T2, T3, T4, respectively low, medium and high doses of NPK fertilizer) and two tapping systems (S/2 d2 with and without ethylene stimulation). Here, we present the results of the first three years on dry rubber yield, latex metabolism assessed with the latex diagnosis method, and latex and rubber properties related to their technological properties. Our results showed a positive effect of fertilization on yield from the first year, but the effect was only statistically significant (p

The impact of NH3-stabilization of latex on its biochemical and physicochemical indicators at various storage times (0–41 days) was investigated. Liquid latex was characterized by pH, dry rubber content, total solid content, surface tension, lipid and protein contents. In addition, lipid and protein profiles were monitored by TLC and SDS-PAGE, respectively. Thin latex films were studied by AFM to unravel their morphology, stiffness and adhesion, whereas infrared and wettability measurements provided information on composition and wetting properties. NH3-stabilization method was shown to rapidly (12 days) and significantly impact lipid and protein contents resulting in drastic changes in the morphology as well as in the mechanical and physicochemical properties of films. The presence of a layer of very high stiffness (GPa) on the rubber surface was noticed which was hypothesized to contain REF1 and SRPP1, the two major latex proteins. This was later confirmed by imaging a film of dry latex extract, known to contain both REF1 and SRPP1 proteins, which revealed stiffness of the order of GPa. As a protein known for its amyloid-forming capacity, REF1 could be the main component responsible for GPa-range stiffness of NH3-stabilized films and might then play a crucial role on the final properties of natural rubber.

International audience; Rubber particle membranes from the Hevea latex contain predominantly two proteins, REF1 and SRPP1 involved in poly(cis-1,4-isoprene) synthesis or rubber quality. The repartition of both proteins on the small or large rubber particles seems to differ, but their role in the irreversible coagulation of the rubber particle is still unknown. In this study we highlighted the different modes of interactions of both recombinant proteins with different classes of lipids extracted from Hevea brasiliensis latex, and defined as phospholipids (PL), glycolipids (GL) and neutral lipids (NL). We combined two biophysical methods, polarization modulated-infrared reflection adsorption spectroscopy (PM-IRRAS) and ellipsometry to elucidate their interactions with monolayers of each class of lipids. REF1 and SRPP1 interactions with native lipids are clearly different; SRPP1 interacts mostly in surface with PL, GL or NL, without modification of its structure. In contrast REF1 inserts deeply in the lipid monolayers with all lipid classes. With NL, REF1 is even able to switch from α-helice conformation to β-sheet structure, as in its aggregated form (amyloid form). Interaction between REF1 and NL may therefore have a specific role in the irreversible coagulation of rubber particles

This paper describes an innovative, low cost and time effective nano-engineering process dedicated to macroelectronics and lab-on-a-chip applications. It consists of the fabrication of vertical nanosheets self-standing on a silicon template, and their embedding in polydimethylsiloxane (PDMS) by a mechanical release during the peel-off of the cured polymer. We demonstrate for the first time the embedding of a multi-stack composed of Au/SiO"2/Au nanosheets in PDMS and discussed in details the influence of each process parameters on the nanosheet composition and dimension. Since the embedded materials are hydrophilic, the formation of micro droplets was observed by condensation of the water vapor, leading to a self-alignment of those micro droplets onto the functional PDMS surface.

An alternative method of micro/nano-transport has been achieved by using motor proteins. Microtubules on a kinesin-coated surface have potential to act as a nano-transport system. When microtubules are used as carriers, either cargo or cargo linkers are attached on the microtubule surface. Such cargo attachments can significantly affect kinesin motion. To deal with the difficulty caused by molecular attachment to the microtubule surface, the cargo loading and transport mechanism should be separated. In this work, we propose to use micromachined needles as cargo carriers which then can be transported on microtubules. Because of the separation of needle functionalization and transport mechanism, functionalization of the needles can proceed without any effect on the microtubule structure, significantly increasing the possible types of cargo. We have fabricated silicon needles in mass numbers using a simple and effective method and have shown that the microtubule-needle composites are transported without affecting the kinesin activity.

The insoluble (macrogel) and soluble fractions of 11 commercial natural rubber (NR) samples (Technically specified rubber) were separated. Nitrogen titrations and lipid extractions enabled a quantitative assessment of the proteins and extractable lipids in each fraction. Swelling was measured in tetrahydrofuran in order to evaluate the crosslink density (Mc –1) of each macrogel. While the soluble fraction had a high lipid concentration, the majority of non-isoprene compounds of the macrogel were found to be proteins, which accounted for 4.6 to 50.8% (w/w) of the macrogel. Indeed, the macrogels contained less than 0.5% (w/w) extractable lipids. However, our results showed that the soluble fraction contained large quantities of proteins (16–66% of the nitrogen content of the raw NR sample), probably structuring microaggregates. An exponential correlation (R2 > 0.96) was found between the crosslink density and the protein concentration of macrogel, suggesting that proteins are involved in the majority of crosslinks in macrogel. (Resume d'auteur)

SummaryPlasma membrane tension and the pressure generated by actin polymerization are two antagonistic forces believed to define the protrusion rate at the leading edge of migrating cells [1–5]. Quantitatively, resistance to actin protrusion is a product of membrane tension and mean local curvature (Laplace’s law); thus, it depends on the local geometry of the membrane interface. However, the role of the geometry of the leading edge in protrusion control has not been yet investigated. Here, we manipulate both the cell shape and substrate topography in the model system of persistently migrating fish epidermal keratocytes. We find that the protrusion rate does not correlate with membrane tension, but, instead, strongly correlates with cell roundness, and that the leading edge of the cell exhibits pinning on substrate ridges—a phenomenon characteristic of spreading of liquid drops. These results indicate that the leading edge could be considered a triple interface between the substrate, membrane, and extracellular medium and that the contact angle between the membrane and the substrate determines the load on actin polymerization and, therefore, the protrusion rate. Our findings thus illuminate a novel relationship between the 3D shape of the cell and its dynamics, which may have implications for cell migration in 3D environments.

We have utilized a cytoskeleton, microtubule (MT), as a template for conductive nanowires. Biotin-labeled MTs were coated with streptavidin-labeled nanogold (NG) particles. Labeling of two modified MTs, biotin-labeled and NG-coated MTs, were evaluated by fluorescent and scanning electron microscopy, which verified the specific binding of NG particles on biotin-labeled MTs. Then, NG-coated MTs were further enlarged by catalytic deposition for better conductivity. Electrical characterization proved that only the enhanced NG-coated MTs have conductivity with dependency on MT and NG concentrations.

In a motor protein system (kinesin/microtubule), individual microtubules were captured and relocated to build a complex nano-scale transport system. Micro tweezers, attached to a micromanipulator arm, were used to capture polarity marked microtubules. Precise relocation of those microtubules provided an oriented track along which kinesin-coated beads move according to the microtubule polarities.

International audience; Puroindolines (PINs), basic and cysteine-rich proteins of wheat endosperm, are composed of two proteins, puroindoline-a (PIN-a) and puroindoline-b (PIN-b). Using a monolayer assay at the air/liquid interface, both PIN-a and PIN-b were studied in pure components and mixed with wheat galactolipids, 1,2-di-O-acyl-3-O-(beta-d-galactopyranosyl)- sn-glycerol (MGDG) and 2-di-O-acyl-3-O-(beta-d-galactopyranosyl-1,6-beta-d-galactopyranosyl)-sn-glycerol (DGDG). Following the adsorption of PINs at the air/liquid interface thanks to surface pressure increases, we concluded that PIN-a displays a more amphipathic character than PIN-b. Compression isotherms combined with ellipsometric measurements showed that the area per molecule is smaller and the protein film is more condensed for PIN-a than for PIN-b. According to the polarization modulation-infrared reflection-absorption spectroscopy data, both proteins display a highly alpha-helical structure and the alpha-helices are oriented rather parallel to the interface. By measuring the overpressure due to PIN adsorption into MGDG and DGDG monolayers, we observed that PIN-a interacts more strongly into lipid films than PIN-b. The observation by atomic force microscopy of mixed protein/lipid films showed that the nature of the lipid plays a significant role in the organization of PINs, particularly for PIN-a. The presence of galactolipids at the interface stabilizes the alpha-helical structure of PINs, but significant changes were observed concerning the orientation of the alpha-helices. They adopt a perfect parallel orientation to the interface in the MGDG monolayer, whereas the bundle of alpha-helices orients normal to the interface in the DGDG film.

International audience; The behavior of the two major galactolipids of wheat endosperm, mono- (MGDG) and di-galactosyldiacylglycerol (DGDG) was studied in aqueous dispersion and at the air/liquid interface. The acyl chains of the pure galactolipids and their binary equimolar mixture are in the fluid or liquid expanded phase. SAXS measurements on liquid-crystalline mesophases associated with the electron density reconstructions show that the DGDG adopts a lamellar phase L(alpha) with parallel orientation of the headgroups with respect to the plane of the bilayer, whereas MGDG forms an inverse hexagonal phase H(II) with a specific organization of galactosyl headgroups. The equimolar mixture shows a different behavior from those previously described with formation of an Im3m cubic phase. In comparing monolayers composed of the pure galactolipids and their equimolar mixtures, PM-IRRAS spectra show significant differences in the optical properties and orientation of galactosyl groups with respect to the interface. Furthermore, Raman and FTIR spectroscopies show that the acyl chains of the galactolipid mixture are more ordered compared to those of the pure components. These results suggest strong interactions between MGDG and DGDG galactosyl headgroups and these specific physical properties of galactolipids are discussed in relation to their biological interest in wheat seed.

In motile cells, membrane pressure at the membrane/cytoplasm interface is balanced by the cytoskeletal forces and hydrostatic pressure. In particular, it is believed that protrusion of the lamellipodia is controlled by the balance of membrane pressure and the pressure generated by actin polymerization, while bleb formation results from the balance of membrane pressure and hydrostatic pressure. However, exact contributions of these forces to the cell shape and motion are not known. Membrane pressure is a product of tension and local curvature of the membrane; thus, to know the membrane pressure one has to know the exact three-dimensional shape of the cell. We have developed a simple approach to measure cell vertical profile and volume, which is based on the displacement of the fluorescent cell-impermeable dye dissolved in the medium. To get insight into the balance of forces at the membrane interface, we measure membrane tension (with tether assay), protrusion velocity, vertical profile, and volume dynamics in migrating fish epidermal keratocytes subjected to cytoskeletal drugs and volume perturbations. Inhibiting myosin-dependent contraction with blebbistatin resulted in irregular cell shape, and reduced protrusion velocity and membrane tension (variable and low tether forces as compared to a force distribution centered around 35-40 pN in control cells).Remarkably, hypoosmotic treatment normalized blebbistatin-treated cells by restoring their tension, protrusion velocity, and shape, and also increased tension and protrusion velocity in control cells. Vertical profile measurements showed that hypoosmotic treatment induced persistent cell swelling and substantial increase in the height of the lamellipodia.We propose that hydrostatic pressure that could be generated due to either myosin dependent contraction or osmotic gradient helps to maintains optimal vertical profile of the lamellipodium and thus cooperates with actin-dependent protrusion.Supported by SNSF, Swiss SystemsX and NCCBI.

The authors demonstrate for the first time the embedding of vertical nanosheets of thermal silicon dioxide into the surface of polydimethylsiloxane (PDMS). The generic process is suitable for embedding a wide range of thin film nanosheets into PDMS-like polymers. The nanosheets are fabricated on a silicon template, and transferred into the PDMS by molding and releasing the nanosheets from the template during the peel-off of the cured PDMS. Since the template is fully fabricated using semiconductor and compatible processes, the presented technique aims at considerably expanding the range of materials that can be embedded in PDMS, thereby increasing the possible applications. As the width of the nanosheets is defined by the thickness of the deposited materials, nanoscale features can be directly embedded without relying on expensive nanolithography tools.

We demonstrate the active transport of liquid cargos in the form of oil-in-water emulsion droplets loaded on kinesin motor proteins moving along oriented microtubules. We analyze the motility properties of the kinesin motors (velocity and run length) and find that the liquid cargo in the form of oil droplets does not alter the motor function of the kinesin molecules. This work provides a novel method for handling only a few molecules/particles encapsulated inside the oil droplets and represents a key finding for the integration of kinesin-based active transport into nanoscale lab-on-a-chip devices. We also investigate the effect of the diameter of the droplets on the motility properties of the kinesin motors. The velocity is approximately constant irrespective of the diameter of the droplets whereas we highlight a strong increase of the run length when the diameter of the droplets increases. We correlate these results with the number of kinesin motors involved in the transport process and find an excellent agreement between our experimental result and a theoretical model.

A hybrid bio-electrical system for direct transportation and electrofusion of oil droplets is proposed. A motor protein system, kinesin/microtubule, was used to transport kinesin-coated oil droplets along microtubules oriented and immobilized on a glass surface. The attachment between the biotinylated-kinesin and the functionalized oil droplet was achieved using the biotin-streptavidin binding. Fusion of the oil droplets was accomplished by applying AC voltage on the electrodes placed inside a flow cell. Together with encapsulation of particles/reactants within oil droplets, this method may lead to the fabrication of a nanoscale reactor.