Fabrication of hydrogel scaffolds via photocrosslinking of methacrylated silk fibroin.

Silk fibroin is a promising biomaterial for tissue engineering due to its valuable mechanical and biological properties. However, being a natural product and a protein, it lacks the processability and uniform quality of an advanced synthetic material. Here we propose a way to overcome this contradiction using novel fibroin photocrosslinkable derivative (FBMA). FBMA was synthesized by methacrylation of native fibroin nucleophilic side groups. It was dissolved in either formic acid (FA) or hexafluoroisopropanol (HFIP), and the obtained solutions were photocrosslinked into hydrogel scaffolds of various structural forms including films, micropatterns, pads and macroporous sponges. UV-exposition of dry FBMA films through a photomask created complex microscaled patterns of the polymer. The nature of the solvent affected the properties of resulting hydrogels. When HFIP was used as the solvent, the resulting hydrogels had a storage modulus ∼4 times higher than that of hydrogels fabricated using FA and ∼20 times higher compared to the reference hydrogel obtained from pristine fibroin. Both FBMA-based hydrogels were biocompatible and supported fibroblast adhesion and growth in vitro. Cells cultivated on FBMA scaffolds produced with HFIP exhibited more spread phenotype at 4 and 24 h of cultivation, consistent with increased stiffness of the hydrogel. Hence, FBMA is an attractive material for fabrication of micropatterned scaffolds of centimeter-scale size with minutely tunable physico-chemical properties via convenient and reproducible technological processes, applicable for rapid prototyping.

Strong alkalinization of Chara cell surface in the area of cell wall incision as an early event in mechanoperception.

Mechanical wounding of cell walls occurring in plants under the impact of pathogens or herbivores can be mimicked by cell wall incision with a glass micropipette. Measurements of pH at the surface of Chara corallina internodes following microperforation of cell wall revealed a rapid (10-30s) localized alkalinization of the apoplast after a lag period of 10-20s. The pH increase induced by incision could be as large as 3 pH units and relaxed slowly, with a halftime up to 20min. The axial pH profile around the incision zone was bell-shaped and localized to a small area, extending over a distance of about 100µm. The pH response was suppressed by lowering cell turgor upon the replacement of artificial pond water (APW) with APW containing 50mM sorbitol. Stretching of the plasma membrane during its impression into the cell wall defect is likely to activate the Ca(2+) channels, as evidenced from sensitivity of the incision-induced alkalinization to the external calcium concentration and to the addition of Ca(2+)-channel blockers, such as La(3+), Gd(3+), and Zn(2+). The maximal pH values attained at the incision site (~10.0) were close to pH in light-dependent alkaline zones of Chara cells. The involvement of cytoskeleton in the origin of alkaline patch was documented by observations that the incision-induced pH transients were suppressed by the inhibitors of microtubules (oryzalin and taxol) and, to a lesser extent, by the actin inhibitor (cytochalasin B). The results indicate that the localized increase in apoplastic pH is an early event in mechanoperception and depends on light, cytoskeleton, and intracellular calcium.

Ca2+ regulates reactive oxygen species production and pH during mechanosensing in Arabidopsis roots.

Mechanical stimulation of plants triggers a cytoplasmic Ca(2+) increase that is thought to link the touch stimulus to appropriate growth responses. We found that in roots of Arabidopsis thaliana, external and endogenously generated mechanical forces consistently trigger rapid and transient increases in cytosolic Ca(2+) and that the signatures of these Ca(2+) transients are stimulus specific. Mechanical stimulation likewise elicited an apoplastic alkalinization and cytoplasmic acidification as well as apoplastic reactive oxygen species (ROS) production. These responses showed the same kinetics as mechanically induced Ca(2+) transients and could be elicited in the absence of a mechanical stimulus by artificially increasing Ca(2+) concentrations. Both pH changes and ROS production were inhibited by pretreatment with a Ca(2+) channel blocker, which also inhibited mechanically induced elevations in cytosolic Ca(2+). In trichoblasts of the Arabidopsis root hair defective2 mutant, which lacks a functional NADPH oxidase RBOH C, touch stimulation still triggered pH changes but not the local increase in ROS production seen in wild-type plants. Thus, mechanical stimulation likely elicits Ca(2+)-dependent activation of RBOH C, resulting in ROS production to the cell wall. This ROS production appears to be coordinated with intra- and extracellular pH changes through the same mechanically induced cytosolic Ca(2+) transient.

A Sec14p-nodulin domain phosphatidylinositol transfer protein polarizes membrane growth of Arabidopsis thaliana root hairs.

Phosphatidylinositol (PtdIns) transfer proteins (PITPs) regulate signaling interfaces between lipid metabolism and membrane trafficking. Herein, we demonstrate that AtSfh1p, a member of a large and uncharacterized Arabidopsis thaliana Sec14p-nodulin domain family, is a PITP that regulates a specific stage in root hair development. AtSfh1p localizes along the root hair plasma membrane and is enriched in discrete plasma membrane domains and in the root hair tip cytoplasm. This localization pattern recapitulates that visualized for PtdIns(4,5)P2 in developing root hairs. Gene ablation experiments show AtSfh1p nullizygosity compromises polarized root hair expansion in a manner that coincides with loss of tip-directed PtdIns(4,5)P2, dispersal of secretory vesicles from the tip cytoplasm, loss of the tip f-actin network, and manifest disorganization of the root hair microtubule cytoskeleton. Derangement of tip-directed Ca2+ gradients is also apparent and results from isotropic influx of Ca2+ from the extracellular milieu. We propose AtSfh1p regulates intracellular and plasma membrane phosphoinositide polarity landmarks that focus membrane trafficking, Ca2+ signaling, and cytoskeleton functions to the growing root hair apex. We further suggest that Sec14p-nodulin domain proteins represent a family of regulators of polarized membrane growth in plants.

UV and blue light signalling: pathways regulating chalcone synthase gene expression in Arabidopsis.

UV-B, UV-A and blue light control a variety of aspects of plant development via distinct photoreceptors and signalling pathways. The known photoreceptors for UV-A/blue light are cryptochrome (cry)1 and cry2, and the phototropism photoreceptor, phototropin. Redox processes are important in cry and phototropin signal transduction. A specific photoreceptor for UV-B has not been identified and there appear to be several possible UV-B signalling pathways. We are investigating the UV and blue light regulation of transcription of the chalcone synthase gene (CHS) in Arabidopsis. Experiments with photoreceptor mutants show that distinct UV-A/blue (cry mediated) and UV-B photoreception systems control CHS expression. Experiments with an Arabidopsis cell suspension culture show that the UV-B and cry1 signalling pathways differ kinetically and pharmacologically. In contrast to some other UV-B responses, the UV-B induction of CHS does not appear to involve oxidative stress signalling. Promoter elements and candidate transcription factors that effect CHS induction have been identified. Interactions within a network of UV-B, cry and phytochrome signalling pathways regulate CHS expression. Synergistic interactions between the UV-B pathway and distinct UV-A and blue-light pathways maximize the response. In addition, specific phytochromes positively control the cry1 pathway via distinct potentiation and coaction effects, and negatively regulate the UV-B pathway.