Alternative method to visualize receptor dynamics in cell membranes

There is a close relation between membrane receptor dynamics and their behavior. Several microscopy techniques have been developed to study protein dynamics in live cells such as the Fluorescence Recovery After Photobleaching (FRAP) or the Single Particle Tracking (SPT). These methodologies require expensive instruments, are time consuming, allow the analysis of a small portion of the cell or an extremely small number of receptors at a time. Here we propose a time-saving approach that allows to visualize the entire receptor pool and its localization in time. This protocol requires an epifluorescence microscope equipped for structured illuminated sectioning and for live cell imaging. It can be applied to characterize membrane receptor and multi-protein complex and their response to activators or inhibitors. Image acquisition and analysis can be performed in two days, while cells and substratum preparation require a few minutes a day for three days

Glucose-6-phosphate dehydrogenase plays a central role in the response of tomato (Solanum lycopersicum) plants to short and long-term drought

The present study was undertaken to investigate the expression, occurrence and activity of glucose 6 phosphate dehydrogenase (G6PDH e EC 1.1.1.49), the key-enzyme of the Oxidative Pentose Phosphate Pathway (OPPP), in tomato plants (Solanum lycopersicum cv. Red Setter) exposed to short- and long-term drought stress. For the first time, drought effects have been evaluated in plants under different growth conditions: in hydroponic laboratory system, and in greenhouse pots under controlled conditions; and in open field, in order to evaluate drought response in a representative agricultural environment. Interestingly, changes observed appear strictly associated to the induction of well known stress response mechanisms, such as the increase of proline synthesis, accumulation of chaperone Hsp70, and ascorbate peroxidase. Results show significant increase in total activity of G6PDH, and specifically in expression and occurrence of cytosolic isoform (cy-G6PDH) in plants grown in any cultivation system upon drought. Intriguingly, the results clearly suggest that abscissic acid (ABA) pathway and signaling cascade (protein phosphatase 2C e PP2C) could be strictly related to increased G6PDH expression, occurrence and activities. We hypothesized for G6PDH a specific role as one of the main reductants’ suppliers to counteract the effects of drought stress, in the light of converging evidences given by young and adult tomato plants under stress of different duration and intensity

β3 Integrin promotes long-lasting activation and polarization of vascular endothelial growth factor receptor 2 by immobilized ligand

OBJECTIVE: During neovessel formation, angiogenic growth factors associate with the extracellular matrix. These immobilized factors represent a persistent stimulus for the otherwise quiescent endothelial cells (ECs), driving directional EC migration and proliferation and leading to new blood vessel growth. Vascular endothelial growth factor receptor 2 (VEGFR2) is the main mediator of angiogenesis. Although VEGFR2 signaling has been deeply characterized, little is known about its subcellular localization during neovessel formation. Aim of this study was the characterization and molecular determinants of activated VEGFR2 localization in ECs during neovessel formation in response to matrix-immobilized ligand. APPROACH AND RESULTS: Here we demonstrate that ECs stimulated by extracellular matrix-associated gremlin, a noncanonical VEGFR2 ligand, are polarized and relocate the receptor in close contact with the angiogenic factor-enriched matrix both in vitro and in vivo. GM1 (monosialotetrahexosylganglioside)-positive planar lipid rafts, β3 integrin receptors, and the intracellular signaling transducers focal adhesion kinase and RhoA (Ras homolog gene family, member A) cooperate to promote VEGFR2 long-term polarization and activation. CONCLUSIONS: A ligand anchored to the extracellular matrix induces VEGFR2 polarization in ECs. Long-lasting VEGFR2 relocation is closely dependent on lipid raft integrity and activation of β3 integrin pathway. The study of the endothelial responses to immobilized growth factors may offer insights into the angiogenic process in physiological and pathological conditions, including cancer, and for a better engineering of synthetic tissue scaffolds to blend with the host vasculature

Transcriptomic and splicing changes underlying tomato responses to combined water and nutrient stress

Tomato is a horticultural crop of high economic and nutritional value. Suboptimal environmental conditions, such as limited water and nutrient availability, cause severe yield reductions. Thus, selection of genotypes requiring lower inputs is a goal for the tomato breeding sector. We screened 10 tomato varieties exposed to water deficit, low nitrate or a combination of both. Biometric, physiological and molecular analyses revealed different stress responses among genotypes, identifying T270 as severely affected, and T250 as tolerant to the stresses applied. Investigation of transcriptome changes caused by combined stress in roots and leaves of these two genotypes yielded a low number of differentially expressed genes (DEGs) in T250 compared to T270, suggesting that T250 tailors changes in gene expression to efficiently respond to combined stress. By contrast, the susceptible tomato activated approximately one thousand and two thousand genes in leaves and roots respectively, indicating a more generalized stress response in this genotype. In particular, developmental and stress-related genes were differentially expressed, such as hormone responsive factors and transcription factors. Analysis of differential alternative splicing (DAS) events showed that combined stress greatly affects the splicing landscape in both genotypes, highlighting the important role of AS in stress response mechanisms. In particular, several stress and growth-related genes as well as transcription and splicing factors were differentially spliced in both tissues. Taken together, these results reveal important insights into the transcriptional and post-transcriptional mechanisms regulating tomato adaptation to growth under reduced water and nitrogen inputs

Bartonella henselae Persistence within Mesenchymal Stromal Cells Enhances Endothelial Cell Activation and Infectibility That Amplifies the Angiogenic Process (*Scutera S and Mitola S co-first authors; Sozzani S and Musso T co-last authors)

Some bacterial pathogens can manipulate the angiogenic response, suppressing or inducing it for their own ends. In humans, Bartonella henselae is associated with cat-scratch disease and vasculoproliferative disorders such as bacillary angiomatosis and bacillary peliosis. Although endothelial cells (ECs) support the pathogenesis of B. henselae, the mechanisms by which B. henselae induces EC activation are not completely clear, as well as the possible contributions of other cells recruited at the site of infection. Mesenchymal stromal cells (MSCs) are endowed with angiogenic potential and play a dual role in infections, exerting antimicrobial properties but also acting as a shelter for pathogens. Here, we delved into the role of MSCs as a reservoir of B. henselae and modulator of EC functions. B. henselae readily infected MSCs and survived in perinuclearly bound vacuoles for up to 8 days. Infection enhanced MSC proliferation and the expression of epidermal growth factor receptor (EGFR), Toll-like receptor 2 (TLR2), and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), proteins that are involved in bacterial internalization and cytokine production. Secretome analysis revealed that infected MSCs secreted higher levels of the proangiogenic factors vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF-7), matrix metallopeptidase 9 (MMP-9), placental growth factor (PIGF), serpin E1, thrombospondin 1 (TSP-1), urokinase-type plasminogen activator (uPA), interleukin 6 (IL-6), platelet-derived growth factor D (PDGF-D), chemokine ligand 5 (CCL5), and C-X-C motif chemokine ligand 8 (CXCL8). Supernatants from B. henselae-infected MSCs increased the susceptibility of ECs to B. henselae infection and enhanced EC proliferation, invasion, and reorganization in tube-like structures. Altogether, these results indicate MSCs as a still underestimated niche for persistent B. henselae infection and reveal MSC-EC cross talk that may contribute to exacerbate bacterium-induced angiogenesis and granuloma formation

Monomeric gremlin is a novel vascular endothelial growth factor receptor-2 antagonist

Angiogenesis plays a key role in various physiological and pathological conditions, including inflammation and tumor growth. The bone morphogenetic protein (BMP) antagonist gremlin has been identified as a novel pro-angiogenic factor. Gremlin promotes neovascular responses via a BMP-independent activation of the vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2). BMP antagonists may act as covalent or non-covalent homodimers or in a monomeric form, while VEGFRs ligands are usually dimeric. However, the oligomeric state of gremlin and its role in modulating the biological activity of the protein remain to be elucidated.Here we show that gremlin is expressed in vitro and in vivo both as a monomer and as a covalently linked homodimer. Mutagenesis of amino acid residue Cys141 prevents gremlin dimerization leading to the formation of gremlinC141A monomers. GremlinC141A monomer retains a BMP antagonist activity similar to the wild-type dimer, but is devoid of a significant angiogenic capacity. Notably, we found that gremlinC141A mutant engages VEGFR2 in a non-productive manner, thus acting as receptor antagonist. Accordingly, both gremlinC141A and wild-type monomers inhibit angiogenesis driven by dimeric gremlin or VEGF-A165. Moreover, by acting as a VEGFR2 antagonist, gremlinC141A inhibits the angiogenic and tumorigenic potential of murine breast and prostate cancer cells in vivo.In conclusion, our data show that gremlin exists in multiple forms endowed with specific bioactivities and provide new insights into the molecular bases of gremlin dimerization. Furthermore, we propose gremlin monomer as a new inhibitor of VEGFR2 signalling during tumor growth

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a Delta9 desaturase gene from either the wild potato species Solanum commersonii or the cyanobacterium Anacystis nidulans, using PEG-mediated DNA uptake by protoplasts. Incorporation of chloroplast antibioticinsensitive point mutations in the transforming DNA was used to select transformants. The presence of the transcript and the Delta9 desaturase protein in transplastomic plants was confirmed by northern and western blot analyses. In comparison with control plants, transplastomic plants showed altered fatty acid profiles and an increase in their unsaturation level both in leaves and seeds. The two transgenes produced comparable results. The results obtained demonstrate the feasibility of using plastid transformation to engineer lipid metabolic pathways in both vegetative and reproductive tissues and suggest an increase of cold tolerance in transplastomic plants showing altered leaf fatty acid profiles. This is the first example of transplastomic plants expressing an agronomically relevant gene produced with the ‘‘binding-type’’ vectors, which do not contain a heterologous marker gene. In fact, the transplastomic plants expressing the S. commersonii gene contain only plant-derived sequences, a clear attraction from a public acceptability perspective

Role of Arabidopsis UV RESISTANCE LOCUS 8 in plant growth reduction under osmotic stress and low levels of UV-B

In high-light environments, plants are exposed to different types of stresses, such as an excess of UV-B, but also drought stress which triggers a common morphogenic adaptive response resulting in a general reduction of plant growth. Here, we report that the Arabidopsis thaliana UV RESISTANCE LOCUS 8 (UVR8) gene, a known regulator of the UV-B morphogenic response, was able to complement a Saccharomyces cerevisiae osmo-sensitive mutant and its expression was induced after osmotic or salt stress in Arabidopsis plants. Under low levels of UV-B, plants overexpressing UVR8 are dwarfed with a reduced root development and accumulate more flavonoids compared to control plants. The growth defects are mainly due to the inhibition of cell expansion. The growth inhibition triggered by UVR8 overexpression in plants under low levels of UV-B was exacerbated by mannitol-induced osmotic stress, but it was not significantly affected by ionic stress. In contrast, uvr8-6 mutant plants do not differ from wild-type plants under standard conditions, but they show an increased shoot growth under high-salt stress. Our data suggest that UVR8-mediated accumulation of flavonoid and possibly changes in auxin homeostasis are the underlying mechanism of the observed growth phenotypes and that UVR8 might have an important role for integrating plant growth and stress signals.This work was supported by the Interuniversity Attraction Poles Programme (IUAP P7/29 'MARS') initiated by the Belgian Science Policy Office, by grants from Ghent University (Bijzonder Onderzoeksfonds Methusalem project no. BOF08/01M00408), and by grants to the MIUR project FIRB Plant-STRESS.Fasano, R.; Gonzalez, N.; Tosco, A.; Dal Piaz, F.; Docimo, T.; Serrano Salom, R.; Grillo, S.... (2014). Role of Arabidopsis UV RESISTANCE LOCUS 8 in plant growth reduction under osmotic stress and low levels of UV-B. Molecular Plant. 7(5):773-791. https://doi.org/10.1093/mp/ssu002S7737917