<i>In Vivo</i> Study of Nuclear-Localized Protein-Protein Interactions in Plant Cells

Recent results suggest that key events controlling light regulated gene expression in plants are import of the phytochrome photoreceptors into the nucleus followed by their binding to transcription factors such as PIF3. Coupled with this, the degradation of positively acting intermediates such as the transcription factor HY5 by COPl and the C0P9 signalosome appears to be an important process whereby photomorphogenesis is repressed in darkness. Genetic analyses in Arabidopsis and tomato have revealed that the nuelear protein DETl also plays a key role in the repression of photomorphogenesis. However, up until now the mechanisms underlying its function have remained obscure. In this thesis through a series of in vitro experiments, persuasive evidence is provided that DETl binds to non-acetylated amino-terminal tails of the core histone H2B in the context of the nucleosome. Furthermore, FRET imaging with GFP variants has been used to prove this interaction within the nucleus of living plant cells. DETl is therefore a novel chromatin interacting protein which binds to nucleosomes via interaction with unmodified H2B tails. Given the dramatic photomorphogenic phenotypes of detl mutants, this result infers that chromatin remodelling plays a heretofore unsuspected role in regulating gene expression during photomorphogenesis. An important part of this thesis was devoted to the establishment of a reliable FRET-based wide-field microscopy system for the detection of the interactions between GFP- (Green Fluorescent Protein) tagged proteins in living cells. Limits of some of the available techniques are shown together with many data proving that the established system is indeed able to reliably detect protein-protein interactions. Moreover, given the emerging importance of ehromatin remodelling in plants, the FRET-based microscopy system has been used with a histone-based eonstruet in an attempt to reveal chromatin dynamics in living cells. Data are presented about changes of chromatin structures, as detected by FRET measurements, occurring during the cell cycle of living BY-2 cells

A Stress Surveillance System Based on Calcium and Nitric Oxide in Marine Diatoms

Diatoms are an important group of eukaryotic phytoplankton, responsible for about 20% of global primary productivity. Study of the functional role of chemical signaling within phytoplankton assemblages is still in its infancy although recent reports in diatoms suggest the existence of chemical-based defense strategies. Here, we demonstrate how the accurate perception of diatom-derived reactive aldehydes can determine cell fate in diatoms. In particular, the aldehyde (2E,4E/Z)-decadienal (DD) can trigger intracellular calcium transients and the generation of nitric oxide (NO) by a calcium-dependent NO synthase-like activity, which results in cell death. However, pretreatment of cells with sublethal doses of aldehyde can induce resistance to subsequent lethal doses, which is reflected in an altered calcium signature and kinetics of NO production. We also present evidence for a DD–derived NO-based intercellular signaling system for the perception of stressed bystander cells. Based on these findings, we propose the existence of a sophisticated stress surveillance system in diatoms, which has important implications for understanding the cellular mechanisms responsible for acclimation versus death during phytoplankton bloom successions

A 14-3-3γ dimer-based scaffold bridges CtBP1-S/BARS to PI(4)KIIIβ to regulate post-Golgi carrier formation

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In vivo study of nuclear-localized protein-protein interactions in plant cells

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The Photomorphogenesis Regulator DET1 Binds the Amino-Terminal Tail of Histone H2B in a Nucleosome Context

AbstractLight provides a major source of information from the environment during plant growth and development [1, 2]. Recent results suggest that the key events controlling light-regulated gene expression in plants are translocation of the phytochrome photoreceptors into the nucleus, followed by their binding to transcription factors such as PIF3. Coupled with this, the degradation of positively acting intermediates such as the transcription factor HY5 by COP1 and the COP9 signalosome appears to be an important process whereby photomorphogenesis is repressed in darkness (e.g., [3–8]). Genetic analyses in Arabidopsis and tomato have revealed that the nuclear protein DET1 also plays a key role in the repression of photomorphogenesis [9–11]. However, the function of this protein has remained a mystery. In a series of in vitro experiments, we provide persuasive evidence that DET1 binds to nonacetylated amino-terminal tails of the core histone H2B in the context of the nucleosome. Furthermore, we have utilized FRET (fluorescence resonance energy transfer) imaging with GFP variants to demonstrate this interaction within the nucleus of living plant cells. Given the dramatic photomorphogenic phenotypes of det1 mutants, we propose that chromatin remodeling [12–14] plays a heretofore unsuspected role in regulating gene expression during photomorphogenesis

Adverse Effect of Metallic Gold and Silver Nanoparticles on Xenopus laevis Embryogenesis

Exposure to metal nanoparticles is potentially harmful, particularly when occurring during embryogenesis. In this study, we tested the effects of commercial AuNPs and AgNPs, widely used in many fields for their features, on the early development of Xenopus laevis, an anuran amphibian key model species in toxicity testing. Through the Frog Embryo Teratogenesis Assay—Xenopus test (FETAX), we ascertained that both nanoparticles did not influence the survival rate but induced morphological anomalies like modifications of head and branchial arch cartilages, depigmentation of the dorsal area, damage to the intestinal brush border, and heart rate alteration. The expression of genes involved in the early pathways of embryo development was also modified. This study suggests that both types of nanoparticles are toxic though nonlethal, thus indicating that their use requires attention and further study to better clarify their activity in animals and, more importantly, in humans

Effetti di nanoparticelle di polistirene sull'embriogenesi di Xenopus laevis

L'impiego di nanoparticelle (NP) utili per diverse applicazioni (cosmetica, abbigliamento, alimentazione e drug delivery) riscuote un vivo interesse. In medicina offrono la possibilità unica di superare la barriera cellulare per dirigere molecole verso targets specifici come le droghe utilizzate in chemioterapia. Per promuovere il giusto sviluppo di tali tecnologie è essenziale chiarire le potenziali conseguenze per la salute umana associate all???esposizione alle NP. La principale preoccupazione riguarda le loro piccole dimensioni e la possibilità che possano essere internalizzate inadeguatamente, in rapporto con le loro caratteristiche fisico-chimiche e con la natura delle cellule target (Shawna et al., 2011; Gorth et al., 2011; Pompa et al., 2011). Le NP di polistirene da noi utilizzate sono considerate materiale ???biologicamente inerte. L'impiego del sistema modello Xenopus laevis, permette di saggiare l???eventuale tossicità delle NP in vivo, valutandone gli effetti sullo sviluppo embrionale. A questo scopo sono state somministrate con due diversi protocolli: la microiniezione in uno dei blastomeri di embrioni allo stadio 2 cellule e il contatto che consiste nell'allevamento di embrioni in 1/10 Ringer contenente NP. Per entrambi i tipi di esperimenti sono state analizzate la morfologia, la percentuale di mortalità e l'espressione di alcuni marcatori embrionali quali: bra (mesoderma presuntivo), myod1 (mesoderma parassiale) e sox9 (creste neurali). Gli embrioni sia microiniettati che contatto presentano malformazioni di testa, intestino e coda, edemi variamente diffusi e mostrano, inoltre, uno sviluppo più lento. Gli embrioni microiniettati presentano un alto tasso di mortalità mentre quelli a contatto hanno un tasso di mortalità simile ai w.t. La microscopia confocale ha evidenziato la presenza di NP nell'intestino, nella faringe e nello strato esterno della retina, non nel cervello. I marcatori usati presentano tutti modificazioni dell'espressione. Questi dati suggeriscono un effetto tossico/teratogenico ma non letale delle NP per gli embrioni trattati alle condizioni precedentemente descritte

Polystyrene nanoparticles affect Xenopus laevis development

Exposing living organisms to nanoparticulates is potentially hazardous, in particular when it takes place during embryogenesis. In this investigation, we have studied the effects of 50-nm-uncoated polystyrene nanoparticles (PSNPs) as a model to investigate the suitability of their possible future employments. We have used the standardized Frog Embryo Teratogenesis Assay-Xenopus test during the early stages of larval development of Xenopus laevis, and we have employed either contact exposure or microinjections. We found that the embryos mortality rate is dose dependent and that the survived embryos showed high percentage of malformations. They display disorders in pigmentation distribution, malformations of the head, gut and tail, edema in the anterior ventral region, and a shorter body length compared with sibling untreated embryos. Moreover, these embryos grow more slowly than the untreated embryos. Expressions of the mesoderm markers, bra (T-box Brachyury gene), myod1 (myogenic differentiation1), and of neural crest marker sox9 (sex SRY (determining region Y-box 9) transcription factor sox9), are modified. Confocal microscopy showed that the nanoparticles are localized in the cytoplasm, in the nucleus, and in the periphery of the digestive gut cells. Our data suggest that PSNPs are toxic and show a potential teratogenic effect for Xenopus larvae. We hypothesize that these effects may be due either to the amount of NPs that penetrate into the cells and/or to the "corona'' effect caused by the interaction of PSNPs with cytoplasm components. The three end-points of our study, i.e., mortality, malformations, and growth inhibition, suggest that the tests we used may be a powerful and flexible bioassay in evaluating pollutants in aquatic embryos

Golgi sorting regulates organization and activity of GPI proteins at apical membranes.

Here we combined classical biochemistry with new biophysical approaches to study the organization of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) with high spatial and temporal resolution at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, after sorting in the Golgi, each GPI-AP reaches the apical surface in homoclusters. Golgi-derived homoclusters are required for their subsequent plasma membrane organization into cholesterol-dependent heteroclusters. By contrast, in nonpolarized MDCK cells, GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form heteroclusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, in contrast to fibroblasts, in polarized epithelial cells, a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and function of GPI-APs at the apical surface