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This work is a collection of three research articles and one review article focused on flotillins (FLOTs) and hypersensitive induced reaction proteins (HIRs) in Arabidopsis thaliana. FLOTs and HIRs are closely related membrane-associated proteins forming two subfamilies both belonging to SPFH domain superfamily. While FLOTs are present in organisms of all evolutionary lineages HIRs are plant specific proteins. The review article sums up the knowledge gained on FLOTs and HIRs from different organisms in terms of cellular localization, interaction with cellular membranes and with other proteins, and physiological functions. The research articles were targeted at three aspects of AtFLOTs and AtHIRs: involvement in response to exogenous stimuli; determination of protein interactors; and subcellular localization and dynamics. The first aspect was approached by transcription measurement of AtFLOTs and phenotypic screen of single loss-of-function mutants of AtFLOTs upon various treatments covering biotic and abiotic stress and phytohormone application. Although we observed changes in transcription none of the treatments provoked a phenotype manifestation in any of AtFLOT mutants. In the second article we focused on interactome of AtFLOT2 and performed co- immunoprecipitation followed by mass spectrometry...

ARP2/3 is an evolutionarily conserved heteroheptameric protein complex. Its main activity lies in the nucleation of dendritic actin filaments that are involved in membrane remodeling. ARP2/3 takes part in plasma membrane remodeling and the formation of cytoplasmic protrusions that serve in the amoeboid motion of mammalian cells and some protists and plays role in exocytosis and endocytosis of animal and yeast cells. The main objective of this work was to find a connection between the ARP2/3 complex and the regulation of the plant endomembrane system. Using TIRF microscopy we visualized the localization of the ARP2/3 complex in the cortical layer of plant cells and compared it to the localization of several endomembrane markers from the Rab family and an exocytotic marker Exo84b. In the vicinity of the plasma membrane, the ARP2/3 complex subunits localized to dynamic dots very similar to the localization of Exo84b protein. Colocalization analysis showed that a small portion of Exo84b marker and ARP2/3 complex signals colocalize and this result was seconded by the biochemical approach of coimmunoprecipitation. Key words: ARP2/3, endomembrane system, cortical layer, RabA1g, RabC1, RaD2a, Exo84b

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No description available.

Angiogenesis is the formation of new blood vessels from pre-existing ones. Unregulated angiogenesis is associated with several diseases such as diabetic retinopathy and tumour growth. Many signal transduction pathways have been implicated in the regulation of angiogenesis such as p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3 kinase (PI3K), extracellular signal-related kinase 1/2 (Erk1/2) and of particular interest the calcineurin/nuclear factor of activated T-cell (NFAT) pathway. Inhibition of calcineurin activity by the drug cyclopsorin A (CsA) has been shown to inhibit processes required for successful angiogenesis such as in vitro cell migration, tube formation and additionally attenuates corneal angiogenesis in vivo. CsA is associated with severe side effects and therefore the identification of an endogenous regulator of this pathway would be beneficial. One possibility is the plasma membrane calcium ATPases (PMCAs). These high affinity calcium extrusion pumps have been shown to interact with calcineurin in mammalian cells and cardiomyocytes and down-regulate the calcineurin/NFAT pathway. This is hypothesised to be due to the interaction between the two proteins which maintains calcineurin in a low calcium micro-environment generated by the calcium removal function of the pump. Interestingly, PMCA4 has been shown to interact with calcineurin in endothelial cells. The aim of our study was to further our understanding of PMCA4s regulation of the calcineurin/NFAT pathway specifically in endothelial cells and establish if PMCA4 has a role in the regulation of angiogenesis. ‘Gain of function’ by adenoviral over-expression of PMCA4 and ‘loss of function’ by either si-RNA mediated knockdown of PMCA4 or isolation of PMCA4-/- MLEC were used as models. Over-expression of PMCA4 in HUVEC resulted in inhibition of the calcineurin/NFAT pathway with the opposite result occurring in the case of the knockout of PMCA4, identifying PMCA4 as a negative-regulator of the calcineurin/NFAT pathway in endothelial cells. Over-expression of PMCA4 significantly attenuated VEGF-induced protein and mRNA expression of the pro-angiogenic proteins RCAN1.4 and Cox-2, endothelial cell migration and in vitro and in vivo tube formation with the opposite result occurring in knockdown or knockout studies, confirming PMCA4 as a down-regulator of angiogenesis. Interestingly, over-expression or knockdown of PMCA4 had no effect on VEGF-induced HUVEC proliferation or Erk1/2 phopshorylation proposing PMCA4 may be a potential inhibitor of angiogenesis without compromising cell survival. Disruption of the interaction between PMCA4 and calcineurin by generation and ectopic expression of an adenovirus encoding the region of PMCA4 that interacts with calcineurin (428-651) (Ad-ID4) resulted in an increase in NFAT activity, RCAN1.4 protein expression and in vitro tube formation. These results identify the mechanism of PMCA4s inhibitory effect of the calcineurin/NFAT pathway and consequently angiogenesis is a result of the interaction between the two proteins. The novel findings of this study establish PMCA4 as a negative-regulator of the calcineurin/NFAT pathway in endothelial cells and angiogenesis. These results are far reaching and highlight a potential role for PMCA4 as a therapeutic target in a variety of diseases that are associated with pathological angiogenesis.

Study of HEK293 cells stably expressing fusion protein between delta opioid receptor (δ-OR) and pertussis toxin-insensitive mutant of Gi1α protein, δ-OR-Gi1α (Cys351 -Ile351 ), provided the following results. Decrease of plasma membrane cholesterol content (cholesterol depletion) induced by cyclic oligosaccharide β-cyclodextrin did not affect binding of specific δ-OR agonist, [3 H]DADLE. Neither the maximum number of binding sites nor the affinity of [3 H]DADLE binding was changed by cholesterol depletion. However, the ability of δ-OR to activate cognate trimeric G proteins was impaired. EC50 value of agonist-stimulated [35 S]GTPγS binding was an order of magnitude higher. This effect was observed in case of both control and pertussis toxin-treated cells. It means that cholesterol depletion markedly reduced the efficiency of functional coupling of δ-OR to endogenously expressed pertussis toxin-sensitive G proteins of Gi/Go family as well as covalently bound Gi1α (Cys351 -Ile351 ) protein. Unchanged plasma membrane cholesterol content is therefore important requirement for proper δ-OR function. Detection of the effect of cholesterol depletion on the functional activity of δ-OR was supported by the analysis of changes in biophysical state of plasma membrane using fluorescent membrane probes,...

La membrane plasmique constitue la première entité séparant la cellule de son environnement. A ce rôle de barrière s'ajoute celui de réguler la. Par conséquent, la membrane plasmique est une zone privilégiée pour le passage d'information. Cependant, son étude reste difficile, ne serait-ce que par l'extraordinaire complexité d'organisation de cet assemblage supramoléculaire.Mon projet de thèse vise à développer de nouvelles approches expérimentales pour explorer plus spécifiquement l'organisation et le rôle de la membrane plasmique d'une cellule dans les mécanismes de transduction de l'information. Deux axes ont été privilégiés : le premier, concerne la description de la dynamique d'organisation de la membrane ; le deuxième concerne l'inter-connectivité et la transmission du signal d'une cellule avec d'autres partenaires.Ce manuscrit se compose de plusieurs parties. Le premier chapitre introduira succinctement les questions biologiques. Dans le second chapitre, je présenterai des méthodes utilisées pour l'étude de la membrane. J'y présenterai aussi une série d'observation que j'ai réalisée sur la diffusion de l'EGFR. Le troisième chapitre sera consacré à la technique de corrélation croisée de fluorescence depuis le montage jusqu'à l'étude du modèle EGFR. Dans la quatrième partie, nous verrons comment les collaborations à l'interface biophysique ont permis des développements innovants sur un système de pinces optiques holographiques. J'y présenterai les applications de ce système à différent modèles d'intérêt biologique. Enfin, je conclurai ce document par une brève discussion autour des résultats obtenus aussi bien d'un point de vue méthodologique que biologique. / The plasma membrane separates the cell from its environment. But it is more than a barrier any cell has to communicate with the outside world. Therefore the plasma membrane plays a prime role in transferring and exchanging information. However, the biological study of the plasma membrane remains difficult due to the extraordinary complexity of it organization.My thesis is a part of an effort to develop new experimental approaches to explore more specifically the organization and the role of the plasma membrane in the signal transduction mechanisms. Two major aspects were followed: the first one concerns the description of the dynamics of membrane organization and of molecular interactions, the second concerns the inter-connectivity and signal transduction between a cell and other biological partners.This manuscript is composed of several parts. The first chapter briefly introduces the biological questions that I tried to answer. In the second chapter, I present the methods commonly used to study the membrane with a dynamic perspective. Additionally, I include a series of observations that I made on the EGF receptor diffusion. The third chapter is devoted to the fluorescence cross-correlation technique to study the assembly of the EGFR. In the fourth part, I demonstrate how scientific collaborations at the interface between biology and physics have led to the development of innovative solutions on a holographic optical tweezers system. I present applications of this system in different biological models. Finally, I conclude this thesis with a brief discussion about my technological and biological results.

Os solos ácidos recobrem, cerca de 40% da área terrestre, constituindo-se em um dos principais fatores limitantes à população à produção vegetal do planeta. No Brasil, os solos ácidos abrangem cerca de dois terços do território nacional. De um modo geral, a acidez do subsolo reduz a profundidade do sistema radicular, aumentando a susceptibilidade à seca e diminuindo o uso de nutrientes pelas plantas. Além da alta atividade de íon hidrogênio (H+), os solos ácidos geralmente apresentam níveis tóxicos de alumínio, sendo que a toxicidade por AI tem sido intensivamente investigada nos últimos anos. No entanto, a toxicidade por AI ocorre apenas a pH baixo e em condições onde a toxicidade por prótons geralmente também se manifesta. Apesar disto, trabalhos envolvendo a toxicidade por prótons são escassos. A sensibilidade de células a pH baixo depende da fase de crescimento e desenvolvimento em que estas se encontram. Em raízes, as células mais sensíveis são as da região de alongamento e em suspensões celulares as células na fase log de crescimento são mais sensíveis do que as células na fase estacionária. Este trabalho faz parte de uma linha de pesquisa que procura explorar as diferenças que existem entre células quanto à sensibilidade a pH baixo para melhor entender a toxicidade e tolerância a prótons. Foram utilizadas células da cultura de tabaco cv. BY-2, um sistema modelo que apresenta diversas vantagens sobre o uso de raízes para a realização deste trabalho. Os objetivos deste estudo foram de a) determinar condições apropriadas para a exposição destas células a acidez; b) caracterizar a resposta destas células a pH baixo, sob influência de diferentes fatores ambientais, quando se encontram em estados distintos de sensibilidade ao pH baixo; e c) verificar se mudanças na sensibilidade das células a pH baixo podem ser decorrentes de alterações na composição da membrana plasmática ou na atividade das ATPases. Vários testes iniciais foram realizados com o intuito de se definir algumas condições experimentais - o tempo de exposição, a composição do meio e o tampão a ser empregado. Optou-se por lavar as células e depois incubá-las por 1h em meio simples com pH desejado e contendo apenas Ca \'Cl IND. 2\' 2mM, KCl 10mM. E o tampão MES ou biftalato (10 mM). O biftalato foi testado porque o tampão MES, usado normalmente no meio de cultura completo, não é eficiente na faixa de pH abaixo de 5,0. O biftalato (pKa = 4,1) praticamente não afetou a viabilidade celular avaliada pela permeabilidade a trypan blue, mas inibiu o crescimento celular no meio de cultura completo. Mesmo assim, os dois tampões foram utilizados em paralelo em diversos experimentos e verificou-se que os resultados foram semelhantes. A viabilidade das células na fase log (2 dias) foi reduzida quando se abaixou o pH de 5,6 a 3,8, sendo que caiu mais acentuadamente até o pH de 4,8. As células da fase estacionaria (7d) foram insensíveis ao baixo pH. A um pH fixo de 4,2 aumentando-se a concentração de Ca \'Cl IND. 2\' para cerca de 8 a 16 mM praticamente aboliu o efeito deletério do pH baixo. Para se ter o mesmo efeito com a adição de KCl, foi necessário adicionar entre 80 e 160 mM. A adição de sacarose também amenizou os efeitos do pH\'sendo praticamente revertido a uma concentração de 100 Mm. Os resultados indicam a importância da força iônica e da osmolaridade da solução, mas o efeito de Ca não parece depender apenas destas duas propriedades. A inibição de ATPases, pelo uso de DCCD, não pareceu ter qualquer relação com a sensibilidade a pH baixo. Tanto células de tabaco na fase log quanto estacionárias foram sensíveis à aplicação de ortovanadato de sódio. Em células da fase estacionária, este efeito mais acentuado a pH 4,2, sugerindo que nestas células, as H+ ATPases do tipo P da membrana plasmática podem ter algum papel na tolerância destas células ao baixo pH. Encontrou-se diferenças no perfil protéico de frações enriquecidas em membrana plasmática entre células da fase log e estacionárias e entre células tratadas ou não a pH baixo. Estas diferenças precisam ser melhor estudadas. / Acid soils account for about 40 % of the surface area of the world and are one of the major factors limiting plant productivity. In Brazil, these soils comprise roughly two-thirds of its total territory. In general, soil acidity is detrimental because it limits the depth of the root system, increasing susceptibility to drought and decreasing the use of nutrients. In addition to the high levels of hydrogen ion activity, acid soils usually have Al toxicity hazards, a topic which has been intensely studied in the past years. However, Al toxicity only occurs at low pH, under conditions in which proton toxicity is also a problem. Despite this, studies of proton toxicity are lacking. The sensitivity of cells to low pH depends on their growth and developmental status. In roots, the most sensitive cells are those of the elongation zone and in cell cultures, cells in the log phase are more sensitive than those of the stationary phase. This study is part of a larger attempt to explore the differences that exist between cells with respect to their sensitivity to low pH to further understand toxicity and tolerance to protons. Cells of tobacco BY-2, a plant cell model system which has several advantages over the use of roots, was used in this study. The objectives were a) to determine the appropriate conditions to expose these cells to low pH; b) characterize the response of these cells to low pH, when different environmental factors are varied and at different stages of cellular sensitivity to acidity; and c) examine if changes in the sensitivity of cells to low pH are due to changes in the composition of plasma membranes or in the activity of ATPases. Several preliminary tests were performed to define the experimental conditions to be used ? the duration of exposure to low pH, the composition of the medium and the buffer to be used. A simple solution containing only CaCl2 2mM, KCl 10 mM and MES or phthalate buffer (10 mM) was chosen to wash and incubate the cells at the pH of interest. Phthalate was tested because MES, the buffer normally used in the culture medium, is not effective at pH values below 5.0. Phthalate (pKa = 4,1) had very little effects on cell viability as evaluated by membrane permeability to trypan blue, but it severely inhibited the growth of the cell culture in complete medium. Nevertheless, both buffers were used in several subsequent experiments, the results of which were found to be similar between both buffers. The viability of log-phase cells (2 day-old) was reduced when the pH was lowered from pH 5,6 to 3.8, but this was sharper down to pH 4,8. Cells in stationary phase (7 day-old) were insensitive to low pH. At a fixed pH of 4,2, increases in the concentration of CaCl2 up to 8 or 16 mM abolished most of the deleterious effects of low pH. To generate the same effect, KCl had to be added at concentrations between 80 and 160 mM. The addition of sucrose also alleviated the effects of low pH. The results suggest the importance of solution ionic strength and osmolarity on sensitivity to low pH, but the effects of Ca do not appear to depend only on these two properties. The inhibition of ATPases, by DCCD, did not appear to bear any relation to cellular sensitivity to low pH. Both log-phase and stationary-phase cells were affected by the addition of sodium orthophosphate. In stationary-phase cells, this effect was more pronounced at pH 4,2, suggesting that in these cells, P-type H+-ATPases of the plasma membrane may play a role in the tolerance of these cells to lo pH. Differences were found in the protein profile of enriched plasma membrane fractions both between log-phase and stationary-phase cells and between cells treated or not with low pH. These differences, however, need to be better examined.