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Plasma membrane H+-ATPases are highly conserved in yeast, other fungi and plants. Their main role is to establish the H+ gradient at the plasma membrane (PM) to maintain the cytosolic pH close to neutral values and fuel the uptake of nutrients through secondary active transport systems. In both plants and yeast, PM H+-ATPases have also been described to promote activation of TORC1, the rapamycin-sensitive kinase complex controlling cell growth. This for instance occurs in yeast upon H+ influx coupled to amino acid uptake. Fungal and plant PM H+-ATPases are self-inhibited by their carboxyterminal tails unless this domain is phosphorylated at specific residues. In the yeast PM H+-ATPase (Pma1), the self-inhibitory action of the carboxyterminal tail is neutralized when the adjacent S911 and T912 are phosphorylated, but the kinase(s) and phosphatase(s) controlling their phosphorylation remain unknown.In this thesis work, with the objective of understanding the molecular events taking place at Pma1`s level upon H+ influx and triggering the signaling cascade towards TORC1, we show that the largely redundant Ptk1 and Ptk2 kinases are responsible for phosphorylating S911-T912 at the carboxyterminal tail of Pma1. We also show that the carboxyterminal tail of Pma1 is not essential to initial TORC1 activation induced by H+-coupled amino acid uptake. However, if not properly phosphorylated by Ptk1 and Ptk2 kinases, the carboxyterminal tail of Pma1 can interfere with TORC1 activation. Additionally, we provide evidence that activated TORC1 exerts negative feedback control on Pma1 by reducing phosphorylation of its carboxyterminal tail.Overall, our results shed important new light on phosphoregulation of the yeast Pma1 H+-ATPase and on its interconnections with TORC1., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrinmediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.

The Golgi apparatus is a major hub for post-translational modifications and for protein sorting. Accordingly, the preservation of Golgi structure and functionalities are everlasting tasks. It is ensured by mechanisms involving the control of luminal chemical composition and of lipid bilayer thickness, and is supported by structural and transport machineries at the cytosolic side of Golgi cisternae. Failings of those mechanisms usually lead to various cellular troubles, including glycosylation defects. In 2012, some congenital disorders of glycosylation were related to mutations of TMEM165 protein in human. As this protein was not characterized at that time, we started in our laboratory the study of Gdt1p, the Saccharomyces cerevisiae ortholog of TMEM165. It was demonstrated that Gdt1p is a Golgi-localized transmembrane protein able to transport Ca2+ and Mn2+ cations. Furthermore, GDT1 deletion is also characterized by Golgi trafficking defects and glycosylation troubles. The predicted topology of the protein matches with the usual topology of exchangers. In this study, we investigated two elements of Gdt1p physiological function, its transport mechanism and its regulation. Starting from the assumption that Gdt1p could exchange H+ ions against Ca2+ and Mn2+ cations across the Golgi membrane, we developed measurement strategies to assess it. A heterologous expression system using Lactococcus lactis was set up for direct H+ transport assay and a Golgi-localized pH sensor was developed for in vivo measurements in S. cerevisiae. Thanks to them, Gdt1p-mediated H+ transport activity was demonstrated. Then, we looked at Gdt1p regulation. We observed that Gdt1p is rapidly downregulated as a response to extracellular Mn2+ addition and that regulation occurs both at transcriptional and post-translational levels. The fast and efficient retrieval of Gdt1p from the Golgi membrane to be sorted to the vacuole probably occurs trough a novel degradation pathway., (SC - Sciences) -- UCL, 2022

Endocytic mechanisms actively regulate plasma membrane composition and sustain fundamental cellular functions. Recently, we identified a clathrin-independent endocytic (CIE) modality mediated by the BAR domain protein endophilin-A3 (endoA3), which controls the cell surface homeostasis of the tumor marker CD166/ALCAM. Deciphering the molecular machinery of endoA3-dependent CIE should therefore contribute to a better understanding of its pathophysiological role, which remains so far unknown. Here,we investigate the role in this mechanism of actin, Rho GTPases and microtubules, which are major actors of CIE processes. We show that the actin cytoskeleton is dynamically associated with endoA3- and CD166-positive endocytic carriers and that its perturbation strongly inhibits the uptake process of CD166. We also reveal that the Rho GTPase Rac1, but not Cdc42, is a master regulator of this endocytic route. Finally, we provide evidence that microtubules and kinesin molecular motors are required to potentiate endoA3-dependent endocytosis. Of note, our study also highlights potential compensation phenomena between endoA3-dependent CIE and macropinocytosis. Altogether, our data deepen our understanding of this CIE modality and further differentiate it from other unconventional endocytic mechanisms.

Competence for DNA transformation is a major strategy for bacterial adaptation and survival. Yet, this successful tactic is energy-consuming, shifts dramatically the metabolism, and transitory impairs the regular cell-cycle. In streptococci, complex regulatory pathways control competence deactivation to narrow its development to a sharp window of time, a process known as competence shut-off. Although characterized in streptococci whose competence is activated by the ComCDE signaling pathway, it remains unclear for those controlled by the ComRS system. In this work, we investigate competence shut-off in the major human gut commensal Streptococcus salivarius. Using a deterministic mathematical model of the ComRS system, we predicted a negative player under the control of the central regulator ComX as involved in ComS/XIP pheromone degradation through a negative feedback loop. The individual inactivation of peptidase genes belonging to the ComX regulon allowed the identification of PepF as an essential oligoendopeptidase in S. salivarius. By combining conditional mutants, transcriptional analyses, and biochemical characterization of pheromone degradation, we validated the reciprocal role of PepF and XIP in ComRS shut-off. Notably, engineering cleavage site residues generated ultra-resistant peptides producing high and long-lasting competence activation. Altogether, this study reveals a proteolytic shut-off mechanism of competence in the salivarius group and suggests that this mechanism could be shared by other ComRS-containing streptococci.

Endocytosis is an essential cellular process required for the uptake of nutrients from the cell environment and the turnover of plasma membrane components(commonly referred as “cargoes”). It regulates fundamental cellular functions as diverse as cell signaling, adhesion, migration, polarity, division, growth or differentiation. In particular, mammalian cells have developed multiple endocytic routes/mechanisms to support the vast array of physiological conditions, functions and cargoes they face. However, while several endocytic mechanisms have been described so far, their biological relevance often remains elusive, especially in pathophysiological contexts such as cancer. In 2016, my lab identified a cell surface cargo whose internalization seemed to rely on a yet to characterize endocytic route: the protein CD166/ALCAM(Activated Leukocyte Cell Adhesion Molecule). CD166 is an immunoglobulin-like protein involved in cell adhesion and migration, which has been linked to cancer progression by numerous studies. This protein has notably received particular attention as a promising target for new immunotherapies. However, virtually nothing was known about its cellular trafficking. In that context, the present thesis aimed at unveiling the molecular machinery that underlies CD166 internalization and that modulates its function at cancer cell surface. Using state-of-the-art microscopy and cell biology techniques, we could show that CD166 is a clathrin- and dynamin-independent cargo internalized via a novel endocytic mechanism that involves the BAR domain protein endophilin-A3 and the extracellular lectin galectin-8. Additionally, we could demonstrate that the actin cytoskeleton and its regulatory GTPase Rac1 dynamically associate with CD166-positive endocytic carriers and that their perturbation strongly inhibits the uptake process. We also provide evidence that microtubules and kinesin molecular motors are required to potentiate the endoA3-dependent endocytosis of CD166. F, (SC - Sciences) -- UCL, 2022

Aquaporins (AQPs) are channels present in all living organisms. They share the hour-glass shape and are involved in many cellular processes. Plasma membrane intrinsic proteins (PIPs) are AQPs responsible for the facilitated diffusion of water and small solutes through plasma membrane (PM). Land plant PIP subfamily is composed of two distinguishable phylogenetic clusters: PIP1 and PIP2. They assemble as homo- or heterotetramers, the monomer being the functional unit. Previous works have demonstrated that PIPs present a dissimilar behavior regarding their subcellular localization and function. PIP2s are considered as efficient water channels and they localize in the PM. On the other hand, when expressed alone, most of the PIP1s are retained in the endoplasmic reticulum (ER) and are not efficient water channels. When co-expressed, PIP1s and PIP2s interact, a process leading to the relocalization of PIP1s from the ER to the PM and to an increase in the cell water permeability. In maize, this synergistic effect was observed for most of ZmPIP1s members, including ZmPIP1;2 when co-expressed with ZmPIP2;5. This unexpected synergy together with localization studies led researchers to further investigate the nature of ZmPIP1 and ZmPIP2 interaction. The heterotetramerization of PIP monomers is the mechanism responsible for the ZmPIP1 trafficking to the PM and for the modulation of the cell membrane permeability. ZmPIP1;1 is a ubiquitous and one of the highest expressed AQP in maize. It is the closest paralog of ZmPIP1;2. Despite their striking similarity, their function does not correlate. Previous studies showed that the co-expression of ZmPIP1;1 with ZmPIP2;5 did not lead to a synergistic effect in oocyte membrane water permeability, while both isoforms were physically interacting and found in the PM. This enigmatic isoform has been considered to be an inactive water channel until nowadays. In this thesis, we aimed at unraveling the role of ZmPIP1;1, using heterologous and h, (SC - Sciences) -- UCL, 2022

Aquaporins (AQPs) are channels facilitating the diffusion of water and small solutes across the cellular membranes. Recent evidence suggests that plant AQPs also facilitate the rapid permeation of hydrogen peroxide (H2O2) and even cations, which are essential for plant physiology. In our project, we aim at establishing molecular and cellular tools to determine whether plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) facilitate the diffusion of H2O2 and cations through the plasma membrane in plant cells and characterize this transport. We prepared genetic constructs to stably co-express in Nicotiana tabacum BY2 suspension cells the fluorescent H2O2 sensor HyPer, with maize PIP2;5, an AQP that, when expressed in yeast, facilitates the membrane diffusion of H2O2. PIP2;5 expression was controlled by a heat shock inducible promoter. After induction of PIP2;5 expression, a HyPer signal was recorded when the cells were incubated with H2O2, which suggests that PIP2;5 facilitates H2O2 transmembrane diffusion. Using this new system, we tested several maize and Arabidopsis PIP2s and showed that they facilitate the H2O2 transmembrane diffusion, but it was not the case for the PIP2;5W85A mutant. We also showed that treatment by abscisic acid and the elicitor flagellin-derived flg22 peptide induced the intracellular H2O2 accumulation in plant cells when PIP2;5 was expressed. These results indicate that plant suspension cells and the HyPer sensor co-expressed with PIPs are a powerful toolkit for evaluating the transport specificity of PIPs in living cells, determining their molecular determinants as well as real-time monitoring H2O2 dynamics in plant single cells. The cation transport ability of PIP2;5 was first tested in a yeast expression system. PIP2;5 increased the Na+ sensitivity of the cells compared with the control yeast. We confirmed this result in BY2 cells using a novel fluorescent photo-switchable Li+ sensor. After induction of PIP2;5 expression and incu, (SC - Sciences) -- UCL, 2021

The maintenance of K+ and Ca2+ homeostasis is crucial for many cellular functions. Potassium is accumulated in cells at high concentrations, while the cytosolic level of calcium, to ensure its signalling function, is kept at low levels and transiently increases in response to stresses. We examined Ca2+ homeostasis and Ca2+ signalling in Saccharomyces cerevisiae strains lacking plasma-membrane K+ influx (Trk1 and Trk2) or efflux (Tok1, Nha1 and Ena1-5) systems. The lack of K+ exporters slightly increased the cytosolic Ca2+, but did not alter the Ca2+ tolerance or Ca2+-stress response. In contrast, the K+-importers Trk1 and Trk2 play important and distinct roles in the maintenance of Ca2+ homeostasis. The presence of Trk1 was vital mainly for the growth of cells in the presence of high extracellular Ca2+, whilst the lack of Trk2 doubled steady-state intracellular Ca2+ levels. The absence of both K+ importers highly increased the Ca2+ response to osmotic or CaCl2 stresses and altered the balance between Ca2+ flux from external media and intracellular compartments. In addition, we found Trk2 to be important for the tolerance to high KCl and hygromycin B in cells growing on minimal media. All the data describe new interconnections between potassium and calcium homeostasis in S. cerevisiae.

Although synthetic pesticides are still used to control insect pests, greater efforts have been made to develop healthier and more environmentally friendly pesticides. Because of their insecticidal properties, botanical essential oils (EOs) are considered as promising alternatives to the use of synthetic insecticides. However, little is known about mechanisms underlying the insecticidal activity of most these natural compounds. In the present study, we evaluated the contact toxicity and the modes of action of the EO from Mentha arvensis against the granary weevil, Sitophilus granarius L. (Coleoptera: Curculionidae), a cosmopolitan insect pest that causes extensive damage to stored cereals. M. arvensis EO caused high contact toxicity in S. granarius adults, resulting in a rapid paralysis and rapid alteration of walking behavior. Our label-free quantitative proteomics approach revealed that M. arvensis EO induced dramatic physiological changes in exposed insects. The majority of the differentially expressed proteins (DEPs) were upregulated and are related to the development and functioning of the muscular and nervous systems, cellular respiration, protein synthesis, and detoxification. These results suggest that M. arvensis essential oil is capable of affecting a variety of biological processes, and shed light on the repair mechanisms put in place in surviving insects to counter the damage inflicted. This work opens new perspectives on the proposed mechanisms of insecticidal activity of a promising EO for controlling pests of stored cereals and may represent a first step in the development of novel bio-rational insecticides.

It is of fundamental interest in evolutionary biology to link the effect of mutations in a protein to the fitness of an organism. One of the best methods to investigate this effect is to perform a deep mutational scan (DMS). This involves creating a library of protein mutants which are subject to selection in diverse conditions and consequently high throughput sequencing is employed to monitor the fate of mutants. In this study, in the first section we introduce a novel method of creating large chromosomally encoded libraries (upto 10^9 variants) by a two-step scarless gene replacement using a cat-oroP cassette in Streptococcus thermophilus. We use this method to create a library of 10^8 variants of alanine racemase (Alr) cloned at its native locus in the chromosome. Then in the second section, the Alr library was grown in two conditions, in presence and absence of selection pressure for function. Growth under functional selection, created a map of residues essential to alanine racemase activity expressed under native conditions. We observe that 44% of substitutions were deleterious for activity. In absence of selection pressure for function, we observe that the majority of substitutions (67%) had neutral effects on fitness. In both conditions, certain substitutions were selected which may be involved in restricting entry to the active site. In the third section, we engineered a S. thermophilus strain by inserting a SARS-CoV-2 protease recognition sequence in the Alr protein. This mutant displayed conditional auxotrophy on expressing the protease. This strain was used to screen libraries of peptidic inhibitors to identify SARS-CoV-2 protease inhibitors., (SC - Sciences) -- UCL, 2021

The coupling between mitochondrial respiration and photosynthesis plays an important role in the energetic physiology of green plants and some secondary-red photosynthetic eukaryotes(diatoms), allowing an efficient CO2 assimilation and optimal growth. - Using the flagellate Euglena gracilis, we first tested if photosynthesis–respiration coupling occurs in this species harbouring secondary green plastids (i.e. originated from an endosymbiosis between a green alga and a phagotrophic euglenozoan). Second, we tested how the trophic state (mixotrophy and photoautotrophy) of the cell alters the mechanisms involved in the photosynthesis–respiration coupling. - Energetic coupling between photosynthesis and respiration was determined by testing the effect of respiratory inhibitors on photosynthesis, and measuring the simultaneous variation of photosynthesis and respiration rates as a function of temperature (i.e. thermal response curves). The mechanism involved in the photosynthesis–respiration coupling was assessed by combining proteomics, biophysical and cytological analyses. - Our work shows that there is photosynthesis–respiration coupling and membrane contacts between mitochondria and chloroplasts in E. gracilis. However, whereas in mixotrophy adjustment of the chloroplast ATP/NADPH ratio drives the interaction, in photoautotrophy the coupling is conditioned by CO2 limitation and photorespiration. This indicates that maintenance of photosynthesis–respiration coupling, through plastic metabolic responses, is key to E. gracilis functioning under changing environmental conditions.