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International audience; Background: Bacterial invasion covers two steps: adhesion and entry per se. The cell signalling response is triggered upon pathogen interaction at the cell surface. This response continues when the pathogen is internalised. It is likely that these two steps activate different molecular machineries. So far, it has not been possible to easily follow in physiological conditions these events separately. We thus developed an approach to uncouple adhesion from entry using atomic force microscopy (AFM)-driven force and fluorescence measurements.Results: We report nanometric-scale, high-resolution, functional dynamic measurements of bacterial interaction with the host cell surface using photonic and adhesion force analyses. We describe how to achieve a precise monitoring of iterative cell–bacterium interactions to analyse host cell signalling responses to infection. By applying this method to Yersinia pseudotuberculosis, we first unveil glycosylphosphatidylinositol-anchored protein domains recruitment to the bacterium cell surface binding site and concomitant cytoskeleton rearrangements using super-resolution fluorescence microscopy. Second, we demonstrate the feasibility of monitoring post-translationally modified proteins, for example, via ubiquitylation, during the first step of infection.Conclusion: We provide an approach to discriminate between cellular signalling response activated at the plasma membrane during host–pathogen interaction and that is triggered during the internalisation of the pathogen within the cell.Significance: This approach adds to the technological arsenal to better understand and fight against pathogens and beyond the scope of microbiology to address conceptual issues of cell surface signalling.

Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry.

// Remi Montagne 1 , Anne Baranzelli 1, 2 , Ghaffar Muharram 3 , Leroy Catherine 1 , Marie Lesaffre 1 , Audrey Vinchent 1 , Zoulika Kherrouche 1 , Elisabeth Werkmeister 1, 4 , Alexis B. Cortot 1, 2 , David Tulasne 1 1 University Lille, CNRS, Institut Pasteur de Lille, UMR 8161-M3T-Mechanisms of Tumorigenesis and Targeted Therapies, F-59000 Lille, France 2 University Lille, CHU Lille, Thoracic Oncology Department, F-59000 Lille, France 3 University Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019-UMR 8204-CIIL-Center for Infection and Immunity of Lille, F-59000 Lille, France 4 BioImaging Center Lille, Lille 59021, France Correspondence to: David Tulasne, email: david.tulasne@ibl.cnrs.fr Keywords: lung cancer, MET, receptor tyrosine kinase, hepatocyte growth factor/scatter factor, proteolytic cleavages Received: September 02, 2016 Accepted: December 26, 2016 Published: January 04, 2017 ABSTRACT The receptor tyrosine kinase MET and its ligand, the hepatocyte growth factor, are essential to embryonic development, whereas deregulation of MET signaling is associated with tumorigenesis leading to various cancers, including lung carcinoma. Mutations in the MET kinase domain lead to constitutive kinase activity and are associated with tumorigenesis. In lung cancer, however, some mutations are found in the juxtamembrane domain, and their functional consequences are unknown. Because the juxtamembrane domain of MET is targeted by several proteolytic cleavages, involved in its degradation during cell death or under steady-state conditions, we evaluated the influence of these mutations on the MET proteolytic cleavages. In stably transfected epithelial cells expressing MET, the juxtamembrane mutations R970C, P991S, and T992I were found not to modify the known caspase or presenilin-dependent regulated intramembrane proteolysis. Yet when overexpressed, the R970C variant caused generation of an as yet undescribed 45-kDa fragment (p45 MET). This fragment was found in the confluent lung cancer cell line NCI-H1437 carrying the R970C mutation and at a lesser extent in cell lines expressing WT MET, suggesting that R970C mutation favors this cleavage. Generation of p45 MET required the activity of the calpain proteases, confirming the involvement of proteolysis. Ectopic expression of reconstituted p45 MET in epithelial cell lines favored cell scattering and invasion indicating active role of this fragment in HGF/SF induced responses. Hence, although the juxtamembrane mutations of MET do not affect its known proteolytic cleavages, the R970C MET variant favors calpain dependent proteolytic cleavage in lung cancer cells.

Toxoplasma gondii possesses an armada of secreted virulent factors that enable parasite invasion and survival into host cells. These factors are contained in specific secretory organelles, the rhoptries, micronemes and dense granules that release their content upon host cell recognition. Dense granules are secreted in a constitutive manner during parasite replication and play a crucial role in modulating host metabolic and immune responses. While the molecular mechanisms triggering rhoptry and microneme release upon host cell adhesion have been well studied, constitutive secretion remains a poorly explored aspect of T. gondii vesicular trafficking. Here, we investigated the role of the small GTPase Rab11A, a known regulator of exocytosis in eukaryotic cells. Our data revealed an essential role of Rab11A in promoting the cytoskeleton driven transport of dense granules and the release of their content into the vacuolar space. Rab11A also regulates transmembrane protein trafficking and localization during parasite replication, indicating a broader role of Rab11A in cargo exocytosis at the plasma membrane. Moreover, we found that Rab11A also regulates extracellular parasite motility and adhesion to host cells. In line with these findings, MIC2 secretion was altered in Rab11A-defective parasites, which also exhibited severe morphological defects. Strikingly, by live imaging we observed a polarized accumulation of Rab11A-positive vesicles and dense granules at the apical pole of extracellular motile and invading parasites suggesting that apically polarized Rab11A-dependent delivery of cargo regulates early secretory events during parasite entry into host cells., Author summary Toxoplasma gondii (T. gondii) is a highly prevalent parasite infecting a wide range of animals as well as humans. T. gondii secretes numerous virulent factors contained in specific organelles, termed the rhoptries, micronemes and dense granules. These factors are released upon host cell recognition and enable parasite invasion and subsequent development into an intracellular vacuole. In particular, dense granules contain critical effectors that modulate intrinsic defenses of infected host cells ensuring parasite survival and dissemination. The mechanisms regulating dense granule secretion have not been elucidated. In this study, we unraveled a novel role for the T. gondii GTPase Rab11A in promoting dense granule transport along the parasite cytoskeleton and their content release into the vacuolar space during parasite replication. We also found that T. gondii Rab11A regulates extracellular parasite motility and adhesion to host cells suggesting a broader role in distinct secretory pathways essential for parasite virulence.

Background Five to 20% of metastatic EGFR-mutated non-small cell lung cancers (NSCLC) develop acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKI) through MET amplification. The effects of MET amplification on tumor and patient phenotype remain unknown. Methods We investigated,in vitro and in vivo, the impact of MET amplification on the biological properties of the HCC827 cell line, derived from an EGFR-mutated NSCLC. We further evaluated the time to new metastases after EGFR-TKI progression in EGFR-mutated NSCLC, exhibiting MET amplification or high MET overexpression. Results MET amplification significantly enhanced proliferation, anchorage independent growth, anoikis resistance, migration, and induced an epithelial to mesenchymal transition. In vivo, MET amplification significantly increased the tumor growth and metastatic spread. Treatment with a MET-TKI reversed this aggressive phenotype. We found that EGFR-mutated NSCLC patients exhibiting MET amplification on a re-biopsy, performed after EGFR-TKI progression, displayed a shorter time to new metastases after EGFR-TKI progression than patients with high MET overexpression but no MET amplification. Conclusion MET amplification increases metastatic spread even in the context of an already pre-existing strong driver mutation such as EGFR mutation. These results prompt development of therapeutic strategies aiming at preventing emergence of MET amplification.

Autophagy is a predominant eukaryotic mechanism for the engulfment of "portions" of cytoplasm allowing their degradation to recycle metabolites. The autophagy is ubiquitous among the life kingdom revealing the importance of this pathway that appears more complex than previously thought. Several reviews have already addressed how to monitor this pathway and have highlighted the existence of new routes such as the LC3-associated phagocytosis (LAP) and the non-canonical autophagy. The principal difference between autophagosomes and LAP vacuoles is that the former has two limiting membranes positives for LC3 whereas the latter has one. Herein, we propose to emphasize the use of correlative light electron microscopy (CLEM) to answer some autophagy's related questions. The structured illumination microscopy (SIM) relatively easy to implement allows to better observe the Atg proteins recruitment and localization during the autophagy process. While LC3 recruitment is performed using light microscopy the ultrastructural morphological analysis of LC3-vacuoles is ascertained by electron microscopy. Hence, these combined and correlated approaches allow to tackle the LAP vs. autophagosome issue.

Atomic force microscopy (AFM) is becoming increasingly used in the biology field. It can give highly accurate topography and biomechanical quantitative data, such as adhesion, elasticity, and viscosity, on living samples. Nowadays, correlative light electron microscopy is a must-have tool in the biology field that combines different microscopy techniques to spatially and temporally analyze the structure and function of a single sample. Here, we describe the combination of AFM with superresolution light microscopy and electron microscopy. We named this technique correlative light atomic force electron microscopy (CLAFEM) in which AFM can be used on fixed and living cells in association with superresolution light microscopy and further processed for transmission or scanning electron microscopy. We herein illustrate this approach to observe cellular bacterial infection and cytoskeleton. We show that CLAFEM brings complementary information at the cellular level, from on the one hand protein distribution and topography at the nanometer scale and on the other hand elasticity at the piconewton scales to fine ultrastructural details.

The interaction of Mycobacterium tuberculosis (Mtb) with pulmonary epithelial cells is critical for early stages of bacillus colonization and during the progression of tuberculosis. Entry of Mtb into epithelial cells has been shown to depend on F‐actin polymerization, though the molecular mechanisms are still unclear. Here, we demonstrate that mycobacterial uptake into epithelial cells requires rearrangements of the actin cytoskeleton, which are regulated by ADP‐ribosylation factor 1 (Arf1) and phospholipase D1 (PLD1), and is dependent on the M3 muscarinic receptor (M3R). We show that this pathway is controlled by Arf GTPase‐activating protein 1 (ArfGAP1), as its silencing has an impact on actin cytoskeleton reorganization leading to uncontrolled uptake and replication of Mtb. Furthermore, we provide evidence that this pathway is critical for mycobacterial entry, while the cellular infection with other pathogens, such as Shigella flexneri and Yersinia pseudotuberculosis, is not affected. Altogether, these results reveal how cortical actin plays the role of a barrier to prevent mycobacterial entry into epithelial cells and indicate a novel role for ArfGAP1 as a restriction factor of host–pathogen interactions.

Atomic force microscopy (AFM) is becoming increasingly used in the biology field. It can give highly accurate topography and biomechanical quantitative data, such as adhesion, elasticity, and viscosity, on living samples. Nowadays, correlative light electron microscopy is a must-have tool in the biology field that combines different microscopy techniques to spatially and temporally analyze the structure and function of a single sample. Here, we describe the combination of AFM with superresolution light microscopy and electron microscopy. We named this technique correlative light atomic force electron microscopy (CLAFEM) in which AFM can be used on fixed and living cells in association with superresolution light microscopy and further processed for transmission or scanning electron microscopy. We herein illustrate this approach to observe cellular bacterial infection and cytoskeleton. We show that CLAFEM brings complementary information at the cellular level, from on the one hand protein distribution and topography at the nanometer scale and on the other hand elasticity at the piconewton scales to fine ultrastructural details.

In this work, we have considered a new multimodality imaging for macroscopy based on Second Harmonic Generation (SHG) method to monitor invasivelessly the matrix collagen. As the triple helicoidally structure of collagen molecules appearing as not centrosymetric, very organized and spatially oriented, collagen fibrils give rise to a very strong SHG signal and can be imaged without any exogenous dye. To integrate a multidimensional scale with a large field of view (non-sliced samples), we have adapted and validated an instrumental coupling between a two photon excitation laser and a macroscope to collect cartography of SHG signal. We introduced an index (F-SHG) based on decay time response measured by TCSPC for respectively Fluorescence (F) and Second Harmonic Generation (SHG) values. For various sample where protein collagen is the major component of extracellular matrix (vessel, skin, carotide vessel, rat femoral head cartilage, mouse tumor, human wharton’s jelly and rat tendon) or not (nacre), we compared the index distribution obtained with MacroSHG. In this work, we showed for the first time that multiscale large field imaging (Macroscopy) combined to Multimodality approaches (SHG-TCSPC) could be an innovative and non-invasive technique to detect and identify some biological interest molecules (collagen) in biomedical topics.

CD81 is a major receptor for Hepatitis C Virus (HCV). It belongs to the tetraspanin family whose members form dynamic clusters with numerous partner proteins and with one another, forming tetraspanin-enriched areas in the plasma membrane. In our study, we combined single-molecule microscopy and biochemistry experiments to investigate the clustering and membrane behaviour of CD81 in the context of cells expressing EWI-2wint, a natural inhibitor of HCV entry. Interestingly, we found that EWI-2wint reduces the global diffusion of CD81 molecules due to a decrease of the diffusion rate of mobile CD81 molecules and an increase in the proportion of confined molecules. Indeed, we demonstrated that EWI-2wint promotes CD81 clustering and confinement in CD81-enriched areas. In addition, we showed that EWI-2wint influences the colocalization of CD81 with Claudin-1 - a co-receptor required for HCV entry. Together, our results indicate that a change in membrane partitioning of CD81 occurs in the presence of EWI-2wint. This study gives new insights on the mechanism by which HCV enters into its target cells, namely by exploiting the dynamic properties of CD81.

Tuberculosis (TB) is still a major global threat, killing more than one million persons each year. With the constant increase of Mycobacterium tuberculosis strains resistant to first- and second-line drugs, there is an urgent need for the development of new drugs to control the propagation of TB. Although screenings of small molecules on axenic M. tuberculosis cultures were successful for the identification of novel putative anti-TB drugs, new drugs in the development pipeline remains scarce. Host-directed therapy may represent an alternative for drug development against TB. Indeed, M. tuberculosis has multiple specific interactions within host phagocytes, which may be targeted by small molecules. In order to enable drug discovery strategies against microbes residing within host macrophages, we developed multiple fluorescence-based HT/CS phenotypic assays monitoring the intracellular replication of M. tuberculosis as well as its intracellular trafficking. What we propose here is a population-based, multi-parametric analysis pipeline that can be used to monitor the intracellular fate of M. tuberculosis and the dynamics of cellular events such as phagosomal maturation (acidification and permeabilization), zinc poisoning system or lipid body accumulation. Such analysis allows the quantification of biological events considering the host-pathogen interplay and may thus be derived to other intracellular pathogens. © 2017 International Society for Advancement of Cytometry.

International audience; The nuclear pore is a key structure in eukaryotes regulating nuclear-cytoplasmic transport as well as a wide range of cellular processes. Here, we report the characterization of the first Toxoplasma gondii nuclear pore protein, named TgNup302, which appears to be the orthologue of the mammalian Nup98-96 protein. We produced a conditional knock-down mutant that expresses TgNup302 under the control of an inducible tetracycline-regulated promoter. Under ATc treatment, a substantial decrease of TgNup302 protein in inducible knock-down (iKD) parasites was observed, causing a delay in parasite proliferation. Moreover, the nuclear protein TgENO2 was trapped in the cytoplasm of ATc-treated mutants, suggesting that TgNup302 is involved in nuclear transport. Fluorescence in situ hybridization revealed that TgNup302 is essential for 18S RNA export from the nucleus to the cytoplasm, while global mRNA export remains unchanged. Using an affinity tag purification combined with mass spectrometry, we identified additional components of the nuclear pore complex, including proteins potentially interacting with chromatin. Furthermore, reverse immunoprecipitation confirmed their interaction with TgNup302, and structured illuminated microscopy confirmed the NPC localization of some of the TgNup302-interacting proteins. Intriguingly, facilitates chromatin transcription complex (FACT) components were identified, suggesting the existence of an NPC-chromatin interaction in T. gondii. Identification of TgNup302-interacting proteins also provides the first glimpse at the NPC structure in Apicomplexa, suggesting a structural conservation of the NPC components between distant eukaryotes.

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aβ peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30mut) did not affect Aβ secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aβ42 secretion in neuron primary cultures, soluble Aβ42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aβ production, thereby contributing to Alzheimer's disease development., Highlights • By transcriptomic analyses, low levels of ADAM30 expression was observed in the brain of AD cases (compared with controls). • Activation of cathepsin D by ADAM30 is required to modulate the APP metabolism in vitro, i.e. decrease in Ab secretion. • In AD-like mice, neuronal ADAM30 expression led to lower soluble Aβ42, amyloid plaques and to rescue long term potentiation The amyloid precursor protein (APP) processing is central in the etiology of Alzheimer's disease (AD). Characterizing the actors of this metabolism is thus an important challenge. We searched for proteins involved in the APP processing and we selected the A disintegrin-like and metalloprotease 30 (ADAM30). Our results revealed an axis in APP metabolism pathway through an ADAM30-dependent cathepsin D activation. When activated following ADAM30 expression, a decrease in Aβ secretion occurred in vitro and in vivo. ADAM30 under-expression observed in AD brains and thus the impairment of the ADAM30-dependent pathway may favor Aβ peptide production and, ultimately, AD development.

International audience; Apicomplexan parasites have an assortment of unique apical secretory organelles (rhoptries and micronemes), which have crucial functions in host infection. Here, we show that a Toxoplasma gondii sortilin-like receptor (TgSORTLR) is required for the subcellular localization and formation of apical secretory organelles.TgSORTLR is a transmembrane protein that resides within Golgi-endosomal related compartments. The lumenal domain specifically interacts with rhoptry and microneme proteins, while the cytoplasmic tail of TgSORTLR recruits cytosolic sorting machinery involved in anterograde and retrograde protein transport. Ectopic expression of the N-terminal TgSORTLR lumenal domain results in dominant negative effects with the mislocalization of both endogenous TgSORTLR as well as rhoptry and microneme proteins. Conditional ablation of TgSORTLR disrupts rhoptry and microneme biogenesis, inhibits parasite motility, and blocks both invasion into and egress from host cells. Thus, the sortilin-like receptor is essential for protein trafficking and the biogenesis of key secretory organelles in Toxoplasma.

International audience; Osteoarthritis is a degenerative pathology leading to degradation of the extracellular matrix (ECM). Similar effects can be visualized when applying mechanical or biochemical constraints on cartilaginous tissue. Here, we characterized modification of the ECM appearing under mechanical compression and/or biochemical action (hypoxia environment, nitric oxide and collagenase action). In recent decades, multiphoton microscopy has proved its interest for observing living, thick and opaque biological tissues. Thus, the main components of the cartilaginous ECM can be observed without fluorescent labeling. In particular, the collagen network emits strong second harmonic generation (SHG) signal which could be collected at half of the excitation wavelength. Combining autofluorescence and SHG signal detection enables to obtain complementary structural information. Here, we proved that multiphoton microscopy represents an appropriate tool for ex vitro cartilage imaging. First, we showed that SHG signal specifically comes from collagen (collagenase digestion). Further, we verified that the use of an appropriate band-pass filter enables to reject the autofluorescence from the ECM. Once this specificity was shown, we followed modification of the cartilage ECM submitted to mechanical or biochemical constraints (compression, enzymatic digestion). By performing textural analysis of SHG images (Haralick's method), we showed the restructuration of the collagen network according to constraints.

Introduction: Treatment of Non-Small-Cell Lung Cancer (NSCLC) harboring an Epidermal Growth Factor Receptor (EGFR) mutation relies on EGFR tyrosine kinase inhibitors (TKI). However all patients treated with EGFR TKI eventually present tumor progression, because of mechanisms of resistance such as amplification of the met receptor gene. There is currently no data on cellular phenotypic changes of tumor cells induced by met amplification in this context. Objective: to determine whether met amplification triggers a more aggressive tumor phenotype in TKI resistant EGFR-mutated NSCLC. Methods: The proliferation and migration capacities were studied in HCC827 cell line, derived from a mutated EGFR NSCLC, and his daughter cell line HCC827 GR6 which is resistant to EGFR TKI through amplification of the met gene. Tumor growth of both strains was analyzed in a mouse model of ectopic xenograft. Results: in vitro, we found a similar proliferation in the two cell lines and a significant increase in migration and anchorage-independant growth in the met amplified cell line. Treatment with PHA-665752, a MET TKI, significantly reduced the migration advantage of met amplified cells. In vivo, we observed a significant increase in tumor growth with the met amplified cell line. In vivo results regarding the metastatic potential of the two cell lines and the impact of EGFR and MET TKI will be presented during the meeting. Conclusion: met amplification confers an aggressive phenotype to TKI resistant EGFR-mutated NSCLC. These results pave the way for an early use of MET inhibitors in association with EGFR TKI in order to prevent emergence of an aggressive tumor clone.

International audience; Various solid tumors including lung or gastric carcinomas display aberrant activation of the Met receptor which correlates with aggressive phenotypes and poor prognosis. Although downstream signaling of Met is well described, its integration at the transcriptional level is poorly understood. We demonstrate here that in cancer cells harboring met gene amplification, inhibition of Met activity with tyrosine kinase inhibitors or specific siRNA drastically decreased expression of ETV1, ETV4 and ETV5, three transcription factors constituting the PEA3 subgroup of the ETS family, while expression of the other members of the family were less or not affected. Similar link between Met activity and PEA3 factors expression was found in lung cancer cells displaying resistance to EGFR targeted therapy involving met gene amplification. Using silencing experiments, we demonstrate that the PEA3 factors are required for efficient migration and invasion mediated by Met, while other biological responses such as proliferation or unanchored growth remain unaffected. PEA3 overexpression or silencing revealed that they participated in the regulation of the MMP2 target gene involved in extracellular matrix remodeling. Our results demonstrated that PEA3-subgroup transcription factors are key players of the Met signaling integration involved in regulation of migration and invasiveness.

Yersinia pseudotuberculosis can replicate inside macrophages by hijacking autophagy and blocking autophagosome acidification. In bone marrow-derived macrophages, the bacteria are mainly observed inside double-membrane vacuoles positive for LC3, a hallmark of autophagy. Here, we address the question of the membrane traffic during internalization of Yersinia investigating the role of vesicle- associated membrane proteins (VAMPs). First, we show that as in epithelial cells, Yersinia pseudotuberculosis replicates mainly in nonacidic LC3-positive vacuoles. Second, in these cells, we unexpectedly found that VAMP3 localizes preferentially to Yersinia-containing vacuoles (YCVs) with single membranes using correlative light-electron microscopy. Third, we reveal the precise kinetics of VAMP3 and VAMP7 association with YCVs positive for LC3. Fourth, we show that VAMP7 knockdown alters LC3′s association with single-and multimembrane-YCVs. Finally, in uninfected epithelial cells stimulated for autophagy, VAMP3 overexpression and knockdown led respectively to a lower and higher number of double-membrane, LC3-positive vesicles. Hence, our results highlight the role that VAMPs play in selection of the pathways leading to generation of ultrastructurally different LC3 compartments and pave the way for determining the full set of docking and fusion proteins involved in Yersinia pseudotuberculosis’ intravesicular life cycle.

16 páginas, 9 figuras. Contiene en material suplementario: Figs. S1–S3 and Tables S1 and S2."This research was originally published in Journal of Biological Chemistry. Fréville A, Landrieu I, García-Gimeno MA, Vicogne J, Montbarbon M, Bertin B, Verger A, Kalamou H, Sanz P, Werkmeister E, Pierrot C, Khalife J. Plasmodium falciparum inhibitor-3 homolog increases protein phosphatase type 1 activity and is essential for parasitic survival. J Biol Chem. 2012 Jan 6;287(2):1306-21. © the American Society for Biochemistry and Molecular Biology", Growing evidence indicates that the protein regulators governing protein phosphatase 1 (PP1) activity have crucial functions because their deletion drastically affects cell growth and division. PP1 has been found to be essential in Plasmodium falciparum, but little is known about its regulators. In this study, we have identified a homolog of Inhibitor-3 of PP1, named PfI3. NMR analysis shows that PfI3 belongs to the disordered protein family. High affinity interaction of PfI3 and PfPP1 is demonstrated in vitro using several methods, with an apparent dissociation constant K(D) of 100 nm. We further show that the conserved (41)KVVRW(45) motif is crucial for this interaction as the replacement of the Trp(45) by an Ala(45) severely decreases the binding to PfPP1. Surprisingly, PfI3 was unable to rescue a yeast strain deficient in I3 (Ypi1). This lack of functional orthology was supported as functional assays in vitro have revealed that PfI3, unlike yeast I3 and human I3, increases PfPP1 activity. Reverse genetic approaches suggest an essential role of PfI3 in the growth and/or survival of blood stage parasites because attempts to obtain knock-out parasites were unsuccessful, although the locus of PfI3 is accessible. The main localization of a GFP-tagged PfI3 in the nucleus of all blood stage parasites is compatible with a regulatory role of PfI3 on the activity of nuclear PfPP1., The NMR facilities were funded by the Région Nord, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry, and the University of Sciences and Technologies of Lille I.This work was supported in part by Inserm, Institut Pasteur de Lille, Université Lille Nord de France, and TGE RMN THC Grant FR-3050, France

Once regarded as an AIDS-defining illness, Pneumocystis pneumonia (PcP) is nowadays prevailing in immunocompromised HIV-negative individuals such as patients receiving immunosuppressive therapies or affected by primary immunodeficiency. Moreover, Pneumocystis clinical spectrum is broadening to non-severely-immunocompromised subjects who could be colonized by the fungus while remaining asymptomatic for PcP, thus being able to transmit the infection by airborne route to susceptible hosts. Although the taxonomical position of the Pneumocystis genus has been clarified, several aspects of its life cycle remain elusive such as its mode of proliferation within the alveolus or its ploidy level. As no long-term culture model exists to grow Pneumocystis organisms in vitro, an option was to use a model of immunosuppressed rat infected with Pneumocystis carinii and sort life cycle stage fractions using a high-through-put cytometer. Subsequently, ploidy levels of the P. carinii trophic and cystic form fractions were measured by flow cytometry. In the cystic form, eight contents of DNA were measured thus strengthening the fact that each mature cyst contains eight haploid spores. Following release, each spore evolves into a trophic form. The majority of the trophic form fraction was haploid in our study. Some less abundant trophic forms displayed two contents of DNA indicating that they could undergo (i) mating/fusion leading to a diploid status or (ii) asexual mitotic division or (iii) both. Even less abundant trophic forms with four contents of DNA were suggestive of mitotic divisions occurring following mating in diploid trophic forms. Of interest, was the presence of trophic forms with three contents of DNA, an unusual finding that could be related to asymmetrical mitotic divisions occurring in other fungal species to create genetic diversity at lower energetic expenses than mating. Overall, ploidy data of P. carinii life cycle stages shed new light on the complexity of its modes of proliferation.

As a comparatively non-destructive imaging technique into living specimens, fluorescence microscopy has a number of strong advantages over alternative imaging modalities (X-ray, MRI, CT-scan, arthro-scan, etc..). The limited analysis in thick tissue has given rise to the development of other techniques, multiphoton excitation microscopy in particular. A need for increased sensitivity and resolution has been driving the development of new sophisticated fluorescence techniques based on microscopies to study: the tissue microstructure in situ (CLSM, SHG) on deeper thick sections of tissue (multiphoton), molecular diffusion (FRAP, FCS) with fluorescent protein variants and molecular interaction (spectral, FRET, FLIM). In this paper, we have considered developments based on near infrared (NIR) femtosecond excitation in the imaging of articular tissue and discussed the technical limitations and perspectives.

Apicomplexan parasites including Toxoplasma gondii have complex life cycles within different hosts and their infectivity relies on their capacity to regulate gene expression. However, little is known about the nuclear factors that regulate gene expression in these pathogens. Here, we report that T. gondii enolase TgENO2 is targeted to the nucleus of actively replicating parasites, where it specifically binds to nuclear chromatin in vivo. Using a ChIP-Seq technique, we provide evidence for TgENO2 enrichment at the 5′ untranslated gene regions containing the putative promoters of 241 nuclear genes. Ectopic expression of HA-tagged TgENO1 or TgENO2 led to changes in transcript levels of numerous gene targets. Targeted disruption of TgENO1 gene results in a decrease in brain cyst burden of chronically infected mice and in changes in transcript levels of several nuclear genes. Complementation of this knockout mutant with ectopic TgENO1-HA fully restored normal transcript levels. Our findings reveal that enolase functions extend beyond glycolytic activity and include a direct role in coordinating gene regulation in T. gondii.

Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.