Frustrated Phagocytosis on Micro-Patterned Immune Complexes to Characterize Lysosome Movements in Live Macrophages

Lysosome mobilization is a key cellular process in phagocytes for bactericidal activities and trans-matrix migration. The molecular mechanisms that regulate lysosome mobilization are still poorly known. Lysosomes are hard to track as they move toward phagosomes throughout the cell volume. In order to anticipate cell regions where lysosomes are recruited to, human and RAW264.7 macrophages were seeded on surfaces that were micro-patterned with immune complexes (ICs) as 4 μm-side squares. Distances between IC patterns were adapted to optimize cell spreading in order to constrain lysosome movements mostly in two dimensions. FcΓ receptors triggered local frustrated phagocytosis, frustrated phagosomes appeared as rings of F-actin dots around the IC patterns as early as 5 min after cells made contact with the substratum. Frustrated phagosomes recruited actin-associated proteins (vinculin, paxillin, and gelsolin). The fusion of lysosomes with frustrated phagosomes was shown by the release of beta-hexosaminidase and the recruitment of Lamp1 to frustrated phagosomes. Lysosomes of RAW264.7 macrophages were labeled with cathepsin-D-mCherry to visualize their movements toward frustrated phagosomes. Lysosomes saltatory movements were markedly slowed down compared to cells layered on non-opsonized patterns. In addition, the linearity of the trajectories and the frequency and duration of contacts of lysosomes with frustrated phagosomes were measured. Our experimental set-up is the first step toward deciphering molecular mechanisms which are involved in lysosome movements in the cytoplasm (speed, directionality, and interaction with phagosomes), and opens the door to approaches such as RNA interference, pharmacological inhibition, or mutant expression

SMF-1, SMF-2 and SMF-3 DMT1 Orthologues Regulate and Are Regulated Differentially by Manganese Levels in C. elegans

Manganese (Mn) is an essential metal that can exert toxic effects at high concentrations, eventually leading to Parkinsonism. A major transporter of Mn in mammals is the divalent-metal transporter (DMT1). We characterize here DMT1-like proteins in the nematode C. elegans, which regulate and are regulated by Mn and iron (Fe) content. We identified three new DMT1-like genes in C. elegans: smf-1, smf-2 and smf-3. All three can functionally substitute for loss of their yeast orthologues in S. cerevisiae. In the worm, deletion of smf-1 or smf-3 led to an increased Mn tolerance, while loss of smf-2 led to increased Mn sensitivity. smf mRNA levels measured by QRT-PCR were up-regulated upon low Mn and down-regulated upon high Mn exposures. Translational GFP-fusions revealed that SMF-1 and SMF-3 strongly localize to partially overlapping apical regions of the gut epithelium, suggesting a differential role for SMF-1 and SMF-3 in Mn nutritional intake. Conversely, SMF-2 was detected in the marginal pharyngeal epithelium, possibly involved in metal-sensing. Analysis of metal content upon Mn exposure in smf mutants revealed that SMF-3 is required for normal Mn uptake, while smf-1 was dispensable. Higher smf-2 mRNA levels correlated with higher Fe content, supporting a role for SMF-2 in Fe uptake. In smf-1 and smf-3 but not in smf-2 mutants, increased Mn exposure led to decreased Fe levels, suggesting that both metals compete for transport by SMF-2. Finally, SMF-3 was post-translationally and reversibly down-regulated following Mn-exposure. In sum, we unraveled a complex interplay of transcriptional and post-translational regulations of 3 DMT1-like transporters in two adjacent tissues, which regulate metal-content in C. elegans

Mineral nutrient concentration influences sunflower infection by broomrape (Orobanche cumana)

L'Orobanche cumana Wallr., plante parasite racinaire, provoque de nombreux dégâts sur les cultures de tournesol (Helianthus annuus L.) dans toute l'Europe. Jusqu'à aujourd'hui, la seule méthode de lutte efficace reste l'utilisation de génotypes résistants. Cependant, les mécanismes de résistance à l'orobanche restent encore méconnus, bien que des études précédentes aient démontré l'existence de plusieurs mécanismes. L'étude de génotypes de tournesol sensible (2603) et résistant (LR1) en culture hydroponique a permis de mettre en évidence que la concentration du milieu de culture modifie le niveau d'infestation du tournesol par l'orobanche. Pour le génotype sensible (2603), le nombre de parasites nécrosés augmente avec la concentration du milieu de culture. Pour le génotype résistant (LR1), l'augmentation de la concentration du milieu de culture diminue l'infestation en diminuant le nombre d'orobanches fixées sur les racines et en limitant leur développement ultérieur. Lorsque l'on cultive les tournesols dans le milieu non dilué, l'allocation de radiocarbone dans la plante se modifie avec un accroissement de la force puits de l'apex caulinaire, alors que l'incorporation de 14C est réduite dans les orobanches. Notre étude démontre qu'en milieu contrôlé, la concentration en nutriments influe directement sur le potentiel de résistance du tournesol à l'orobanche

Mineral nutrient concentration influences sunflower infection by broomrape (<i>Orobanche cumana</i>)

Orobanche cumana Wallr., a root parasitic angiosperm, causes severe yield losses in Helianthus annuus  L. (sunflower) in Europe. Until now, the only effective method of controlling this parasite has been the use of resistant sunflower genotypes. Broomrape resistance is, however, poorly understood even though previous studies have revealed several defence mechanisms. The study of a susceptible (2603) and a resistant (LR1) sunflower genotype in hydroponic co-culture showed that the degree of infection by broomrape is influenced by the concentration of nutrients in the growth medium. For the susceptible genotype, an increase in broomrape necrosis was observed when the nutrient concentration was increased. In the resistant genotype LR1, the rate of infection was reduced by increasing the concentration of mineral nutrients, measured as a decrease in broomrape attachments and a lack of underground stem development. When sunflowers were cultivated in full-strength medium, these findings correlated with a lower 14C incorporation in broomrape and a change in carbon allocation to host plant organs with a reinforced “shoot apex sink strength”. Results demonstrated that in controlled conditions, the nutrient concentration directly affects sunflower resistance potential towards broomrape. </jats:p

Analysis of resistance criteria of sunflower recombined inbred lines against Orobanche cumana Wallr.

International audienc

Several mechanisms are involved in resistance of Helianthus to Orobanche cumana Wallr.

DOI: 10.1006/anbo.2001.1520International audienc

Contribution of the GTPase Domain to the Subcellular Localization of Dynamin in the Nematode<i>Caenorhabditis elegans</i>

Caenorhabditis elegans dynamin is expressed at high levels in neurons and at lower levels in other cell types, consistent with the important role that dynamin plays in the recycling of synaptic vesicles. Indirect immunofluorescence showed that dynamin is concentrated along the dorsal and ventral nerve cords and in the synapse-rich nerve ring. Green fluorescent protein (GFP) fused to the N terminus of dynamin is localized to synapse-rich regions. Furthermore, this chimera was detected along the apical membrane of intestinal cells, in spermathecae, and in coelomocytes. Dynamin localization was not affected by disrupting axonal transport of synaptic vesicles in the unc-104 (kinesin) mutant. To investigate the alternative mechanisms that dynamin might use for translocation to the synapse, we systematically tested the localization of different protein domains by fusion to GFP. Localization of each chimera was measured in one specific neuron, the ALM. The GTPase, a middle domain, and the putative coiled coil each contribute to synaptic localization. Surprisingly, the pleckstrin homology domain and the proline-rich domain, which are known to bind to coated-pit constituents, did not contribute to synaptic localization. The GFP-GTPase chimera was most strongly localized, although the GTPase domain has no known interactions with proteins other than with dynamin itself. Our results suggest that different dynamin domains contribute to axonal transport and the sequestration of a pool of dynamin molecules in synaptic cytosol.</jats:p