Fast Dynamic Color Switching in Temperature-Responsive Plasmonic Films

This research was supported by UK Engineering and Physical Sciences Research Council grants EP/G060649/1 and EP/L027151/1, and ERC grant LINASS 320503. F.B. thanks the supports from the Winton Programme for the Physics of Sustainability.This is the final version of the article. It first appeared from Wiley via https://doi.org/10.1002/adom.20160009

Interactome and F-Actin Interaction Analysis of Dictyostelium discoideum Coronin A

Coronin proteins are evolutionary conserved WD repeat containing proteins that have been proposed to carry out different functions. In; Dictyostelium; , the short coronin isoform, coronin A, has been implicated in cytoskeletal reorganization, chemotaxis, phagocytosis and the initiation of multicellular development. Generally thought of as modulators of F-actin, coronin A and its mammalian homologs have also been shown to mediate cellular processes in an F-actin-independent manner. Therefore, it remains unclear whether or not coronin A carries out its functions through its capacity to interact with F-actin. Moreover, the interacting partners of coronin A are not known. Here, we analyzed the interactome of coronin A as well as its interaction with F-actin within cells and in vitro. Interactome analysis showed the association with a diverse set of interaction partners, including fimbrin, talin and myosin subunits, with only a transient interaction with the minor actin10 isoform, but not the major form of actin, actin8, which was consistent with the absence of a coronin A-actin interaction as analyzed by co-sedimentation from cells and lysates. In vitro, however, purified coronin A co-precipitated with rabbit muscle F-actin in a coiled-coil-dependent manner. Our results suggest that an in vitro interaction of coronin A and rabbit muscle actin may not reflect the cellular interaction state of coronin A with actin, and that coronin A interacts with diverse proteins in a time-dependent manner

The Leishmania donovani Lipophosphoglycan Excludes the Vesicular Proton-ATPase from Phagosomes by Impairing the Recruitment of Synaptotagmin V

We recently showed that the exocytosis regulator Synaptotagmin (Syt) V is recruited to the nascent phagosome and remains associated throughout the maturation process. In this study, we investigated the possibility that Syt V plays a role in regulating interactions between the phagosome and the endocytic organelles. Silencing of Syt V by RNA interference revealed that Syt V contributes to phagolysosome biogenesis by regulating the acquisition of cathepsin D and the vesicular proton-ATPase. In contrast, recruitment of cathepsin B, the early endosomal marker EEA1 and the lysosomal marker LAMP1 to phagosomes was normal in the absence of Syt V. As Leishmania donovani promastigotes inhibit phagosome maturation, we investigated their potential impact on the phagosomal association of Syt V. This inhibition of phagolysosome biogenesis is mediated by the virulence glycolipid lipophosphoglycan, a polymer of the repeating Galβ1,4Manα1-PO4 units attached to the promastigote surface via an unusual glycosylphosphatidylinositol anchor. Our results showed that insertion of lipophosphoglycan into ganglioside GM1-containing microdomains excluded or caused dissociation of Syt V from phagosome membranes. As a consequence, L. donovani promatigotes established infection in a phagosome from which the vesicular proton-ATPase was excluded and which failed to acidify. Collectively, these results reveal a novel function for Syt V in phagolysosome biogenesis and provide novel insight into the mechanism of vesicular proton-ATPase recruitment to maturing phagosomes. We also provide novel findings into the mechanism of Leishmania pathogenesis, whereby targeting of Syt V is part of the strategy used by L. donovani promastigotes to prevent phagosome acidification

Malarial Hemozoin Activates the NLRP3 Inflammasome through Lyn and Syk Kinases

The intraerythrocytic parasite Plasmodium—the causative agent of malaria—produces an inorganic crystal called hemozoin (Hz) during the heme detoxification process, which is released into the circulation during erythrocyte lysis. Hz is rapidly ingested by phagocytes and induces the production of several pro-inflammatory mediators such as interleukin-1β (IL-1β). However, the mechanism regulating Hz recognition and IL-1β maturation has not been identified. Here, we show that Hz induces IL-1β production. Using knockout mice, we showed that Hz-induced IL-1β and inflammation are dependent on NOD-like receptor containing pyrin domain 3 (NLRP3), ASC and caspase-1, but not NLRC4 (NLR containing CARD domain). Furthermore, the absence of NLRP3 or IL-1β augmented survival to malaria caused by P. chabaudi adami DS. Although much has been discovered regarding the NLRP3 inflammasome induction, the mechanism whereby this intracellular multimolecular complex is activated remains unclear. We further demonstrate, using pharmacological and genetic intervention, that the tyrosine kinases Syk and Lyn play a critical role in activation of this inflammasome. These findings not only identify one way by which the immune system is alerted to malarial infection but also are one of the first to suggest a role for tyrosine kinase signaling pathways in regulation of the NLRP3 inflammasome

Étude du rôle de la Synaptotagmine V au cours de la phagocytose et de la maturation du phagosome

De par leur rôle central au sein de l'immunité innée et leur participation à l'immunité acquise, l'étude des fonctions du macrophage est cruciale afin de pouvoir comprendre et contrôler l'invasion de l'organisme par de nombreux microorganismes pathogènes. Parmi les effecteurs de 1'activité microbicide mis en jeu par les macrophages pour éliminer les microorganismes, la phagocytose tient un rôle prépondérant en éliminant les microbes et en permettant le développement de l'immunité adaptative via la présentation antigénique. L'identification et la régulation des molécules impliquées dans ce processus d'internalisation ne sont que partiellement connues. Les résultats obtenus au cours de ces travaux nous ont conduit à l'identification d'un nouvel acteur de la phagocytose, la Synaptotagmine V (Syt V), qui contrôle le processus phagocytique à deux niveaux : lors de la formation du phagosome puis lors de sa maturation. Ainsi, ces travaux ont permis de proposer un rôle de la Syt V dans l'apport membranaire nécessaire à la formation du phagosome, ainsi qu'un mécanisme de régulation du recrutement de la pompe à proton V-ATPase et de la protéase cathepsine D au phagosome dépendant de la Syt V. Dans une seconde partie, nous avons mis en relation le caractère régulateur de la Syt V pour la biogénèse du phagolysosome, avec l'altération de cette biogénèse par le promastigote du parasite Leishmania donovani. Nous avons alors pu observer que le LPG de Leishmania donovani empêchait l'acidification du phagosome et cela en inhibant le recrutement de la Syt V à la membrane du phagosome. Enfin nos résultats proposent une explication à 1'observation de plusieurs études, démontrant que les promastigotes WT de L. donovani sont moins phagocytés que les mutants déficients pour le LPG. Nos résultats suggèrent que l'effet inhibiteur du LPG sur le recrutement de la Syt V est très précoce et qu'il pourrait ainsi empêcher l'apport membranaire dépendant de la Syt V lors de la phagocytose de particule de grandes tailles

Initiation of multicellular differentation in Dictyostelium discoideum regulated by coronin A

Many biological systems respond to environmental changes by activating intracellular signaling cascades, resulting in an appropriate response. One such system is represented by the social amoeba Dictyostelium discoideum. When food sources become scarce, these unicellular cells can initiate a cAMP-driven multicellular aggregation program to ensure long-term survival. On starvation, the cells secrete conditioned medium factors that initiate cAMP signal transduction by inducing expression of genes such as cAMP receptors and adenylate cyclase. The mechanisms involved in the activation of the first pulses of cAMP release have been unclear. We here show a crucial role for the evolutionarily conserved protein coronin A in the initiation of the cAMP response. On starvation, coronin A-deficient cells failed to up-regulate the expression of cAMP-regulated genes, thereby failing to initiate development, despite a normal prestarvation response. Of importance, external addition of cAMP to coronin A-deficient cells resulted in normal chemotaxis and aggregate formation, thereby restoring the developmental program and suggesting a functional cAMP relay in the absence of coronin A. These results suggest that coronin A is dispensable for cAMP sensing, chemotaxis, and development per se but is part of a signal transduction cascade essential for system initiation leading to multicellular development in Dictyostelium

Large scale phagosome preparation

Phagocytosis is the process by which cells engulf and destroy large particles such as pathogens or apoptotic cells. In this way, macrophages play a pivotal role in the resolution of microbial infections. However, many microorganisms have evolved efficient strategies to preempt the weaponry of macrophages. A better understanding of the components engaged in the phagosome formation and maturation is necessary to devise novel approaches aimed at counteracting these microbial strategies. Recently, large-scale approaches have been used to improve our understanding of phagosome functional properties by the identification of hundreds of proteins and by studying each of them. Presently, purification of pathogen-containing phagosomes presents several technical challenges, whereas the use of latex beads to isolate phagosomes presents many advantages because this system can mimic host-pathogen interactions during phagocytosis. This system thus remains the best approach to advance our knowledge of phagosome biology, notably when used in conjunction with functional approaches. In this chapter, we outline an approach for the isolation of large-scale phagosome preparations with high degrees of purity.</br

induit un défaut d’acidification du phagosome

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The Exocytosis Regulator Synaptotagmin V Controls Phagocytosis in Macrophages

Synaptotagmins (Syts) play a key role in the regulation of Ca2+-triggered exocytosis and membrane fusion events, two crucial events associated to the phagocytic process. In the present study, we investigated the role of Syt V, a regulator of focal exocytosis, in phagocytosis. In macrophages, Syt V is localized on recycling endosomes and on filopodia-like structures and is recruited to the nascent phagosomes independently of the phagocytic receptor engaged. Silencing of Syt V by RNA interference revealed a role for this protein for phagocytosis, particularly under conditions of high membrane demand. In contrast, silencing of Syt V had no effect on the recruitment of the lysosomal marker LAMP1 to phagosomes, indicating that phagosome maturation is not regulated by Syt V. Collectively, these results illustrate the importance of Syt V in the regulation of an important innate function of macrophages. Furthermore, our results are consistent with the concept that focal exocytosis of endocytic organelles is a key event in phagocytosis and suggest that Syt V regulates this process.</br

The exocytosis regulator Synaptotagmin V controls phagocytosis in macrophages

Phagocytosis is initiated through binding of a particle by receptors that trigger actin polymerization at the site of contact. Previous studies showed a focalized exocytosis of membrane from internal vesicles at the phagocytic cup. Components of SNAREs are essential for this process. Synaptotagmins (Syt) are a large family of membrane proteins that contains two Ca2+-C2 domains which can bind phospholipids as well as SNARE components. Although they were identified and widely studied in neuronal cells for their role in the regulation of neurotransmitters exocytosis, few studies have demonstrated the expression of synaptotagmin isoforms in macrophages. Several signaling molecules, including members of the protein kinase C (PKC) superfamily participate in the regulation of actin polymerisation and phagolysosome biogenesis. Using a proteomic approach, we identified Syt V as a new potential partner to PKC-a in regulating phagocytosis. We showed that Syt V is expressed in macrophages and that a large part is localized on recycling endosomes. Moreover, we observed the recruitment of Syt V to phagosomes containing various particles of wich Leishmania donovani indicating that Syt V is recruited independently of the phagocytic receptors involved. We also demonstrated an early recruitment of Syt V in macrophages and an accumulation throughout the maturation process notably for both promastigote and amastigote forms of Leishmania donovani. Silencing of Syt V by RNAi revealed a key role for this protein in the regulation of phagocytosis. Collectively, these results showed for the first time the importance of Syt V in the regulation of an important innate function of macrophages and suggest that Syt V acts as a positive modulator of exocytosis with a key role in the regulation of focal exocytosis during phagocytosis.</p