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1.
Int J Mol Sci ; 21(4)2020 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-32098122

RESUMEN

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.


Asunto(s)
Actinas/metabolismo , Dictyostelium/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Conejos
2.
Microbiology (Reading) ; 157(Pt 9): 2619-2628, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21680635

RESUMEN

Regulators of membrane fusion play an important role in phagocytosis, as they regulate the focal delivery of endomembrane that is required for optimal internalization of large particles. During internalization of Leishmania promastigotes, the surface glycolipid lipophosphoglycan (LPG) is transferred to the macrophage membrane and modifies its fusogenic properties. In this study, we investigated the impact of LPG on the recruitment of the exocytosis regulator synaptotagmin V (Syt V) at the area of internalization and on the early steps of phagocytosis. Using Leishmania donovani LPG-defective mutants and LPG-coated particles, we established that LPG reduces the phagocytic capacity of macrophages and showed that it causes exclusion of Syt V from the nascent phagosome. Silencing of Syt V inhibited phagocytosis to the same extent as LPG, and these effects were not cumulative, consistent with a Syt V-dependent mechanism for the inhibition of phagocytosis by LPG. Previous work has revealed that LPG-mediated exclusion of Syt V from phagosomes prevents the recruitment of the vacuolar ATPase and acidification. Thus, whereas exclusion of Syt V from phagosomes in the process of formation may be beneficial for the creation of a hospitable intracellular niche, it reduces the phagocytic capacity of macrophages. We propose that the cost associated with a reduced internalization rate may be compensated by increased survival, and could lead to a greater overall parasite fitness.


Asunto(s)
Glicoesfingolípidos/metabolismo , Leishmania donovani/fisiología , Macrófagos/inmunología , Fagocitosis/inmunología , Sinaptotagminas/metabolismo , Animales , Línea Celular , Femenino , Leishmania donovani/crecimiento & desarrollo , Leishmania donovani/inmunología , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Fagosomas/inmunología
3.
PLoS Pathog ; 5(10): e1000628, 2009 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-19834555

RESUMEN

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 Galbeta1,4Manalpha1-PO(4) 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.


Asunto(s)
Glicoesfingolípidos/farmacología , Leishmania donovani/química , Fagosomas/efectos de los fármacos , ATPasas de Translocación de Protón/metabolismo , Sinaptotagminas/metabolismo , Animales , Animales Modificados Genéticamente , Células Cultivadas , Femenino , Glicoesfingolípidos/aislamiento & purificación , Glicoesfingolípidos/metabolismo , Leishmania donovani/genética , Leishmania donovani/metabolismo , Leishmania donovani/fisiología , Macrófagos/metabolismo , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Fagosomas/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Vesículas Transportadoras/efectos de los fármacos , Vesículas Transportadoras/metabolismo
4.
PLoS Pathog ; 5(8): e1000559, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19696895

RESUMEN

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-1beta (IL-1beta). However, the mechanism regulating Hz recognition and IL-1beta maturation has not been identified. Here, we show that Hz induces IL-1beta production. Using knockout mice, we showed that Hz-induced IL-1beta 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-1beta 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.


Asunto(s)
Proteínas Portadoras/fisiología , Hemoproteínas/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Familia-src Quinasas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Adaptadoras de Señalización CARD , Proteínas de Unión al Calcio/inmunología , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Caspasa 1/inmunología , Caspasa 1/metabolismo , Catepsina B/metabolismo , Proteínas del Citoesqueleto/inmunología , Proteínas del Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Femenino , Proteínas HSP90 de Choque Térmico/metabolismo , Hemoproteínas/inmunología , Hemoproteínas/metabolismo , Humanos , Inflamación/inmunología , Interleucina-1beta/biosíntesis , Interleucina-1beta/metabolismo , Malaria/inmunología , Malaria/metabolismo , Malaria/parasitología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Infiltración Neutrófila/inmunología , Fagocitosis , Fosforilación/inmunología , Plasmodium chabaudi/química , Plasmodium chabaudi/metabolismo , Potasio/metabolismo , Proteómica , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/inmunología , Quinasa Syk
5.
J Immunol ; 181(8): 5289-95, 2008 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-18832684

RESUMEN

Synaptotagmins (Syts) play a key role in the regulation of Ca(2+)-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.


Asunto(s)
Exocitosis/inmunología , Macrófagos Peritoneales/inmunología , Fusión de Membrana/inmunología , Fagocitosis/inmunología , Seudópodos/inmunología , Sinaptotagminas/inmunología , Animales , Línea Celular , Exocitosis/genética , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/inmunología , Macrófagos Peritoneales/citología , Fusión de Membrana/genética , Ratones , Fagocitosis/genética , Fagosomas/genética , Fagosomas/inmunología , Seudópodos/genética , Interferencia de ARN , Sinaptotagminas/genética
6.
FEBS Lett ; 2020 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-32298460

RESUMEN

Coronin proteins are widely expressed among eukaryotic organisms. Most coronins consist of a WD-repeat domain followed by a C-terminal coiled coil. Dictyostelium discoideum expresses a single short coronin coronin A, which has been implicated in both actin modulation and multicellular differentiation. Whether coronin A's coiled coil is important for functionality, as well as the oligomeric state of coronin A is not known. Here, we show that the coiled-coil domain in Dictyostelium coronin A functions in homodimerization, is dispensable for coronin A stability and localization but essential for multicellular differentiation. These results allow a better understanding of the role for the coiled-coil domain of coronin A in oligomerization and demonstrate that its presence is essential for multicellular differentiation.

7.
Methods Mol Biol ; 531: 329-46, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19347326

RESUMEN

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.


Asunto(s)
Biología Molecular/métodos , Fagosomas/metabolismo , Animales , Western Blotting , Electroforesis en Gel Bidimensional , Técnica del Anticuerpo Fluorescente , Inmunoprecipitación , Ratones , Proteínas/aislamiento & purificación , Colorantes de Rosanilina , Tinción con Nitrato de Plata
8.
J Vis Exp ; (112)2016 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-27403805

RESUMEN

Dictyostelium discoideum amoeba are found in soil, feeding on bacteria. When food sources become scarce, they secrete factors to initiate a multicellular development program, during which single cells chemotax towards aggregation centers(1-4). This process is dependent on the release of cyclic adenosine monophosphate (cAMP)(5). cAMP is produced in waves through the concerted action of adenylate cyclase and phosphodiesterases, and binds to G protein-coupled cAMP receptors(6,7). A widely used assay to analyze the mechanisms involved in the developmental cycle of the lower eukaryote Dictyostelium discoideum is based on the observation of cell aggregation in submerged conditions(8,9). This protocol describes the analysis of the role of coronin A in the developmental cycle by starvation in tissue-culture plates submerged in balanced salt solution (BSS)(10). Coronin A is a member of the widely conserved protein family of coronins that have been implicated in a wide variety of activities(11,12). Dictyostelium cells lacking coronin A are unable to form multicellular aggregates, and this defect can be rescued by supplying pulses of cAMP, suggesting that coronin A acts upstream of the cAMP cascade(10). The techniques described in these studies provide robust tools to investigate functions of proteins during the initial stages of the developmental cycle of Dictyostelium discoideum upstream of the cAMP cascade. Therefore, utilizing this aggregation assay may allow the further study of coronin A function and advance our understanding of coronin biology.


Asunto(s)
Dictyostelium , Adenilil Ciclasas , Agregación Celular , AMP Cíclico
10.
PLoS One ; 9(2): e88682, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24520414

RESUMEN

The sensing of mechanical forces modulates several cellular responses as adhesion, migration and differentiation. Transient elevations of calcium concentration play a key role in the activation of cells following mechanical stress, but it is still unclear how eukaryotic cells convert a mechanical signal into an ion flux. In this study, we used the model organism Dictyostelium discoideum to assess systematically the role of individual calcium channels in mechanosensing. Our results indicate that PKD2 is the major player in the cell response to rheotaxis (i.e., shear-flow induced mechanical motility), while other putative calcium channels play at most minor roles. Mutant pkd2 KO cells lose the ability to orient relative to a shear flow, whereas their ability to move towards a chemoattractant is unaffected. PKD2 is also important for calcium-induced lysosome exocytosis: WT cells show a transient, 2-fold increase in lysosome secretion upon sudden exposure to high levels of extracellular calcium, but pkd2 KO cells do not. In Dictyostelium, PKD2 is specifically localized at the plasma membrane, where it may generate calcium influxes in response to mechanical stress or extracellular calcium changes.


Asunto(s)
Quimiotaxis , Dictyostelium/citología , Dictyostelium/metabolismo , Proteínas Protozoarias/metabolismo , Reología , Secuencia de Aminoácidos , Calcio/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Quimiotaxis/efectos de los fármacos , Dictyostelium/efectos de los fármacos , Exocitosis/efectos de los fármacos , Ácido Fólico/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Mecanotransducción Celular/efectos de los fármacos , Datos de Secuencia Molecular , Filogenia , Transporte de Proteínas/efectos de los fármacos , Proteínas Protozoarias/química , Reología/efectos de los fármacos , Resistencia al Corte , Estrés Fisiológico/efectos de los fármacos
11.
Mol Biol Cell ; 25(5): 688-701, 2014 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-24403600

RESUMEN

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.


Asunto(s)
Dictyostelium/citología , Proteínas de Microfilamentos/fisiología , Proteínas Protozoarias/fisiología , Transducción de Señal , Adaptación Fisiológica , Quimiotaxis , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Eliminación de Gen , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Transcriptoma
12.
Microbes Infect ; 11(2): 215-22, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19070677

RESUMEN

Lipophosphoglycan (LPG), the major surface glycoconjugate on Leishmania donovani promastigotes, is crucial for the establishment of infection inside macrophages. LPG comprises a polymer of repeating Galbeta1,4Manalpha-PO(4) attached to a lysophosphatidylinositol membrane anchor. LPG is transferred from the parasite to the host macrophage membrane during phagocytosis and induces periphagosomal F-actin accumulation correlating with an inhibition of phagosomal maturation. The biophysical properties of LPG suggest that it may be intercalated into membrane rafts of the host-cell membrane. The aim of this study was to investigate if the effects of LPG on phagosomal maturation are mediated via action on membrane rafts. We show that LPG accumulates in rafts during phagocytosis of L. donovani and that disruption of membrane rafts abolished the effects of LPG on periphagosomal F-actin and phagosomal maturation, indicating that LPG requires intact membrane rafts to manipulate host-cell functions. We conclude that LPG associates with membrane rafts in the host cell and exert its actions on host-cell actin and phagosomal maturation through subversion of raft function.


Asunto(s)
Glicoesfingolípidos/inmunología , Glicoesfingolípidos/metabolismo , Leishmania donovani/inmunología , Macrófagos/inmunología , Microdominios de Membrana/metabolismo , Fagosomas/inmunología , Actinas/metabolismo , Animales , Células Cultivadas , Humanos , Macrófagos/microbiología , Fagosomas/microbiología
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