RESUMO
A Paramecium cell has as many types of membrane interactions as mammalian cells, as established with monoclonal antibodies by R. Allen and A. Fok. Since then, we have identified key players, such as SNARE proteins, Ca2+ -regulating proteins, including Ca2+ -channels, Ca2+ -pumps, Ca2+ -binding proteins of different affinity, etc., at the molecular level, probed their function and localized them at the light and electron microscopy level. SNARE proteins, in conjunction with a synaptotagmin-like Ca2+ -sensor protein, mediate membrane fusion. This interaction is additionally regulated by monomeric GTPases whose spectrum in Tetrahymena and Paramecium has been established by A. Turkewitz. As known from mammalian cells, GTPases are activated on membranes in conjunction with lumenal acidification by an H+ -ATPase. For these complex molecules, we found in Paramecium an unsurpassed number of 17 a-subunit paralogs which connect the polymeric head and basis part, V1 and V0. (This multitude may reflect different local functional requirements.) Together with plasmalemmal Ca2+ -influx channels, locally enriched intracellular InsP3 -type (InsP3 R, mainly in osmoregulatory system) and ryanodine receptor-like Ca2+ -release channels (ryanodine receptor-like proteins, RyR-LP), this complexity mediates Ca2+ signals for most flexible local membrane-to-membrane interactions. As we found, the latter channel types miss a substantial portion of the N-terminal part. Caffeine and 4-chloro-meta-cresol (the agent used to probe mutations of RyRs in man during surgery in malignant insomnia patients) initiate trichocyst exocytosis by activating Ca2+ -release channels type CRC-IV in the peripheral part of alveolar sacs. This is superimposed by Ca2+ -influx, that is, a mechanism called "store-operated Ca2+ -entry" (SOCE). For the majority of key players, we have mapped paralogs throughout the Paramecium cell, with features in common or at variance in the different organelles participating in vesicle trafficking. Local values of free Ca2+ -concentration, [Ca2+ ]i , and their change, for example, upon exocytosis stimulation, have been registered by flurochromes and chelator effects. In parallel, we have registered release of Ca2+ from alveolar sacs by quenched-flow analysis combined with cryofixation and X-ray microanalysis.
Assuntos
Paramecium , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Humanos , Mamíferos , Paramecium/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas SNARE/metabolismoRESUMO
From the very dawn of biological evolution, ATP was selected as a multipurpose energy-storing molecule. Metabolism of ATP required intracellular free Ca(2+) to be set at exceedingly low concentrations, which in turn provided the background for the role of Ca(2+) as a universal signalling molecule. The early-eukaryote life forms also evolved functional compartmentalization and vesicle trafficking, which used Ca(2+) as a universal signalling ion; similarly, Ca(2+) is needed for regulation of ciliary and flagellar beat, amoeboid movement, intracellular transport, as well as of numerous metabolic processes. Thus, during evolution, exploitation of atmospheric oxygen and increasingly efficient ATP production via oxidative phosphorylation by bacterial endosymbionts were a first step for the emergence of complex eukaryotic cells. Simultaneously, Ca(2+) started to be exploited for short-range signalling, despite restrictions by the preset phosphate-based energy metabolism, when both phosphates and Ca(2+) interfere with each other because of the low solubility of calcium phosphates. The need to keep cytosolic Ca(2+) low forced cells to restrict Ca(2+) signals in space and time and to develop energetically favourable Ca(2+) signalling and Ca(2+) microdomains. These steps in tandem dominated further evolution. The ATP molecule (often released by Ca(2+)-regulated exocytosis) rapidly grew to be the universal chemical messenger for intercellular communication; ATP effects are mediated by an extended family of purinoceptors often linked to Ca(2+) signalling. Similar to atmospheric oxygen, Ca(2+) must have been reverted from a deleterious agent to a most useful (intra- and extracellular) signalling molecule. Invention of intracellular trafficking further increased the role for Ca(2+) homeostasis that became critical for regulation of cell survival and cell death. Several mutually interdependent effects of Ca(2+) and ATP have been exploited in evolution, thus turning an originally unholy alliance into a fascinating success story.This article is part of the themed issue 'Evolution brings Ca(2+) and ATP together to control life and death'.
Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Células Eucarióticas/fisiologia , Evolução Molecular , Transdução de Sinais , Sinalização do Cálcio , Comunicação CelularRESUMO
The lipid raft proteins reggie-1 and -2 (flotillins) are implicated in membrane protein trafficking but exactly how has been elusive. We find that reggie-1 and -2 associate with the Rab11a, SNX4, and EHD1-decorated tubulovesicular recycling compartment in HeLa cells and that reggie-1 directly interacts with Rab11a and SNX4. Short hairpin RNA-mediated down-regulation of reggie-1 (and -2) in HeLa cells reduces association of Rab11a with tubular structures and impairs recycling of the transferrin-transferrin receptor (TfR) complex to the plasma membrane. Overexpression of constitutively active Rab11a rescues TfR recycling in reggie-deficient HeLa cells. Similarly, in a Ca(2+) switch assay in reggie-depleted A431 cells, internalized E-cadherin is not efficiently recycled to the plasma membrane upon Ca(2+) repletion. E-cadherin recycling is rescued, however, by overexpression of constitutively active Rab11a or SNX4 in reggie-deficient A431 cells. This suggests that the function of reggie-1 in sorting and recycling occurs in association with Rab11a and SNX4. Of interest, impaired recycling in reggie-deficient cells leads to de novo E-cadherin biosynthesis and cell contact reformation, showing that cells have ways to compensate the loss of reggies. Together our results identify reggie-1 as a regulator of the Rab11a/SNX4-controlled sorting and recycling pathway, which is, like reggies, evolutionarily conserved.
Assuntos
Caderinas/metabolismo , Proteínas de Membrana/metabolismo , Receptores da Transferrina/metabolismo , Nexinas de Classificação/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Caderinas/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Regulação para Baixo , Células HeLa , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Filogenia , Transporte ProteicoRESUMO
The reggie/flotillin proteins are implicated in membrane trafficking and, together with the cellular prion protein (PrP), in the recruitment of E-cadherin to cell contact sites. Here, we demonstrate that reggies, as well as PrP down-regulation, in epithelial A431 cells cause overlapping processes and abnormal formation of adherens junctions (AJs). This defect in cell adhesion results from reggie effects on Src tyrosine kinases and epidermal growth factor receptor (EGFR): loss of reggies reduces Src activation and EGFR phosphorylation at residues targeted by Src and c-cbl and leads to increased surface exposure of EGFR by blocking its internalization. The prolonged EGFR signaling at the plasma membrane enhances cell motility and macropinocytosis, by which junction-associated E-cadherin is internalized and recycled back to AJs. Accordingly, blockage of EGFR signaling or macropinocytosis in reggie-deficient cells restores normal AJ formation. Thus, by promoting EGFR internalization, reggies restrict the EGFR signaling involved in E-cadherin macropinocytosis and recycling and regulate AJ formation and dynamics and thereby cell adhesion.
Assuntos
Junções Aderentes/metabolismo , Caderinas/metabolismo , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Junções Aderentes/ultraestrutura , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Endocitose , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Membrana/genética , Fosfoproteínas/metabolismo , Fosforilação , Príons/genética , Príons/metabolismo , Processamento de Proteína Pós-Traducional , Transporte Proteico , Interferência de RNA , Transdução de Sinais , beta Catenina/metabolismoRESUMO
The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.
Assuntos
Eimeria/química , Organelas/química , Organelas/metabolismo , Sequência de Aminoácidos , Eimeria/genética , Eimeria/metabolismo , Eimeria/ultraestrutura , Dados de Sequência Molecular , Organelas/genética , Organelas/ultraestrutura , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de SequênciaRESUMO
Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to humans. They possess an acidic matrix that contains several cations bound to phosphates, which are mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. Calcium uptake occurs through a Ca(2+)/H(+) countertransporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. We report the purification and characterization of an acidocalcisome fraction from T. gondii tachyzoites after subcellular fractionation and further discontinuous iodixanol gradient purification. Proton and calcium transport activities in the fraction were characterized by fluorescence microscopy and spectrophotometric methods using acridine orange and arsenazo III, respectively. This work will facilitate the understanding of the function of acidocalcisomes in Apicomplexan parasites, as we can now isolate highly purified fractions that could be used for proteomic analysis to find proteins that may clarify the biogenesis of these organelles.
Assuntos
Cálcio/metabolismo , Organelas/metabolismo , Prótons , Toxoplasma/metabolismo , Trifosfato de Adenosina/metabolismo , Biomarcadores/metabolismo , Cloreto de Cálcio/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Organelas/efeitos dos fármacos , Organelas/ultraestrutura , Polifosfatos/metabolismo , Bombas de Próton/metabolismo , Cloreto de Sódio/farmacologia , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Toxoplasma/citologia , Toxoplasma/efeitos dos fármacos , Ácidos Tri-Iodobenzoicos/farmacologiaRESUMO
Due to their well-defined pathways of vesicle trafficking and manyfold mutants ciliates have served as good model systems. Further studies required the development of databases, now available for Paramecium and Tetrahymena. A variety of key players have been identified and characterized based on BLAST search, domain analysis, localization, and gene-silencing studies. They include NSF (N-ethylmaleimide sensitive factor), SNAREs (soluble NSF attachment protein [SNAP] receptors), the H(+)-ATPase (V-ATPase) and actin, while Arf (ADP-ribosylation factor) and Rab-type small GTPases, COPs (coatamer proteins) and many others remain to be elucidated. The number of SNAREs, H(+)-ATPase subunits, and actins ever found within one cell type are unexpectedly high and most of the manifold vesicle types seem to be endowed with specific molecular components pertinent to trafficking. As in higher eukaryotes, multifactorial targeting likely occurs. It appears that, in parallel to higher organisms, ciliates have evolved a similar structural and molecular complexity of vesicle trafficking.
Assuntos
Actinas/metabolismo , Membrana Celular/metabolismo , Cilióforos/citologia , Cilióforos/fisiologia , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas SNARE/metabolismo , Sequência de Aminoácidos , Animais , Transporte Biológico/fisiologia , Cálcio/metabolismo , Cilióforos/classificação , Endocitose/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Exocitose/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Humanos , Fusão de Membrana/fisiologia , Modelos Moleculares , Dados de Sequência Molecular , Paramecium/citologia , Paramecium/fisiologia , Fagocitose/fisiologia , Filogenia , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Tetrahymena/citologia , Tetrahymena/fisiologia , Vesículas Transportadoras/metabolismoRESUMO
The vacuolar H(+)-ATPase (V-ATPase), a multisubunit, adenosine triphosphate (ATP)-driven proton pump, is essential for numerous cellular processes in all eukaryotes investigated so far. While structure and catalytic mechanism are similar to the evolutionarily related F-type ATPases, the V-ATPase's main function is to establish an electrochemical proton potential across membranes using ATP hydrolysis. The holoenzyme is formed by two subcomplexes, the transmembraneous V(0) and the cytoplasmic V(1) complexes. Sequencing of the whole genome of the ciliate Paramecium tetraurelia enabled the identification of virtually all the genes encoding V-ATPase subunits in this organism and the studying of the localization of the enzyme and roles in membrane trafficking and osmoregulation. Surprisingly, the number of V-ATPase genes in this free-living protozoan is strikingly higher than in any other species previously studied. Especially abundant are V(0)-a-subunits with as many as 17 encoding genes. This abundance creates the possibility of forming a large number of different V-ATPase holoenzymes by combination and has functional consequences by differential targeting to various organelles.
Assuntos
Isoenzimas/metabolismo , Paramecium/enzimologia , Paramecium/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Isoenzimas/genética , Paramecium/citologia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
The causative agent of transmissible spongiform encephalopathies (TSE) is PrPSc, an infectious, misfolded isoform of the cellular prion protein (PrPC). The localisation and trafficking of PrPSc and sites of conversion from PrPC to PrPSc are under debate, particularly since most published work did not discriminate between PrPC and PrPSc. Here we describe the localisation of PrPC and PrPSc in a scrapie-infected neuroblastoma cell line, ScN2a, by light and electron microscopic immunolocalisation. After eliminating PrPC with proteinase K, PrPSc was detected at the plasma membrane, endocytosed via clathrin-coated pits and delivered to early endosomes. Finally, PrPSc was detected in late endosomes/lysosomes. As we detected PrPSc at the cell surface, in early endosomes and in late endosomes/lysosomes, i.e. locations where PrPC is also present, our data imply that the conversion process could take place at the plasma membrane and/or along the endocytic pathway. Finally, we observed the release of PrPC/PrPSc via exocytotic pathways, i.e. via exosomes and as an opaque electron-dense mass which may represent a mechanism of intercellular spreading of infectious prions.
Assuntos
Neuroblastoma , Proteínas PrPSc , Scrapie , Animais , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Membrana Celular/química , Membrana Celular/ultraestrutura , Concanavalina A/metabolismo , Endocitose , Endopeptidase K/metabolismo , Endossomos/metabolismo , Endossomos/ultraestrutura , Exossomos/química , Exossomos/metabolismo , Exossomos/ultraestrutura , Guanidinas/química , Humanos , Imuno-Histoquímica , Isotiocianatos/química , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos , Neuroblastoma/química , Neuroblastoma/ultraestrutura , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Desnaturação Proteica , Scrapie/metabolismoRESUMO
The reggie/flotillin proteins oligomerize and associate into clusters which form scaffolds for membrane microdomains. Besides their localization at the plasma membrane, the reggies/flotillins reside at various intracellular compartments; however, the trafficking pathways used by reggie-1/flotillin-2 remain unclear. Here, we show that trafficking of reggie-1/flotillin-2 is BFA sensitive and that deletion mutants of reggie-1/flotillin-2 accumulate in the Golgi complex in HeLa, Jurkat and PC12 cells, suggesting Golgi-dependent trafficking of reggie-1/flotillin-2. Using total internal reflection fluorescence microscopy, we observed fast cycling of reggie-1/flotillin-2-positive vesicles at the plasma membrane, which engaged in transient interactions with the plasma membrane only. Reggie-1/flotillin-2 cycling was independent of clathrin, but was inhibited by cholesterol depletion and microtubule disruption. Cycling of reggie-1/flotillin-2 was negatively correlated with cell-cell contact formation but was stimulated by serum, epidermal growth factor and by cholesterol loading mediated by low density lipoproteins. However, reggie-1/flotillin-2 was neither involved in endocytosis of the epidermal growth factor itself nor in endocytosis of GPI-GFPs or the GPI-anchored cellular prion protein (PrP(c)). Reggie-2/flotillin-1 and stomatin-1 also exhibited cycling at the plasma membrane similar to reggie-1/flotillin-2, but these vesicles and microdomains only partially co-localized with reggie-2/flotillin-1. Thus, regulated vesicular cycling might be a general feature of SPFH protein-dependent trafficking.
Assuntos
Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Animais , Colesterol/metabolismo , Endocitose , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Células HeLa , Humanos , Células Jurkat , Microdomínios da Membrana/química , Microdomínios da Membrana/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia de Fluorescência , Microtúbulos/metabolismo , Mutação , Proteínas do Tecido Nervoso , Células PC12 , Príons/metabolismo , Estrutura Terciária de Proteína , Transporte Proteico , RatosRESUMO
Acidocalcisomes are acidic calcium stores found in diverse organisms, being conserved from bacteria to man. They posses an acidic matrix that contains several cations bound to phosphates, mainly present in the form of short and long polyphosphate chains. Their matrix is acidified through the action of proton pumps such as a vacuolar proton ATPase and a vacuolar proton pyrophosphatase. The calcium uptake occurs through a Ca2+/H+ counter transporting ATPase located in the membrane of the organelle. Acidocalcisomes have been identified in a variety of microorganisms, including Apicomplexan parasites such as Plasmodium and Eimeria species, and in Toxoplasma gondii. In this paper, we review the structural, biochemical and physiological aspects of acidocalcisomes in Apicomplexan parasites and discuss their functional roles in the maintenance of intracellular ion homeostasis.
Assuntos
Apicomplexa/ultraestrutura , Cálcio/metabolismo , Organelas/fisiologia , Animais , Apicomplexa/fisiologia , Concentração de Íons de Hidrogênio , Organelas/química , Organelas/ultraestrutura , Fósforo/análise , Fósforo/química , Plasmodium/fisiologia , Plasmodium/ultraestrutura , Toxoplasma/fisiologia , Toxoplasma/ultraestruturaRESUMO
The reggies/flotillins are oligomeric scaffolding proteins for membrane microdomains. We show here that reggie-1/flotillin-2 microdomains are organized along cortical F-actin in several cell types. Interaction with F-actin is mediated by the SPFH domain as shown by in vivo co-localization and in vitro binding experiments. Reggie-1/flotillin-2 microdomains form independent of actin, but disruption or stabilization of the actin cytoskeleton modulate the lateral mobility of reggie-1/flotillin-2 as shown by FRAP. Furthermore, reggie/flotillin microdomains can efficiently be immobilized by actin polymerisation, while exchange of reggie-1/flotillin-2 molecules between microdomains is enhanced by actin disruption as shown by tracking of individual microdomains using TIRF microscopy.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos , Animais , Linhagem Celular Tumoral , Humanos , Proteínas de Membrana/genética , Camundongos , Ligação Proteica , Transporte ProteicoRESUMO
BACKGROUND: A Paramecium tetraurelia pilot genome project, the subsequent sequencing of a Megabase chromosome as well as the Paramecium genome project aimed at gaining insight into the genome of Paramecium. These cells display a most elaborate membrane trafficking system, with distinct, predictable pathways in which actin could participate. Previously we had localized actin in Paramecium; however, none of the efforts so far could proof the occurrence of actin in the cleavage furrow of a dividing cell, despite the fact that actin is unequivocally involved in cell division. This gave a first hint that Paramecium may possess actin isoforms with unusual characteristics. The genome project gave us the chance to search the whole Paramecium genome, and, thus, to identify and characterize probably all actin isoforms in Paramecium. RESULTS: The ciliated protozoan, P. tetraurelia, contains an actin multigene family with at least 30 members encoding actin, actin-related and actin-like proteins. They group into twelve subfamilies; a large subfamily with 10 genes, seven pairs and one trio with > 82% amino acid identity, as well as three single genes. The different subfamilies are very distinct from each other. In comparison to actins in other organisms, P. tetraurelia actins are highly divergent, with identities topping 80% and falling to 30%. We analyzed their structure on nucleotide level regarding the number and position of introns. On amino acid level, we scanned the sequences for the presence of actin consensus regions, for amino acids of the intermonomer interface in filaments, for residues contributing to ATP binding, and for known binding sites for myosin and actin-specific drugs. Several of those characteristics are lacking in several subfamilies. The divergence of P. tetraurelia actins and actin-related proteins between different P. tetraurelia subfamilies as well as with sequences of other organisms is well represented in a phylogenetic tree, where P. tetraurelia sequences only partially cluster. CONCLUSION: Analysis of different features on nucleotide and amino acid level revealed striking differences in isoforms of actin and actin-related proteins in P. tetraurelia, both within the organism and in comparison to other organisms. This diversification suggests unprecedented specification in localization and function within a unicellular eukaryote.
Assuntos
Actinas/química , Actinas/genética , Família Multigênica/genética , Paramecium tetraurellia/química , Paramecium tetraurellia/genética , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteína 2 Relacionada a Actina/química , Proteína 2 Relacionada a Actina/genética , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/química , Proteína 3 Relacionada a Actina/genética , Proteína 3 Relacionada a Actina/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sequência Consenso/genética , Filogenia , Projetos Piloto , Ligação Proteica/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
T cell activation after contact with an antigen-presenting cell depends on the regulated assembly of the T cell receptor signaling complex, which involves the polarized assembly of a stable, raft-like macrodomain surrounding engaged T cell receptors. Here we show that the preformed reggie/flotillin caps present in resting T cells act as priming platforms for macrodomain assembly. Preformed reggie-1/flotillin-2 caps are exceptionally stable, as shown by fluorescence recovery after photobleaching (FRAP). Upon T cell stimulation, signaling molecules are recruited to the stable reggie/flotillin caps. Importantly, a trans-negative reggie-1/flotillin-2 deletion mutant, which interferes with assembly of the preformed reggie/flotillin cap, impairs raft polarization and macrodomain formation after T cell activation. Accordingly, expression of the trans-negative reggie-1 mutant leads to the incorrect positioning of the guanine nucleotide exchange factor Vav, resulting in defects in cytoskeletal reorganization. Thus, the preformed reggie/flotillin caps are stable priming platforms for the assembly of multiprotein complexes controlling actin reorganization during T cell activation.
Assuntos
Proteínas de Membrana/metabolismo , Linfócitos T/metabolismo , Actinas/metabolismo , Animais , Citoesqueleto/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Humanos , Células Jurkat , Ativação Linfocitária , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/genética , Mutação , Células PC12 , Ratos , Transdução de SinaisRESUMO
Acidocalcisomes are acidic calcium storage organelles found in several microorganisms. They are characterized by their acidic nature, high electron density, high content of polyphosphates and several cations. Electron microscopy contrast tuned images of Herpetomonas sp. showed the presence of several electron dense organelles ranging from 100 to 300 nm in size. In addition, X-ray element mapping associated with energy-filtering transmission electron microscopy showed that most of the cations, namely Na, Mg, P, K, Fe and Zn, are located in their matrix. Using acridine orange as an indicator dye, a pyrophosphate-driven H+ uptake was measured in cells permeabilized by digitonin. This uptake has an optimal pH of 6.5-6.7 and was inhibited by sodium fluoride (NaF) and imidodiphosphate (IDP), two H+-pyrophosphatase inhibitors. H+ uptake was not promoted by ATP. Addition of 50 microM Ca2+ induced the release of H+, suggesting the presence of a Ca2+/H+ countertransport system in the membranes of the acidic compartments. Na+ was unable to release protons from the organelles. The pyrophosphate-dependent H+ uptake was dependent of ion K+ and inhibited by Na+ Herpetomonas sp. immunolabeled with monoclonal antibodies raised against a Trypanosoma cruzi V-H+-pyrophosphatase shows intense fluorescence in cytoplasmatic organelles of size and distribution similar to the electron-dense vacuoles. Together, these results suggest that the electron dense organelles found in Herpetomonas sp. are homologous to the acidocalcisomes described in other trypanosomatids. They possess a vacuolar H+-pyrophosphatase and a Ca2+/H+ antiport. However, in contrast to the other trypanosomatids so far studied, we were not able to measure any ATP promoted H+ transport in the acidocalcisomes of this parasite.
Assuntos
Pirofosfatase Inorgânica/metabolismo , Trypanosomatina/metabolismo , Animais , Transporte Biológico , Imunofluorescência , Pirofosfatase Inorgânica/antagonistas & inibidores , Pirofosfatase Inorgânica/química , Microscopia Eletrônica , Organelas/química , Organelas/metabolismo , Organelas/ultraestrutura , Trypanosomatina/ultraestruturaRESUMO
Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites, and recently found in other unicellular eukaryotes. The aim of this study was to identify the presence of acidocalcisomes in the plant trypanosomatid Phytomonas françai. Electron-dense organelles of P. françai were shown to contain large amounts of oxygen, sodium, magnesium, phosphorus, potassium, calcium, iron, and zinc as determined by X-ray microanalysis, either in situ or when purified using iodixanol gradient centrifugation or by elemental mapping. The presence of iron is not common in other acidocalcisomes. In situ, but not when purified, these organelles showed an elongated shape differing from previously described acidocalcisomes. However, these organelles also possessed a vacuolar H+-pyrophosphatase (V-H+-PPase) as determined by biochemical methods and by immunofluorescence microscopy using antibodies against the enzyme. Together, these results suggest that the electron-dense organelles of P. françai are homologous to the acidocalcisomes described in other trypanosomatids, although with distinct morphology and elemental content.
Assuntos
Cálcio/metabolismo , Ferro/metabolismo , Manihot/parasitologia , Organelas/ultraestrutura , Trypanosomatina/ultraestrutura , Animais , Microscopia Eletrônica de Transmissão , Organelas/metabolismoRESUMO
Hematopoietic cells have long been defined as round, nonpolar cells that show uniform distribution of cell surface-associated molecules. However, recent analyses of the immunological synapse and the importance of lipid microdomains in signaling have shed new light on the aspect of lymphocyte polarization during the activation processes, but none of the molecules implicated so far in either the activation process or the microdomain residency are known to have a preferential localization in nonactivated cells. Chemical crosslinking and fluorescence resonance energy transfer methods have allowed the visualization of certain glycosylphosphatidylinositol-anchored proteins in lipid rafts but so far no microdomain resident protein has been shown to exist as visible stable platforms in the membrane. We report here that two lipid microdomain resident proteins, flotillins/reggies, form preassembled platforms in hematopoietic cells. These platforms recruit signaling molecules upon activation through lipid rafts. The preassembled platforms significantly differ from the canonical cholesterol-dependent "lipid rafts," as they are resistant to cholesterol-disrupting agents. Most evidence for the functional relevance of microdomains in living cells remains indirect. Using laser scanning confocal microscopy, we show that these proteins exist as stable, microscopically patent domains localizing asymmetrically to one pole of the cell. We present evidence that the asymmetric concentration of these microdomain resident proteins is built up during cytokinesis.
Assuntos
Linfócitos B/ultraestrutura , Polaridade Celular/fisiologia , Ativação Linfocitária/fisiologia , Microdomínios da Membrana/fisiologia , Proteínas de Membrana/fisiologia , Linfócitos T/ultraestrutura , beta-Ciclodextrinas , Antígenos CD/análise , Linfócitos B/metabolismo , Divisão Celular , Toxina da Cólera/farmacologia , Colesterol/análise , Ciclodextrinas/farmacologia , Detergentes/farmacologia , Proteínas de Fluorescência Verde , Humanos , Ionóforos/farmacologia , Células Jurkat/metabolismo , Células Jurkat/ultraestrutura , Proteínas Luminescentes/análise , Lipídeos de Membrana/análise , Microdomínios da Membrana/química , Microdomínios da Membrana/efeitos dos fármacos , Proteínas de Membrana/análise , Nocodazol/farmacologia , Octoxinol/farmacologia , Proteínas Recombinantes de Fusão/análise , Linfócitos T/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/ultraestruturaRESUMO
A Paramecium cell has a stereotypically patterned surface, with regularly arranged cilia, dense-core secretory vesicles and subplasmalemmal calcium stores. Less strikingly, there is also a patterning of molecules; for instance, some ion channels are restricted to certain regions of the cell surface. This design may explain very effective and selective responses, such as that to Ca(2+) upon stimulation. It enables the cell to respond to a Ca(2+) signal precisely secretion (exocytosis) or by changing its ciliary activity. These responses depend on the location and/or type of signal, even though these two target structures co-exist side-by-side, and normally only limited overlap occurs between the different functions. Furthermore, the patterning of exocytotic sites and the possibility of synchronous exocytosis induction in the sub-second time range have considerably facilitated analyses, and thus led to new concepts of exocytotic membrane fusion. It has been possible to dissect complicated events like overlapping Ca(2+) fluxes produced from external sources and from internal stores. Since molecular genetic approaches have become available for Paramecium, many different gene products have been identified only some of which are known from "higher" eukaryotes. Although a variety of basic cellular functions are briefly addressed to demonstrate the uniqueness of this unicellular organism, this article focuses on exocytosis regulation.
Assuntos
Sinalização do Cálcio/fisiologia , Exocitose/fisiologia , Paramecium/fisiologia , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Endocitose/fisiologia , Corantes Fluorescentes/metabolismo , Técnica de Fratura por Congelamento , Paramecium/ultraestrutura , Proteínas Recombinantes de Fusão/metabolismoRESUMO
During exocytosis of dense-core secretory vesicles (trichocysts) in Paramecium, the protein pp63/parafusin (pp63/pf) is transiently dephosphorylated. We report here the structures of two crystal forms of one isoform of this protein which has a high degree of homology with rabbit phosphoglucomutase, whose structure has been reported. As expected, both proteins possess highly similar structures, showing the same four domains forming two lobes with an active-site crevice in between. The two X-ray structures that we report here were determined after crystallization in the presence of sulfate and tartrate, and show the lobes arranged as a closed and an open conformation, respectively. While both conformations possess a bound divalent cation, only the closed (sulfate-bound) conformation shows bound sulfate ions in the "phosphate-transfer site" near the catalytic serine residue and in the "phosphate-binding site". Comparison with the open form shows that the latter dianion is placed in the centre of three arginine residues, one contributed by subunit II and two by subunit IV, suggesting that it causes a contraction of the arginine triangle, which establishes the observed conformational closure of the lobes. It is therefore likely that the closed conformation forms only when a phosphoryl group is bound to the phosphate-binding site. The previously published structure of rabbit phosphoglucomutase is intermediate between these two conformers. Several of the known reversible phosphorylation sites of pp63/pf-1 are at positions critical for transition between the conformations and for binding of the ligands and thus give hints as to possible roles of pp63/pf-1 in the course of exocytosis.