RESUMO
Macroautophagy (hereafter "autophagy") is a lysosomal degradation pathway that is important for learning and memory, suggesting critical roles for autophagy at the neuronal synapse. Little is known, however, about the molecular details of how autophagy is regulated with synaptic activity. Here, we used live-cell confocal microscopy to define the autophagy pathway in primary hippocampal neurons under various paradigms of synaptic activity. We found that synaptic activity regulates the motility of autophagic vacuoles (AVs) in dendrites. Stimulation of synaptic activity dampens AV motility, whereas silencing synaptic activity induces AV motility. Activity-dependent effects on dendritic AV motility are local and reversible. Importantly, these effects are compartment specific, occurring in dendrites and not in axons. Most strikingly, synaptic activity increases the presence of degradative autolysosomes in dendrites and not in axons. On the basis of our findings, we propose a model whereby synaptic activity locally controls AV dynamics and function within dendrites that may regulate the synaptic proteome.
Assuntos
Autofagia , Movimento Celular , Dendritos/fisiologia , Hipocampo/fisiologia , Neurônios/fisiologia , Sinapses/fisiologia , Vacúolos/fisiologia , Animais , Autofagossomos/fisiologia , Axônios/fisiologia , Hipocampo/citologia , Lisossomos/fisiologia , Camundongos , Neurônios/citologia , Ratos , Ratos Sprague-DawleyRESUMO
MAIN CONCLUSION: Different autophagy pathways are a driver of vacuolar biogenesis and are development stage specific during the extrafloral nectary development in Citharexylum myrianthum. Plant autophagy plays an important role in various developmental processes such as seed germination, pollen maturation and leaf senescence. However, studies that address the evidence of autophagy and its role in the development of plant glands are scarce and largely restricted to laticifers. Regarding nectary, studies have repeatedly pointed to signs of degradation associated with the end of the secretory cycle, without exploring autophagy. Likewise, the relationship between autophagy and biogenesis of vacuoles remains an unexplored issue. In this study, using conventional and microwave fixation in association with ultracytochemical methods for transmission electron microscopy, we investigated the occurrence of autophagy and its implication in the differentiation of extrafloral nectary in Citharexylum myrianthum (Verbenaceae) under natural conditions, focusing on the vacuole biogenesis. We described a variety of vacuole types associated with the stage of nectary epidermis development, which differs with respect to origin, function and nature of the products to be stored. Three distinct autophagy pathways were detected: macroautophagy, microautophagy (both restricted to the undifferentiated epidermal cells, at the presecretory stage) and megaautophagy (circumscribed to the differentiated epidermal cells, at the postsecretory stage). Our study clearly demonstrated that the vacuole variety and autophagy processes in the nectary epidermal cells are development specific. This study highlights the role of autophagy in vacuole biogenesis and its implications for the development of nectary and opens new venues for future studies on regulation mechanisms for autophagy in plant secretory structures under normal conditions.
Assuntos
Autofagia , Néctar de Plantas/metabolismo , Verbenaceae/fisiologia , Microscopia Eletrônica de Transmissão , Vacúolos/fisiologia , Vacúolos/ultraestrutura , Verbenaceae/ultraestruturaRESUMO
Improving carbon fixation in order to enhance crop yield is a major goal in plant sciences. By quantitative trait locus (QTL) mapping, it has been demonstrated that a vacuolar invertase (vac-Inv) plays a key role in determining the radical length in Arabidopsis. In this model, variation in vac-Inv activity was detected in a near isogenic line (NIL) population derived from a cross between two divergent accessions: Landsberg erecta (Ler) and Cape Verde Island (CVI), with the CVI allele conferring both higher Inv activity and longer radicles. The aim of the current work is to understand the mechanism(s) underlying this QTL by analyzing structural and functional differences of vac-Inv from both accessions. Relative transcript abundance analyzed by quantitative real-time PCR (qRT-PCR) showed similar expression patterns in both accessions; however, DNA sequence analyses revealed several polymorphisms that lead to changes in the corresponding protein sequence. Moreover, activity assays revealed higher vac-Inv activity in genotypes carrying the CVI allele than in those carrying the Ler allele. Analyses of purified recombinant proteins showed a similar K m for both alleles and a slightly higher V max for that of Ler. Treatment of plant extracts with foaming to release possible interacting Inv inhibitory protein(s) led to a large increase in activity for the Ler allele, but no changes for genotypes carrying the CVI allele. qRT-PCR analyses of two vac-Inv inhibitors in seedlings from parental and NIL genotypes revealed different expression patterns. Taken together, these results demonstrate that the vac-Inv QTL affects root biomass accumulation and also carbon partitioning through a differential regulation of vac-Inv inhibitors at the mRNA level.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , beta-Frutofuranosidase/fisiologia , Alelos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Conformação Proteica , Locos de Características Quantitativas/genética , Locos de Características Quantitativas/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Plântula/crescimento & desenvolvimento , Análise de Sequência de DNA , Vacúolos/enzimologia , Vacúolos/fisiologia , beta-Frutofuranosidase/genéticaRESUMO
Toxoplasma gondii is an obligate intracellular protozoan parasite, the causative agent of toxoplasmosis, one of the most widespread zoonoses in the world. During the host immune response, tissue cysts are formed, allowing the maintenance of the parasite within the host cell. Autophagy, a degradation process of cellular components, is critical for cellular homeostasis. Recently, it has been proposed that autophagy participates in host-pathogen interactions. Autophagic inducers (rapamycin or glucose plus serum deprivation) inhibited infection and parasite proliferation in a clinically relevant model of primary skeletal muscle cells (SkMC). The ultrastructural analysis showed in SkMC submitted to autophagic stimuli the presence of structures suggestive of autophagosomes close to the parasitophorous vacuole containing degraded parasites. Fluorescence microscopy results pointed out the increase in LC3 puncta in these cells after incubation with autophagic inducers. In the present study, SkMC autophagy controlled the proliferation of tachyzoites inside the cell, data reinforced by ultrastructural evidences and increased LC3 expression.
Assuntos
Autofagia/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Proteínas Associadas aos Microtúbulos/metabolismo , Músculo Esquelético/parasitologia , Toxoplasma/ultraestrutura , Toxoplasmose/parasitologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Biomarcadores/metabolismo , Células Cultivadas , Feminino , Glucose/metabolismo , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Músculo Esquelético/ultraestrutura , Sirolimo/farmacologia , Toxoplasma/fisiologia , Toxoplasmose/tratamento farmacológico , Toxoplasmose/imunologia , Vacúolos/parasitologia , Vacúolos/fisiologia , Vacúolos/ultraestruturaRESUMO
BACKGROUND: A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. The two main routes, the secretory and the endocytic pathways have pivotal functions in uni- and multi-cellular organisms. Protein delivery and targeting includes cargo recognition, vesicle formation and fusion. Developing new tools to modulate protein trafficking allows better understanding the endomembrane system mechanisms and their regulation. The compound Sortin2 has been described as a protein trafficking modulator affecting targeting of the vacuolar protein carboxypeptidase Y (CPY), triggering its secretion in Saccharomyces cerevisiae. RESULTS: A reverse chemical-genetics approach was used to identify key proteins for Sortin2 bioactivity. A genome-wide Sortin2 resistance screen revealed six yeast deletion mutants that do not secrete CPY when grown at Sortin2 condition where the parental strain does: met18, sla1, clc1, dfg10, dpl1 and yjl175w. Integrating mutant phenotype and gene ontology annotation of the corresponding genes and their interactome pointed towards a high representation of genes involved in the endocytic process. In wild type yeast endocytosis towards the vacuole was faster in presence of Sortin2, which further validates the data of the genome-wide screen. This effect of Sortin2 depends on structural features of the molecule, suggesting compound specificity. Sortin2 did not affect endocytic trafficking in Sortin2-resistant mutants, strongly suggesting that the Sortin2 effects on the secretory and endocytic pathways are linked. CONCLUSIONS: Overall, the results reveal that Sortin2 enhances the endocytic transport pathway in Saccharomyces cerevisiae. This cellular effect is most likely at the level where secretory and endocytic pathways are merged. Them Sortin2 specificity over the endomembrane system places it as a powerful biological modulator for cell biology.
Assuntos
Alcanossulfonatos/farmacologia , Endocitose/fisiologia , Proteínas de Plantas/fisiologia , Transporte Proteico , Rodanina/análogos & derivados , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Transporte Biológico , Fenótipo , Transporte Proteico/genética , Rodanina/farmacologia , Via Secretória , Vacúolos/fisiologiaRESUMO
BACKGROUND: A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. The two main routes, the secretory and the endocytic pathways have pivotal functions in uni- and multi-cellular organisms. Protein delivery and targeting includes cargo recognition, vesicle formation and fusion. Developing new tools to modulate protein trafficking allows better understanding the endomembrane system mechanisms and their regulation. The compound Sortin2 has been described as a protein trafficking modulator affecting targeting of the vacuolar protein carboxypeptidase Y (CPY), triggering its secretion in Saccharomyces cerevisiae. RESULTS: A reverse chemical-genetics approach was used to identify key proteins for Sortin2 bioactivity. A genome-wide Sortin2 resistance screen revealed six yeast deletion mutants that do not secrete CPY when grown at Sortin2 condition where the parental strain does: met18, sla1, clc1, dfg10, dpl1 and yjl175w. Integrating mutant phenotype and gene ontology annotation of the corresponding genes and their interactome pointed towards a high representation of genes involved in the endocytic process. In wild type yeast endocytosis towards the vacuole was faster in presence of Sortin2, which further validates the data of the genome-wide screen. This effect of Sortin2 depends on structural features of the molecule, suggesting compound specificity. Sortin2 did not affect endocytic trafficking in Sortin2-resistant mutants, strongly suggesting that the Sortin2 effects on the secretory and endocytic pathways are linked. CONCLUSIONS: Overall, the results reveal that Sortin2 enhances the endocytic transport pathway in Saccharomyces cerevisiae. This cellular effect is most likely at the level where secretory and endocytic pathways are merged. Them Sortin2 specificity over the endomembrane system places it as a powerful biological modulator for cell biology.
Assuntos
Proteínas de Plantas/fisiologia , Rodanina/análogos & derivados , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Alcanossulfonatos/farmacologia , Transporte Proteico/genética , Endocitose/fisiologia , Fenótipo , Rodanina/farmacologia , Vacúolos/fisiologia , Transporte Biológico , Via SecretóriaRESUMO
Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.
Assuntos
Gotículas Lipídicas/parasitologia , Ativação de Macrófagos/fisiologia , Macrófagos Peritoneais/parasitologia , Toxoplasma/fisiologia , Vacúolos/parasitologia , Animais , Bovinos , Interações Hospedeiro-Parasita , Indometacina/farmacologia , Gotículas Lipídicas/fisiologia , Macrófagos Peritoneais/química , Macrófagos Peritoneais/fisiologia , Macrófagos Peritoneais/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C3H , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Óxido Nítrico/biossíntese , Cultura Primária de Células , Prostaglandinas E/antagonistas & inibidores , Prostaglandinas E/biossíntese , Vacúolos/fisiologiaRESUMO
Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.
Assuntos
Animais , Bovinos , Masculino , Camundongos , Gotículas Lipídicas/parasitologia , Ativação de Macrófagos/fisiologia , Macrófagos Peritoneais/parasitologia , Toxoplasma/fisiologia , Vacúolos/parasitologia , Interações Hospedeiro-Parasita , Indometacina/farmacologia , Gotículas Lipídicas/fisiologia , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Macrófagos Peritoneais/química , Macrófagos Peritoneais/fisiologia , Macrófagos Peritoneais/ultraestrutura , Óxido Nítrico/biossíntese , Cultura Primária de Células , Prostaglandinas E/antagonistas & inibidores , Prostaglandinas E/biossíntese , Vacúolos/fisiologiaRESUMO
Aging of long-lived post-mitotic cells is characterized as a progressive and irreversible reduction of functional activity. In such cells, mitochondria are organelles critical for bioenergetic supply, whose turnover is mediated by an autophagic-lysosomal pathway. In human teeth, odontoblasts are post-mitotic cells responsible for sensory function and dentin preservation. Here, human odontoblasts were processed for immunohistochemistry with antibodies against mitochondrial (MTCO2) and lysosomal (LAMP2) markers, and comparatively analyzed in two age groups (young-adult and adult) with light and electron microscopy. Selective engulfment of mitochondrial profiles into autophagic vacuoles is common in young-adult odontoblasts, suggesting a microautophagic pathway. With age, the odontoblast layer is reduced in width, and mitochondrial elements converge around large clusters of autofluorescent lipofuscin deposits. Age-related changes in odontoblasts are observed as a long-term process in which the progressive accumulation of intralysosomal debris limits the autophagic turnover of mitochondrial components, causing an eventual decline in physiological cell functions, which leads to increased vulnerability under stress conditions.
Assuntos
Autofagia/fisiologia , Senescência Celular/fisiologia , Lipofuscina/metabolismo , Mitocôndrias/fisiologia , Odontoblastos/citologia , Adolescente , Adulto , Idoso , Humanos , Lipofuscina/análise , Lisossomos/fisiologia , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Odontoblastos/metabolismo , Vacúolos/fisiologia , Adulto JovemRESUMO
BACKGROUND: Although the motile sperm organelle morphology examination (MSOME) was developed only as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in evaluation of semen quality, with potential clinical repercussions. The present study aimed to evaluate individual variations in the motile sperm organelle morphology examination (MSOME) analysis after a time interval. METHODS: Two semen samples were obtained from 240 men from an unselected group of couples undergoing infertility investigation and treatment. Mean time interval between the two semen evaluations was 119+/-102 days. No clinical or surgical treatment was realized between the two observations. Spermatozoa were analyzed at greater than or equal to 8400x magnification by inverted microscope equipped with DIC/Nomarski differential interference contrast optics. At least 200 motile spermatozoa per semen sample were evaluated and percentages of normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV/one or more vacuoles occupying>50% of the sperm nuclear area) were determined. A spermatozoon was classified as morphologically normal when it exhibited a normal nucleus (smooth, symmetric and oval nucleus, width 3.28+/-0.20 microm, length 4.75+/-0.20 microm/absence of vacuoles occupying>4% of nuclear area) as well as acrosome, post-acrosomal lamina, neck and tail, besides not presenting cytoplasm around the head. One examiner, blinded to subject identity, performed the entire study. RESULTS: Mean percentages of morphologically normal and LNV spermatozoa were identical in the two MSOME analyses (1.6+/-2.2% vs. 1.6+/-2.1% P=0.83 and 25.2+/-19.2% vs. 26.1+/-19.0% P=0.31, respectively). Regression analysis between the two samples revealed significant positive correlation for morphologically normal and for LNV spermatozoa (r=0.57 95% CI:0.47-0.65 P<0.0001 and r=0.50 95% CI:0.38-0.58 P<0.0001, respectively). CONCLUSIONS: The significant positive correlation and absence of differences between two sperm samples evaluated after a time interval with respect to normal morphology and LNV spermatozoa indicated that MSOME seems reliable (at least for these two specific sperm forms) for analyzing semen. The present result supports the future use of MSOME as a routine method for semen analysis.
Assuntos
Análise do Sêmen/métodos , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/ultraestrutura , Vacúolos/patologia , Vacúolos/ultraestrutura , Adulto , Humanos , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Tamanho das Organelas/fisiologia , Organelas/ultraestrutura , Controle de Qualidade , Análise do Sêmen/normas , Vacúolos/fisiologiaRESUMO
BACKGROUND: Nuclear magnetic resonance studies of banana fragments during ripening show an increase on the water transverse relaxation time (T2) and a decrease in water self-diffusion coefficient (D). As T(2) and D are normally directly correlated, we studied these two properties in intact bananas during ripening, in an attempt to rule out the effect of injury on the apparent discrepancies in the behavior of T(2) and D. RESULTS: The results show that injury in bananas causes a decrease in T2 of the water in vacuoles (T(2vac)). They also show that T(2vac) increased and D decreased during ripening, ruling out the injury effect. To explain the apparent discrepancies, we propose a new hypothesis for the increase in T2 values, based on the reduction of Fe3+ ions to Fe2+ by galacturonic acid, produced by the hydrolysis of pectin and a decrease in internal oxygen concentration during ripening. CONCLUSION: As injury alters T2 values it is necessary to use intact bananas to study relaxation times during ripening. The novel interpretation for the increase in T(2vac) based on reduction of Fe+3 and O2 concentration is an alternative mechanism to that based on the hydrolysis of starch in amyloplasts.
Assuntos
Cátions/metabolismo , Frutas/fisiologia , Ácidos Hexurônicos/metabolismo , Musa/fisiologia , Oxigênio/fisiologia , Pectinas/metabolismo , Água/fisiologia , Compostos Férricos/metabolismo , Compostos Ferrosos/metabolismo , Manipulação de Alimentos , Hidrólise , Espectroscopia de Ressonância Magnética/métodos , Vacúolos/fisiologiaRESUMO
Toxoplasma gondii invades and destroys nucleated cells of warm blooded hosts in a process which involves several steps: recognition, adhesion, penetration, multiplication inside a parasitophorous vacuole (PV) and egress. The last one is the least understood. Parasite egress from LLC-MK2 cells infected with the RH strain of T. gondii was artificially triggered with 4BrA23187 calcium ionophore. The combination of videomicroscopy, field emission scanning electron microscopy (FESEM), and transmission electron microscopy (TEM) showed that egress does not result from host cell rupture due to overloading with tachyzoites. Videomicroscopy showed that upon calcium ionophore administration parasite rosettes disassemble, the contour of the parasitophorous vacuole disappears and each tachyzoite takes a separate route to the extracellular medium. FESEM and TEM showed the fragmentation of the intravacuolar network, the fragmentation of parasitophorous vacuole membrane and individual tachyzoites with extruded conoids migrating through the cytosol, tightly surrounded by remnants of parasitophorous vacuole membrane or free in the cytosol. Both videomicroscopy and FESEM showed that a single parasite can cross the host cell membrane without disrupting it, while a large number of parasites, egressing simultaneously, rupture the membrane and the cell as a whole. These data suggest that invasion and egress share less similarities than previously believed.
Assuntos
Cálcio/fisiologia , Membrana Celular/parasitologia , Toxoplasma/fisiologia , Toxoplasmose/parasitologia , Vacúolos/parasitologia , Animais , Calcimicina/farmacologia , Linhagem Celular , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Ionóforos/farmacologia , Macaca mulatta , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Microscopia de Vídeo , Toxoplasma/ultraestrutura , Vacúolos/fisiologia , Vacúolos/ultraestruturaRESUMO
BACKGROUND: Previous studies have suggested that the addition of heparin to a preservation solution attenuated the autonomic dysfunction observed in rat jejunum and in addition that hypothermic hyperbaric oxygenation may play a role as a preservation technique. However, these studies did not address the lesion indices of the autonomic enteric neurons. We sought to investigate whether the autonomic enteric neurons are injured during cold ischemic preservation and whether administration of heparin or hyperbaric oxygenation prevents this lesion. METHODS: Jejunal segments (2 cm; n = 20) of Wistar rats (12-16 weeks old) were maintained in lactated Ringer's solution without or with heparin (H- and H+, respectively) at 4 degrees C under normobaric conditions. Other jejunal segments (n = 10) were maintained at 4 degrees C in H- under hyperbaric oxygenation conditions (HBO). After preservation for 12 hours, H-, H+, and HBO preparations fixed in 10% formaldehyde were stained with hematoxylin and eosin. The lesion indices were expressed as the mean number of affected neurons (karyorhexis, nuclear dislocation, cytoplasmic vacuolisation) per 100 neurons present in intramural ganglia. Statistical analysis was performed using the Mann-Whitney test (P < .05). RESULTS: The histologic studies showed that enteric autonomic neurons were damaged in H- jejunal segments. The lesion indices observed were: karyorhexis 90/100, nuclear dislocation 85/100, and cytoplasmic vacuolization 82/100. The autonomic neurons in H+ and HBO segments seemed to be normal and significantly well-preserved (P < .001). CONCLUSION: Hypothermic hyperbaric oxygenation and heparin prevented lesions in cold ischemic preservation of enteric autonomic neurons.
Assuntos
Heparina/uso terapêutico , Oxigenoterapia Hiperbárica , Neurônios/fisiologia , Preservação de Órgãos/métodos , Traumatismo por Reperfusão/prevenção & controle , Animais , Sistema Nervoso Autônomo/efeitos dos fármacos , Sistema Nervoso Autônomo/fisiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Citoplasma/efeitos dos fármacos , Citoplasma/fisiologia , Jejuno/efeitos dos fármacos , Jejuno/inervação , Masculino , Neurônios/citologia , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Vacúolos/efeitos dos fármacos , Vacúolos/fisiologiaRESUMO
Leaf proteins, and in particular the photosynthetic proteins of plastids, are extensively degraded during senescence. Although this involves massive amounts of protein, the mechanisms responsible for chloroplast protein degradation are largely unknown. Degradation within the plastid itself is supported by the observation that chloroplasts contain active proteases, and that chloroplasts isolated from senescing leaves can cleave Rubisco to release partially digested fragments. It is less clear whether chloroplasts can complete Rubisco degradation. Chloroplastic proteases are likely involved in the breakdown of the D1 and LHCII proteins of photosystem II. Small senescence-associated vacuoles (SAVs) with high-proteolytic activity develop in senescing leaf cells, and there is evidence that SAVs contain chloroplast proteins. Thus, an extra-plastidic pathway involving SAVs might participate in the degradation of some chloroplast proteins. Plastidic and extra-plastidic pathways might cooperate in the degradation of chloroplast proteins, or they might represent alternative, redundant pathways for photosynthetic protein degradation.
Assuntos
Senescência Celular , Cloroplastos/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Autofagia/fisiologia , Modelos Biológicos , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Folhas de Planta/crescimento & desenvolvimento , Ribulose-Bifosfato Carboxilase/metabolismo , Vacúolos/metabolismo , Vacúolos/fisiologiaRESUMO
[Leishmania(L.)] amazonensis amastigotes reside in macrophages within spacious parasitophorous vacuoles (PVs) which may contain numerous parasites. After sporadic fusion events were detected by time-lapse cinemicrography, PV fusion was examined in two different models. In single infections, it was inferred from the reduction in PV numbers per cell. In a reinfection model, macrophages infected with unlabeled amastigotes were reinfected with GFP-transfected- or carboxyfluorescein diacetate succinimidyl ester-labeled parasites, and fusion was detected by the colocalization of labeled and unlabeled amastigotes in the same PVs. The main findings were: (1) as expected, fusion frequency increased with the multiplicity of infection; (2) most fusion events took place in the first 24h of infection or reinfection, prior to the multiplication of incoming parasites; (3) resident and incoming parasites multiplied at similar rates in fused PVs. The model should be useful in studies of parasite and host cell factors and mechanisms involved in PV fusogenicity.
Assuntos
Leishmania mexicana/fisiologia , Leishmaniose Cutânea/parasitologia , Macrófagos/parasitologia , Vacúolos/parasitologia , Animais , Feminino , Leishmaniose Cutânea/patologia , Macrófagos/ultraestrutura , Fusão de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Recidiva , Vacúolos/fisiologia , Vacúolos/ultraestruturaRESUMO
Toxoplasma gondii resides in a nonfusogenic parasitophorous vacuole (PV), which provides a safe environment for parasite survival and replication. In this work, we used the freeze-fracture technique to analyze the PV during different times of T. gondii infection in an epithelial cell line. After a short time of interaction with host cell, T. gondii PV membrane already showed a significant quantity of intramembranous particles (IMPs)-293IMPs/microm(2). The IMP density evaluated did not vary until 6h of interaction. As the PV area enlarged with the progression of infection, the density of these particles increased, reaching a stable quantity in the order of 1100particles/microm(2). The IMPs were heterogeneous in size and were found distributed without any special pattern throughout the time of infection studied. The membrane lining the PV presented circular figures, which resembled vesicle fusion areas or attachments of the membranous tubular network, regions free from particles and small depressions, demonstrating to be a dynamic structure. IMPs were found in tubulo-vesicular structures present in the intravacuolar matrix, although rarely observed in elements of the intravacuolar network.
Assuntos
Técnica de Fratura por Congelamento/métodos , Toxoplasma/ultraestrutura , Vacúolos/ultraestrutura , Animais , Linhagem Celular , Células Epiteliais/parasitologia , Interações Hospedeiro-Parasita , Rim/citologia , Rim/parasitologia , Camundongos , Microscopia Eletrônica , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/patogenicidade , Vacúolos/fisiologiaRESUMO
Históricamente, la apoptosis y la necrosis han sido consideradas como las dos formas fundamentales de muerte celular. Sin embargo, evidencias recientes sugieren que la muerte celular programada no está confinada sólo a la apoptosis sino que las células disponen de distintos mecanismos de autodestrucción, entre los que se cuenta la autofagia. Esta última se define como un proceso dinámico y programado que procede con el secuestro de proteínas citoplasmáticas y organelos enteros dentro de vacuolas de doble membrana, que se contactan y se fusionan con los lisosomas, formando los autolisosomas. Los elementos capturados en las vacuolas son degradados por proteasas lisosomales y removidos de la célula por exocitosis. La autofagia se describió inicialmente como un proceso fisiológico clave para la sobrevida celular en respuesta al estrés derivado de la privación de nutrientes. Además, la autofagia también se ha observado en algunas patologías cardiovasculares, especialmente aquellas asociadas a procesos de isquemia/reperfusión. En esta revisión se sintetiza el conocimiento actual de la autofagia, sus implicancias y proyecciones en el área cardiovascular.
Assuntos
Humanos , Apoptose , Miócitos Cardíacos/fisiologia , Necrose , Vacúolos/fisiologiaRESUMO
Colletotrichum acutatum may develop one or more secondary conidia after conidial germination and before mycelial growth. Secondary conidia formation and germination were influenced by conidia concentration. Concentrations greater than 1x105 conidia/mL were associated with germination decrease and with secondary conidia emergence. Secondary conidia can form either alone or simultaneously with germ tubes and appressoria. Confocal analysis showed numerous lipid bodies stored inside ungerminated conidia, which diminished during germ tube and appressoria formation, with or without secondary conidia formation. They were also reduced during secondary conidia formation alone. While there was a decrease inside germinated conidia, lipid bodies appeared inside secondary conidia since the initial stages. Intense vacuolization inside primary germinated conidia occurred at the same time as the decrease in lipid bodies, which were internalized and digested by vacuoles. During these events, small acidic vesicles inside secondary conidia were formed. Considering that the conidia were maintained in distilled water, with no exogenous nutrients, it is clear that ungerminated conidia contain enough stored lipids to form germ tubes, appressoria, and the additional secondary conidia replete with lipid reserves. These results suggested a very complex and well-balanced regulation that makes possible the catabolic and anabolic pathways of these lipid bodies.
Assuntos
Colletotrichum/fisiologia , Metabolismo dos Lipídeos/fisiologia , Vacúolos/fisiologia , Colletotrichum/ultraestrutura , Microscopia ConfocalRESUMO
The aim of the present study was analyze, by histological and morphometrical studies, mandibular glands of Melipona bicolor queens collected from monogynic and polygynic colonies and compare their level of development. The results showed that the glands ofphysogastric queens from monogynic colony present a higher level of activity in relation to the queens of polygynic colonies; this is explained by the fact that just a unique queen controls the monogynic colony. In the polygynic colonies, the queens may divide such control to each other.
Assuntos
Abelhas/citologia , Abelhas/crescimento & desenvolvimento , Feromônios/metabolismo , Glândulas Salivares/citologia , Glândulas Salivares/crescimento & desenvolvimento , Animais , Comportamento Animal/fisiologia , Forma Celular/fisiologia , Tamanho Celular , Citoplasma/fisiologia , Citoplasma/ultraestrutura , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Feminino , Mandíbula/citologia , Mandíbula/fisiologia , Glândulas Salivares/metabolismo , Vesículas Secretórias/fisiologia , Vesículas Secretórias/ultraestrutura , Comportamento Social , Vacúolos/fisiologia , Vacúolos/ultraestruturaRESUMO
Coxiella burnetii is a Gram-negative obligate intracellular bacterium that infects a wide range of hosts including humans, causing Q fever, a disease characterized by high fever and flu-like symptoms. After its internalization the Coxiella-containing phagosomes interact with intracellular compartments and generate a large replicative vacuole that displays certain characteristics of a phagolysosome. We have shown that this bacterially-customized replicative vacuole also has the hallmarks of an autophagosomal compartment. Furthermore, in a recent publication we have reported that induction of autophagy is beneficial for the replication and survival of Coxiella. Different morphological forms of this bacterium have been described during its developmental cycle. Here we present additional data and discuss a model indicating that induction of autophagy favors the differentiation of the Coxiella small cell variants to the metabolically active large cells variants. We postulate that nutrient acquisition, likely by fusion with the nutrient-rich autophagic vacuoles, triggers the development of the large cell variants which actively multiply in the host cell.