RESUMEN
In many phenomena of biological systems, not a majority, but a minority of cells act on the entire multicellular system causing drastic changes in the system properties. To understand the mechanisms underlying such phenomena, it is essential to observe the spatiotemporal dynamics of a huge population of cells at sub-cellular resolution, which is difficult with conventional tools such as microscopy and flow cytometry. Here, we describe an imaging system named AMATERAS that enables optical imaging with an over-one-centimeter field-of-view and a-few-micrometer spatial resolution. This trans-scale-scope has a simple configuration, composed of a low-power lens for machine vision and a hundred-megapixel image sensor. We demonstrated its high cell-throughput, capable of simultaneously observing more than one million cells. We applied it to dynamic imaging of calcium ions in HeLa cells and cyclic-adenosine-monophosphate in Dictyostelium discoideum, and successfully detected less than 0.01% of rare cells and observed multicellular events induced by these cells.
Asunto(s)
Células/citología , Microscopía Fluorescente/métodos , Animales , Encéfalo/citología , Calcio/análisis , AMP Cíclico/análisis , Dictyostelium/química , Dictyostelium/ultraestructura , Perros , Entosis , Células Epiteliales/ultraestructura , Diseño de Equipo , Proteínas Fluorescentes Verdes , Células HeLa/química , Células HeLa/ultraestructura , Humanos , Interneuronas/ultraestructura , Proteínas Luminiscentes , Células de Riñón Canino Madin Darby , Ratones , Microscopía Fluorescente/instrumentación , Neuronas/ultraestructura , Semiconductores , Proteína Fluorescente RojaRESUMEN
The Dictyostelium discoideum-Mycobacterium marinum host-pathogen system is a well-established and powerful alternative model system to study mycobacterial infections. In this chapter, we will describe three microscopy methods that allow the precise identification and quantification of very diverse phenotypes arising during infection of D. discoideum with M. marinum. First, at the lowest end of the scale, we use the InfectChip, a microfluidic device that enables the long-term monitoring of the integrated history of the infection course at the single-cell level. We use single-cell analysis to precisely map and quantitate the various fates of the host and the pathogen during infection. Second, a high-content microscopy setup was established to study the infection dynamics with high-throughput imaging of a large number of cells at the different critical stages of infection. The large datasets are then fed into a deep image analysis pipeline allowing the development of complex phenotypic analyses. Finally, as part of its life cycle, single D. discoideum amoebae aggregate by chemotaxis to form multicellular structures, which represent ordered assemblies of hundreds of thousands of cells. This transition represents a challenge for the monitoring of infection at multiple scales, from single cells to a true multicellular organism. In order to visualize and quantitate the fates of host cells and bacteria during the developmental cycle in a controlled manner, we can adjust the proportion of infected cells using live FAC-sorting. Then, cells are plated in defined humidity conditions on optical glass plates in order to image large fields, using tile scans, with the help of a spinning disc confocal microscope.
Asunto(s)
Dictyostelium/microbiología , Interacciones Huésped-Patógeno , Dispositivos Laboratorio en un Chip , Microscopía Electrónica/métodos , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Mycobacterium marinum/crecimiento & desarrollo , Análisis de la Célula Individual/métodos , Dictyostelium/ultraestructura , Infecciones por Mycobacterium no Tuberculosas/microbiologíaRESUMEN
After a cell divides into two daughter cells, the total cell surface area of the daughter cells should increase to the original size to maintain cell size homeostasis in a single cell cycle. Previously, three models have been proposed to explain the regulation of cell size homeostasis: sizer, timer, and adder models. Here, we precisely measured the total cell surface area of Dictyostelium cells in a whole cell cycle by using the agar-overlay method, which eliminated the influence of surface membrane reservoirs, such as microvilli and membrane wrinkles. The total cell surface area exponentially increased during interphase, slightly decreased at metaphase, and then increased by approximately 20% during cytokinesis. From the analysis of the added surface area, we concluded that the cell size was regulated by the adder or near-adder model in interphase. This adder model is not caused by a simple cell membrane addition, but is more dynamic due to the rapid cell membrane turnover. We propose a 'dynamic adder model' to explain cell size homeostasis in interphase.
Asunto(s)
Tamaño de la Célula , Dictyostelium/genética , Homeostasis/genética , Modelos Biológicos , Ciclo Celular/genética , División Celular/genética , Dictyostelium/ultraestructura , Interfase/genéticaRESUMEN
Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1-3 (ATL1-3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER-associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the "Legionella-containing vacuole" (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila.
Asunto(s)
Dictyostelium/fisiología , Retículo Endoplásmico/ultraestructura , GTP Fosfohidrolasas/metabolismo , Legionella pneumophila/fisiología , Proteínas Protozoarias/metabolismo , Vacuolas/microbiología , Dictyostelium/crecimiento & desarrollo , Dictyostelium/microbiología , Dictyostelium/ultraestructura , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Retículo Endoplásmico Rugoso/microbiología , Retículo Endoplásmico Rugoso/fisiología , GTP Fosfohidrolasas/genética , Homeostasis , Interacciones Huésped-Patógeno , Legionella pneumophila/crecimiento & desarrollo , Movimiento , Muramidasa/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Protozoarias/genética , Vacuolas/fisiologíaRESUMEN
Multilamellar bodies (MLBs), structures composed of concentric membrane layers, are known to be produced by different protozoa, including species of ciliates, free-living amoebae, and Dictyostelium discoideum social amoebae. Initially believed to be metabolic waste, potential roles like cell communication and food storage have been suggested for D. discoideum MLBs, which could be useful for the multicellular development of social amoebae and as a food source. However, among dictyostelids, this phenomenon has only been observed with D. discoideum, and mainly with laboratory strains grown in axenic conditions. It was thought that other social amoebae may also produce MLBs. Four environmental social amoeba isolates were characterized. All strains belong to the Dictyostelium genus, including some likely to be Dictyostelium giganteum. They have distinctive phenotypes comprising their growth rate on Klebsiella aerogenes lawns and the morphology of their fruiting bodies. They all produce MLBs like those produced by a D. discoideum laboratory strain when grown on K. aerogenes lawns, as revealed by analysis using the H36 antibody in epifluorescence microscopy as well as by transmission electron microscopy. Consequently, this study shows that MLBs are produced by various dictyostelid species, which further supports a role for MLBs in the lifestyle of amoebae.
Asunto(s)
Dictyostelium/fisiología , Ambiente , Estructuras Celulares/metabolismo , Estructuras Celulares/ultraestructura , Dictyostelium/crecimiento & desarrollo , Dictyostelium/ultraestructura , FenotipoRESUMEN
The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.
Asunto(s)
Dictyostelium/genética , MAP Quinasa Quinasa Quinasa 3/genética , Proteínas Protozoarias/genética , Seudópodos/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Animales , Sistemas CRISPR-Cas , Adhesión Celular , Línea Celular Tumoral , Quimiotaxis/genética , Dictyostelium/metabolismo , Dictyostelium/ultraestructura , Edición Génica/métodos , Regulación de la Expresión Génica , MAP Quinasa Quinasa Quinasa 3/metabolismo , Melanocitos/metabolismo , Melanocitos/ultraestructura , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Mutación , Células 3T3 NIH , Fenotipo , Fosforilación , Ploidias , Proteínas Protozoarias/metabolismo , Seudópodos/genética , Seudópodos/ultraestructura , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Apoptosis Inducing Factor (AIF), a nuclear encoded mitochondrial inter-membrane space flavoprotein with intrinsic NADH oxidase activity, plays an important role in inducing cell death mechanisms. In response to cell death signals, it undergoes mitochondrio-nuclear translocation leading to DNA fragmentation. In addition to its role in cell death, AIF has a pro-survival role, wherein it contributes to the maintenance of mitochondrial structure and function in a coordinated manner. However, its exact mechanism of controlling mitochondrial homeostasis is unclear. The current study aims to explore the protective functions of AIF by its downregulation and overexpression in Dictyostelium discoideum. Constitutive AIF downregulated (dR) cells exhibited compromised oxidative phosphorylation along with elevated levels of cellular ROS. Interestingly, constitutive AIF dR cells showed amelioration in the activity of the ETC complexes upon antioxidant treatment, strengthening AIF's role as an ROS regulator, by virtue of its oxidoreductase property. Also, constitutive AIF dR cells showed lower transcript levels of the various subunits of ETC. Moreover, loss of AIF affected mtDNA content and mitochondrial fusion-fission mechanism, which subsequently caused morphometric mitochondrial alterations. Constitutive AIF overexpressed (OE) cells also showed higher cellular ROS and mitochondrial fission genes transcript levels along with reduced mitochondrial fusion genes transcript levels and mtDNA content. Thus, the results of the current study provide a paradigm where AIF is implicated in cell survival by maintaining mitochondrial bioenergetics, morphology and fusion-fission mechanism in D. discoideum, an evolutionarily significant model organism for mitochondrial diseases.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Citoprotección , Dictyostelium/citología , Proteínas Protozoarias/metabolismo , Factor Inductor de la Apoptosis/genética , Citoprotección/genética , ADN Mitocondrial/genética , Dictyostelium/genética , Dictyostelium/metabolismo , Dictyostelium/ultraestructura , Transporte de Electrón , Regulación de la Expresión Génica , Glutatión/metabolismo , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Dinámicas Mitocondriales/genética , Consumo de Oxígeno/genética , Proteínas Protozoarias/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
OBJECTIVE: Rhizoctonia solani is a soil-borne fungal pathogen of many important crop plants. In rice, R. solani causes sheath blight disease, which results in devastating grain yield and quality losses. Few methods are available to control this pathogen and classic single gene resistance mechanisms in rice plants have not been identified. We hypothesize that alternate means of control are available in the environment including free-living amoebae. Amoebae are soil-, water- and air-borne microorganisms that are predominantly heterotrophic. Many amoeba species are mycophagous, and several harm their prey using mechanisms other than phagocytosis. Here, we used light and scanning electron microscopy to survey the interactions of R. solani with four amoeba species, with the goal of identifying amoebae species with potential for biocontrol. RESULTS: We observed a wide range of responses during interactions of R. solani with four different free-living amoebae. Two Acanthamoeba species encyst in co-cultures with R. solani at higher rates than medium without R. solani. Vermamoeba vermiformis (formerly Hartmanella vermiformis) attach to R. solani mycelium and are associated with mycelial shriveling and perforations of fungal cell walls, indicating an antagonistic interaction. No phenotypic changes were observed in co-cultures of Dictyostelium discoideum and R. solani.
Asunto(s)
Acanthamoeba/fisiología , Antibiosis , Hartmannella/fisiología , Micelio/ultraestructura , Control Biológico de Vectores/métodos , Rhizoctonia/ultraestructura , Acanthamoeba/microbiología , Acanthamoeba/ultraestructura , Agentes de Control Biológico/metabolismo , Agentes de Control Biológico/farmacología , Pared Celular/química , Pared Celular/efectos de los fármacos , Pared Celular/ultraestructura , Técnicas de Cocultivo , Dictyostelium/microbiología , Dictyostelium/fisiología , Dictyostelium/ultraestructura , Hartmannella/microbiología , Hartmannella/ultraestructura , Micelio/efectos de los fármacos , Micelio/crecimiento & desarrollo , Micelio/patogenicidad , Oryza/microbiología , Enfermedades de las Plantas/prevención & control , Rhizoctonia/efectos de los fármacos , Rhizoctonia/crecimiento & desarrollo , Rhizoctonia/patogenicidadRESUMEN
Integrated with both a historical perspective and an evolutionary angle, this opinion article presents a brief and personal view of the emergence of cellular microbiology research. From the very first observations of phagocytosis by Goeze in 1777 to the exhaustive analysis of the cellular defence mechanisms performed in modern laboratories, the studies by cell biologists and microbiologists have converged into an integrative research field distinct from, but fully coupled to immunity: cellular microbiology. In addition, this brief article is thought as a humble patchwork of the motivations that have guided the research in my group over a quarter century.
Asunto(s)
Dictyostelium/inmunología , Mycobacterium marinum/inmunología , Fagocitosis/inmunología , Animales , Dictyostelium/crecimiento & desarrollo , Dictyostelium/microbiología , Dictyostelium/ultraestructura , Historia del Siglo XVIII , Historia del Siglo XIX , Historia del Siglo XXI , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata , Microbiología/historia , Mycobacterium marinum/crecimiento & desarrollo , Mycobacterium marinum/patogenicidad , Fagosomas/inmunología , Fagosomas/microbiología , Fagosomas/ultraestructuraRESUMEN
Actin waves are dynamic supramolecular structures involved in cell migration, cytokinesis, adhesion, and neurogenesis. Although wave-like propagation of actin networks is a widespread phenomenon, the actin architecture underlying wave propagation remained unknown. In situ cryo-electron tomography of Dictyostelium cells unveils the wave architecture and provides evidence for wave progression by de novo actin nucleation. Subtomogram averaging reveals the structure of Arp2/3 complex-mediated branch junctions in their native state, and enables quantitative analysis of the 3D organization of branching within the waves. We find an excess of branches directed toward the substrate-attached membrane, and tent-like structures at sites of branch clustering. Fluorescence imaging shows that Arp2/3 clusters follow accumulation of the elongation factor VASP. We propose that filament growth toward the membrane lifts up the actin network as the wave propagates, until depolymerization of oblique filaments at the back causes the collapse of horizontal filaments into a compact layer.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Moléculas de Adhesión Celular/metabolismo , Dictyostelium/ultraestructura , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Protozoarias/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/química , Dictyostelium/metabolismo , Tomografía con Microscopio Electrónico , Modelos Moleculares , Proteínas Protozoarias/químicaRESUMEN
Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.
Asunto(s)
Dictyostelium/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Animales , Permeabilidad de la Membrana Celular , Dictyostelium/ultraestructura , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Oocitos/metabolismo , Oocitos/ultraestructura , XenopusRESUMEN
Chemotactic signals are relayed to neighboring cells through the secretion of additional chemoattractants. We previously showed in Dictyostelium discoideum that the adenylyl cyclase A, which synthesizes the chemoattractant cyclic adenosine monophosphate (cAMP), is present in the intraluminal vesicles of multivesicular bodies (MVBs) that coalesce at the back of cells. Using ultrastructural reconstructions, we now show that ACA-containing MVBs release their contents to attract neighboring cells. We show that the released vesicles are capable of directing migration and streaming and are central to chemotactic signal relay. We demonstrate that the released vesicles not only contain cAMP but also can actively synthesize and release cAMP to promote chemotaxis. Through proteomic, pharmacological, and genetic approaches, we determined that the vesicular cAMP is released via the ABCC8 transporter. Together, our findings show that extracellular vesicles released by Ddiscoideum cells are functional entities that mediate signal relay during chemotaxis and streaming.
Asunto(s)
Quimiotaxis , Dictyostelium/metabolismo , Vesículas Extracelulares/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/fisiología , Movimiento Celular , AMP Cíclico/metabolismo , Dictyostelium/ultraestructura , Espectrometría de Masas , Proteoma , Transducción de SeñalRESUMEN
Programed cell death or apoptosis is a key developmental process that maintains tissue homeostasis in multicellular organisms. Inositol polyphosphates (InsPs) are key signaling molecules known to regulate a variety of cellular processes including apoptosis in such organisms. The signaling role of InsPs in unicellular organisms such as Dictyostelium discoideum (D. discoideum) is not well understood. We investigated whether InsPs also play any role in apoptosis in D. discoideum and whether InsPs-mediated apoptosis follows a mechanism similar to that present in higher multicellular eukaryotes. We measured known apoptotic markers in response to exogenously administered InsP6, the major InsPs in the cell. We found that InsP6 was able to cause cell death in D. discoideum cell culture in a dose- and time-dependent manner as determined by cytotoxicity assays. Fluorescence staining with acridine orange/ethidium bromide and flow cytometry results confirmed that the cell death in D. discoideum by InsP6 was due to apoptotic changes. Poly(ADP-ribose) expression, a known apoptotic marker used in D. discoideum, was also increased following InsP6 treatment suggesting a role for InsP6-mediated apoptosis in this organism. InsP6-mediated cell death was accompanied by production of reactive oxygen species and a decrease in mitochondrial membrane potential. Additionally, we studied the effects of InsP6 on the developmental life cycle of D. discoideum, the process likely affected by apoptosis. In conclusion, our studies provide evidence that InsP6-mediated cell death process is conserved in D. discoideum and plays an important signaling role in its developmental life cycle.
Asunto(s)
Apoptosis/fisiología , Dictyostelium/metabolismo , Estadios del Ciclo de Vida/fisiología , Ácido Fítico/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Dictyostelium/ultraestructuraRESUMEN
Cell migration requires the coordination of an excitable signal transduction network involving Ras and PI3K pathways with cytoskeletal activity. We show that expressing activated Ras GTPase-family proteins in cells lacking PTEN or other mutations which increase cellular protrusiveness transforms cells into a persistently activated state. Leading- and trailing-edge markers were found exclusively at the cell perimeter and the cytosol, respectively, of the dramatically flattened cells. In addition, the lifetimes of dynamic actin puncta were increased where they overlapped with actin waves, suggesting a mechanism for the coupling between these two networks. All of these phenotypes could be reversed by inhibiting signal transduction. Strikingly, maintaining cells in this state of constant activation led to a form of cell death by catastrophic fragmentation. These findings provide insight into the feedback loops that control excitability of the signal transduction network, which drives migration.
Asunto(s)
Dictyostelium/fisiología , Proteínas Protozoarias/fisiología , Transducción de Señal/fisiología , Citoesqueleto de Actina/fisiología , Citoesqueleto de Actina/ultraestructura , Adhesión Celular , Movimiento Celular , Forma de la Célula , Quimiotaxis , Dictyostelium/genética , Dictyostelium/ultraestructura , Activación Enzimática , Microscopía Fluorescente , Microscopía de Contraste de Fase , Mutación Missense , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/fisiología , Fenotipo , Proteínas Protozoarias/genética , Proteínas Recombinantes/metabolismo , Proteínas de Unión al GTP rap1/genética , Proteínas de Unión al GTP rap1/fisiologíaRESUMEN
Eukaryotic cells chemotax in a wide range of chemoattractant concentration gradients, and thus need inhibitory processes that terminate cell responses to reach adaptation while maintaining sensitivity to higher-concentration stimuli. However, the molecular mechanisms underlying inhibitory processes are still poorly understood. Here, we reveal a locally controlled inhibitory process in a GPCR-mediated signaling network for chemotaxis in Dictyostelium discoideum We identified a negative regulator of Ras signaling, C2GAP1, which localizes at the leading edge of chemotaxing cells and is activated by and essential for GPCR-mediated Ras signaling. We show that both C2 and GAP domains are required for the membrane targeting of C2GAP1, and that GPCR-triggered Ras activation is necessary to recruit C2GAP1 from the cytosol and retains it on the membrane to locally inhibit Ras signaling. C2GAP1-deficient c2gapA- cells have altered Ras activation that results in impaired gradient sensing, excessive polymerization of F actin, and subsequent defective chemotaxis. Remarkably, these cellular defects of c2gapA- cells are chemoattractant concentration dependent. Thus, we have uncovered an inhibitory mechanism required for adaptation and long-range chemotaxis.
Asunto(s)
Quimiotaxis/genética , Dictyostelium/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Protozoarias/genética , Proteínas ras/genética , Actinas/genética , Actinas/metabolismo , Adaptación Fisiológica , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Quimiotaxis/efectos de los fármacos , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Dictyostelium/efectos de los fármacos , Dictyostelium/genética , Dictyostelium/ultraestructura , Proteínas Activadoras de GTPasa/deficiencia , Regulación de la Expresión Génica , Transporte de Proteínas , Proteínas Protozoarias/metabolismo , Transducción de Señal , Proteínas ras/metabolismoRESUMEN
Autophagy is a highly conserved cellular degradation pathway which is crucial for various cellular processes. The autophagic process is subdivided in the initiation, autophagosome maturation and lysosomal degradation phases and involves more than forty core and accessory autophagy-related (ATG) proteins. Autophagy 8 (ATG8, in mammals LC3) is a well-established marker of autophagy and is linked to the autophagic membrane from initiation until fusion with the lysosome. We generated single and double knock-out mutants of the two Dictyostelium paralogues, ATG8a and ATG8b, as well as strains that expressed RFP-ATG8a and/or GFP-ATG8b, RFP-ATG8b, RFP-GFP-ATG8a or RFP-GFP-ATG8b in different knock-out mutants. The ATG8b¯ mutant displayed only subtle phenotypic changes in comparison to AX2 wild-type cells. In contrast, deletion of ATG8a resulted in a complex phenotype with delayed development, reduced growth, phagocytosis and cell viability, an increase in ubiquitinylated proteins and a concomitant decrease in proteasomal activity. The phenotype of the ATG8a¯/b¯ strain was, except for cell viability, in all aforementioned aspects more severe, showing that both proteins function in parallel during most analysed cellular processes. Immunofluorescence analysis of knock-out strains expressing either RFP-GFP-ATG8a or RFP-GFP-ATG8b suggests a crucial function for ATG8b in autophagosome-lysosome fusion. Quantitative analysis of strains expressing RFP-ATG8a, RFP-ATG8b, or RFP-ATG8a and GFP-ATG8b revealed that ATG8b generally localised to small and large vesicles, whereas ATG8a preferentially co-localised with ATG8b on large vesicles, indicating that ATG8b associated with nascent autophagosomes before ATG8a, which is supported by previous results (Matthias et al., 2016). Deconvoluted confocal fluorescence images showed that ATG8b localised around ATG8a and was presumably mainly present on the outer membrane of the autophagosome while ATG8a appears to be mainly associated with the inner membrane. In summary, our data show that ATG8a and ATG8b have distinct functions and are involved in canonical as well as non-canonical autophagy. The data further suggest that ATG8b predominantly acts as adapter for the autophagy machinery at the outer and ATG8a as cargo receptor at the inner membrane of the autophagosome.
Asunto(s)
Autofagosomas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Autofagia/genética , Dictyostelium/genética , Proteínas Protozoarias/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/deficiencia , Dictyostelium/metabolismo , Dictyostelium/ultraestructura , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Lisosomas/metabolismo , Fusión de Membrana/genética , Microscopía Fluorescente , Modelos Moleculares , Fenotipo , Complejo de la Endopetidasa Proteasomal/metabolismo , Conformación Proteica , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
Apoptosis Inducing Factor (AIF), a phylogenetically conserved mitochondrial inter-membrane space flavoprotein has an important role in caspase independent cell death. Nevertheless, AIF is also essential for cell survival. It is required for mitochondrial organization and energy metabolism. Upon apoptotic stimulation, AIF induces DNA fragmentation after its mitochondrio-nuclear translocation. Although it executes critical cellular functions in a coordinated manner, the exact mechanism still remains obscure. The present study aims to understand AIF's role in cell survival, growth and development by its down-regulation in an interesting unicellular eukaryote, D. discoideum which exhibits multicellularity upon starvation. Constitutive AIF down-regulated (dR) cells exhibited slower growth and delayed developmental morphogenesis. Also, constitutive AIF dR cells manifested high intracellular ROS, oxidative DNA damage and calcium levels with lower ATP content. Interestingly, constitutive AIF dR cells showed amelioration in cell growth upon antioxidant treatment, strengthening its role as ROS regulator. Under oxidative stress, AIF dR cells showed early mitochondrial membrane depolarization followed by AIF translocation from mitochondria to nucleus and exhibited necrotic cell death as compared to paraptoptic cell death of control cells. Thus, the results of this study provide an exemplar where AIF is involved in growth and development by regulating ROS levels and maintaining mitochondrial function in D. discoideum, an evolutionarily significant model organism exhibiting caspase independent apoptosis.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Evolución Biológica , Dictyostelium/citología , Dictyostelium/metabolismo , Adenosina Trifosfato/metabolismo , Anexina A5/metabolismo , Calcio/metabolismo , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Citosol/efectos de los fármacos , Citosol/metabolismo , Dictyostelium/crecimiento & desarrollo , Dictyostelium/ultraestructura , Regulación hacia Abajo/efectos de los fármacos , Citometría de Flujo , Fluoresceína-5-Isotiocianato/metabolismo , Fluorometría , Glucosa/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , NAD/metabolismo , Estrés Oxidativo/efectos de los fármacos , Propidio/metabolismo , Transporte de Proteínas/efectos de los fármacos , ARN sin Sentido/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Coloración y EtiquetadoRESUMEN
Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."
Asunto(s)
Proteínas de Capping de la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/genética , Dictyostelium/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas Protozoarias/genética , Seudópodos/metabolismo , Proteínas de Capping de la Actina/metabolismo , Proteína 2 Relacionada con la Actina/genética , Proteína 2 Relacionada con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteína 3 Relacionada con la Actina/genética , Proteína 3 Relacionada con la Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Quimiotaxis/genética , Secuencia Conservada , Dictyostelium/genética , Dictyostelium/ultraestructura , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cinética , Ratones , Mutación , Fosforilación , Pinocitosis/genética , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Protozoarias/metabolismo , Seudópodos/genética , Seudópodos/ultraestructura , Alineación de Secuencia , Transducción de SeñalRESUMEN
The impact of cellular individuality on host-microbe interactions is increasingly appreciated but studying the temporal dynamics of single-cell behavior in this context remains technically challenging. Here we present a microfluidic platform, InfectChip, to trap motile infected cells for high-resolution time-lapse microscopy. This approach allows the direct visualization of all stages of infection, from bacterial uptake to death of the bacterium or host cell, over extended periods of time. We demonstrate the utility of this approach by co-culturing an established host-cell model, Dictyostelium discoideum, with the extracellular pathogen Klebsiella pneumoniae or the intracellular pathogen Mycobacterium marinum. We show that the outcome of such infections is surprisingly heterogeneous, ranging from abortive infection to death of the bacterium or host cell. InfectChip thus provides a simple method to dissect the time-course of host-microbe interactions at the single-cell level, yielding new insights that could not be gleaned from conventional population-based measurements.
Asunto(s)
Rastreo Celular/instrumentación , Técnicas de Cocultivo/instrumentación , Interacciones Huésped-Patógeno , Dispositivos Laboratorio en un Chip , Modelos Biológicos , Fagocitosis , Análisis de la Célula Individual/instrumentación , Animales , Células Cultivadas , Células Inmovilizadas , Diseño Asistido por Computadora , Dictyostelium/citología , Dictyostelium/inmunología , Dictyostelium/fisiología , Dictyostelium/ultraestructura , Dimetilpolisiloxanos/química , Diseño de Equipo , Humanos , Interpretación de Imagen Asistida por Computador , Klebsiella pneumoniae/citología , Klebsiella pneumoniae/inmunología , Klebsiella pneumoniae/fisiología , Klebsiella pneumoniae/ultraestructura , Microscopía Confocal , Microscopía Electrónica de Rastreo , Mycobacterium marinum/citología , Mycobacterium marinum/inmunología , Mycobacterium marinum/fisiología , Mycobacterium marinum/ultraestructura , Fagocitos/citología , Fagocitos/inmunología , Fagocitos/microbiología , Fagocitos/parasitología , Imagen de Lapso de TiempoRESUMEN
In a 3D environment, motile cells accommodate their protruding and retracting activities to geometrical cues. Dictyostelium cells migrating on a perforated film explored its holes by forming actin rings around their border and extending protrusions through the free space. The response was initiated when an actin wave passed a hole, and the rings persisted only in the PIP3-rich territories surrounded by a wave. To reconstruct actin structures from cryo-electron tomograms, actin rings were identified by cryo-correlative light and electron microscopy, and thin wedges of relevant regions were obtained by cryo-focused ion-beam milling. Retracting stages were distinguished from protruding ones by the accumulation of myosin-II. Early actin rings consisted of filaments pointing upright from the membrane, entangled with a meshwork of filaments close to the membrane. Branches identified at later stages suggested that formin-based nucleation of filaments was followed by Arp2/3-mediated network stabilization, which prevented buckling of the force-generating filaments.