Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 7.446
Filtrar
1.
Elife ; 102021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34415238

RESUMO

Using a self-generated hypoxic assay, we show that the amoeba Dictyostelium discoideum displays a remarkable collective aerotactic behavior. When a cell colony is covered, cells quickly consume the available oxygen (O2) and form a dense ring moving outwards at constant speed and density. To decipher this collective process, we combined two technological developments: porphyrin-based O2 -sensing films and microfluidic O2 gradient generators. We showed that Dictyostelium cells exhibit aerotactic and aerokinetic response in a low range of O2 concentration indicative of a very efficient detection mechanism. Cell behaviors under self-generated or imposed O2 gradients were modeled using an in silico cellular Potts model built on experimental observations. This computational model was complemented with a parsimonious 'Go or Grow' partial differential equation (PDE) model. In both models, we found that the collective migration of a dense ring can be explained by the interplay between cell division and the modulation of aerotaxis.


Assuntos
Quimiotaxia , Dictyostelium/fisiologia , Oxigênio/metabolismo , Anaerobiose
2.
Nat Commun ; 12(1): 5044, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413298

RESUMO

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS.


Assuntos
Mutação , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ribossomos/genética , Ribossomos/patologia , Síndrome de Shwachman-Diamond/genética , Síndrome de Shwachman-Diamond/patologia , Adolescente , Adulto , Animais , Fenômenos Biológicos , Células Cultivadas , Criança , Pré-Escolar , Dictyostelium , Drosophila , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células Germinativas , Humanos , Lactente , Simulação de Dinâmica Molecular , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Proteínas/genética , Proteínas/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Síndrome de Shwachman-Diamond/metabolismo , Adulto Jovem
3.
J Microbiol ; 59(9): 848-853, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34449058

RESUMO

Rap small GTPases are involved in diverse signaling pathways associated with cell growth, proliferation, and cell migration. There are three Rap proteins in Dictyostelium, RapA, RapB, and RapC. RapA is a key regulator in the control of cell adhesion and migration. Recently RapA and RapC have been reported to have opposite functions in the regulation of cellular processes. In this study, we demonstrate that the C-terminus of RapC, which is not found in RapA, is essential for the opposite functions of RapC and is able to reverse the functions of RapA when fused to the tail of RapA. Cells lacking RapC displayed several defective phenotypes, including spread morphology, strong adhesion, and decreased cell migration compared to wild-type cells. These phenotypes were rescued by full-length RapC, but not by RapC missing the C-terminus. Furthermore, recombinant RapA fused with the C-terminus of RapC completely recovered the phenotypes of rapC null cells, indicating that the functions of RapA were modified to become similar to those of RapC by the C-terminus of RapC with respect to cell morphology, cell adhesion and migration, cytokinesis, and development. These results suggest that the C-terminal residues of RapC are able to suppress and change the functions of other Ras proteins in Ras oncogenic signaling pathways.


Assuntos
Dictyostelium/enzimologia , Proteínas de Protozoários/metabolismo , Proteínas ras/metabolismo , Motivos de Aminoácidos , Dictyostelium/química , Dictyostelium/genética , Regulação da Expressão Gênica , Ligação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas ras/genética
4.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200865

RESUMO

A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN- molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described.


Assuntos
Corantes/química , Dictyostelium/enzimologia , Heme/química , Peróxido de Hidrogênio/química , Peroxidase/química , Peroxidase/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , Heme/metabolismo , Ligação de Hidrogênio , Oxirredução
5.
Methods Mol Biol ; 2314: 183-203, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34235653

RESUMO

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.


Assuntos
Dictyostelium/microbiologia , Interações Hospedeiro-Patógeno , Dispositivos Lab-On-A-Chip , Microscopia Eletrônica/métodos , Infecções por Mycobacterium não Tuberculosas/diagnóstico , Mycobacterium marinum/crescimento & desenvolvimento , Análise de Célula Única/métodos , Dictyostelium/ultraestrutura , Infecções por Mycobacterium não Tuberculosas/microbiologia
6.
Int J Mol Sci ; 22(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34298920

RESUMO

Protein dimerization plays a crucial role in the regulation of numerous biological processes. However, detecting protein dimers in a cellular environment is still a challenge. Here we present a methodology to measure the extent of dimerization of GFP-tagged proteins in living cells, using a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis of single-color fluorescence fluctuation data. We named this analysis method brightness and diffusion global analysis (BDGA) and adapted it for biological purposes. Using cell lysates containing different ratios of GFP and tandem-dimer GFP (diGFP), we show that the average brightness per particle is proportional to the fraction of dimer present. We further adapted this methodology for its application in living cells, and we were able to distinguish GFP, diGFP, as well as ligand-induced dimerization of FKBP12 (FK506 binding protein 12)-GFP. While other analysis methods have only sporadically been used to study dimerization in living cells and may be prone to errors, this paper provides a robust approach for the investigation of any cytosolic protein using single-color fluorescence fluctuation spectroscopy.


Assuntos
Multimerização Proteica/fisiologia , Proteínas/metabolismo , Células Cultivadas , Citosol/metabolismo , Dictyostelium/metabolismo , Difusão , Dimerização , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Ligantes , Fótons , Espectrometria de Fluorescência/métodos
7.
Environ Sci Technol ; 55(13): 8709-8720, 2021 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-34138552

RESUMO

Amoebas are protists that are widespread in water and soil environments. Some species are pathogenic, inducing potentially lethal effects on humans, making them a major threat to public health. Nonpathogenic amoebas are also of concern because they have the potential to carry a mini-microbiome of bacteria, either transiently or via more long-term stable transport. Due to their resistance to disinfection processes, the physical removal of amoeba by filtration is necessary to prevent their propagation throughout drinking water distribution networks and occurrence in tap water. In this study, a model amoeba species Dictyostelium discoideum was used to study the transport and retention behavior of amoeba spores in porous media. The key factors affecting the transport behavior of amoeba spores in fully saturated media were comprehensively evaluated, with experiments performed using a quartz crystal microbalance with dissipation monitoring (QCM-D) and parallel plate chamber system. The effects of ionic strength (IS) on the deposition of spores were found to be in contrast to the predicted Derjaguin-Landau-Verwey-Overbeek (DLVO) theory that more deposition is observed under lower-IS conditions. The presence of extracellular polymeric substances (EPS) was found to be the main contributor to deposition behavior. Overall, these results provide plausible evidence for the presence of amoeba in tap water. Furthermore, this is one of the first studies to examine the mechanisms affecting the fate of amoeba spores in porous media, providing a significant baseline for future research to minimize the safety risk presented by amoeba in drinking water systems.


Assuntos
Amoeba , Dictyostelium , Matriz Extracelular de Substâncias Poliméricas , Humanos , Porosidade , Esporos de Protozoários
8.
BMC Genomics ; 22(1): 444, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34126926

RESUMO

BACKGROUND: Autophagy is an evolutionary ancient mechanism that sequesters substrates for degradation within autolysosomes. The process is driven by many autophagy-related (ATG) proteins, including the core members ATG9 and ATG16. However, the functions of these two core ATG proteins still need further elucidation. Here, we applied RNAseq and tandem mass tag (TMT) proteomic approaches to identify differentially expressed genes (DEGs) and proteins (DEPs) in Dictyostelium discoideum ATG9‾, ATG16‾ and ATG9‾/16‾ strains in comparison to AX2 wild-type cells. RESULT: In total, we identified 332 (279 up and 53 down), 639 (487 up and 152 down) and 260 (114 up and 146 down) DEGs and 124 (83 up and 41 down), 431 (238 up and 193 down) and 677 (347 up and 330 down) DEPs in ATG9‾, ATG16‾ and ATG9‾/16‾ strains, respectively. Thus, in the single knock-out strains, the number of DEGs was higher than the number of DEPs while in the double knock-out strain the number of DEPs was higher. Comparison of RNAseq and proteomic data further revealed, that only a small proportion of the transcriptional changes were reflected on the protein level. Gene ontology (GO) analysis revealed an enrichment of DEPs involved in lipid metabolism and oxidative phosphorylation. Furthermore, we found increased expression of the anti-oxidant enzymes glutathione reductase (gsr) and catalase A (catA) in ATG16‾ and ATG9‾/16‾ cells, respectively, indicating adaptation to excess reactive oxygen species (ROS). CONCLUSIONS: Our study provides the first combined transcriptome and proteome analysis of ATG9‾, ATG16‾ and ATG9‾/16‾ cells. Our results suggest, that most changes in protein abundance were not caused by transcriptional changes, but were rather due to changes in protein homeostasis. In particular, knock-out of atg9 and/or atg16 appears to cause dysregulation of lipid metabolism and oxidative phosphorylation.


Assuntos
Dictyostelium , Autofagia/genética , Dictyostelium/genética , Proteômica , Proteínas de Protozoários/genética , RNA
9.
J Cell Sci ; 134(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34169317

RESUMO

Allorecognition and tissue formation are interconnected processes that require signaling between matching pairs of the polymorphic transmembrane proteins TgrB1 and TgrC1 in Dictyostelium. Extracellular and intracellular cAMP signaling are essential to many developmental processes. The three adenylate cyclase genes, acaA, acrA and acgA are required for aggregation, culmination and spore dormancy, respectively, and some of their functions can be suppressed by activation of the cAMP-dependent protein kinase PKA. Previous studies have suggested that cAMP signaling might be dispensable for allorecognition and tissue formation, while others have argued that it is essential throughout development. Here, we show that allorecognition and tissue formation do not require cAMP production as long as PKA is active. We eliminated cAMP production by deleting the three adenylate cyclases and overexpressed PKA-C to enable aggregation. The cells exhibited cell polarization, tissue formation and cooperation with allotype-compatible wild-type cells, but not with incompatible cells. Therefore, TgrB1-TgrC1 signaling controls allorecognition and tissue formation, while cAMP is dispensable as long as PKA-C is overexpressed.


Assuntos
Dictyostelium , Adenilil Ciclases/genética , AMP Cíclico , Proteínas Quinases Dependentes de AMP Cíclico/genética , Dictyostelium/genética , Proteínas de Protozoários/genética
10.
J Phys Chem B ; 125(24): 6513-6521, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34105970

RESUMO

Human cardiac ß myosin undergoes the cross-bridge cycle as part of the force-generating mechanism of cardiac muscle. The recovery stroke is considered one of the key steps of the kinetic cycle as it is the conformational rearrangement required to position the active site residues for hydrolysis of ATP and interaction with actin. We explored the free-energy surface of the transition and investigated the effect of the genetic cardiomyopathy causing mutations R453C, I457T, and I467T on this step using metadynamics. This work extends previous studies on Dictyostelium myosin II with engineered mutations. Here, like previously, we generated an unbiased thermodynamic ensemble of reactive trajectories for the chemical step using transition path sampling. Our methodologies were able to predict the changes to the dynamics of the recovery stroke as well as predict the pathway of breakdown of ATP to ADP and HPO42- with the stabilization of the metaphosphate intermediate. We also observed clear differences between the Dictyostelium myosin II and human cardiac ß myosin for ATP hydrolysis as well as predict the effect of the mutation I467T on the chemical step.


Assuntos
Cardiomiopatias , Dictyostelium , Acidente Vascular Cerebral , Actinas , Trifosfato de Adenosina , Dictyostelium/genética , Humanos , Hidrólise , Mutação Puntual , Miosinas Ventriculares
11.
PLoS One ; 16(5): e0250710, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34043641

RESUMO

The phospholipid phosphatidylserine (PS) is a key signaling molecule and binding partner for many intracellular proteins. PS is normally found on the inner surface of the cell membrane, but PS can be flipped to the outer surface in a process called PS exposure. PS exposure is important in many cell functions, yet the mechanisms that control PS exposure have not been extensively studied. Copines (Cpn), found in most eukaryotic organisms, make up a family of calcium-dependent phospholipid binding proteins. In Dictyostelium, which has six copine genes, CpnA strongly binds to PS and translocates from the cytosol to the plasma membrane in response to a rise in calcium. Cells lacking the cpnA gene (cpnA-) have defects in adhesion, chemotaxis, membrane trafficking, and cytokinesis. In this study we used both flow cytometry and fluorescent microscopy to show that cpnA- cells have increased adhesion to beads and bacteria and that the increased adhesion was not due to changes in the actin cytoskeleton or cell surface proteins. We found that cpnA- cells bound higher amounts of Annexin V, a PS binding protein, than parental cells and showed that unlabeled Annexin V reduced the increased cell adhesion property of cpnA- cells. We also found that cpnA- cells were more sensitive to Polybia-MP1, which binds to external PS and induces cell lysis. Overall, this suggests that cpnA- cells have increased PS exposure and this property contributes to the increased cell adhesion of cpnA- cells. We conclude that CpnA has a role in the regulation of plasma membrane lipid composition and may act as a negative regulator of PS exposure.


Assuntos
Dictyostelium/efeitos dos fármacos , Dictyostelium/genética , Mutação , Fosfatidilserinas/farmacologia , Adesão Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dictyostelium/citologia , Proteínas de Protozoários/genética
12.
Methods Mol Biol ; 2304: 193-205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028718

RESUMO

Macropinocytosis and phagocytosis are the processes by which eukaryotic cells use their plasma membrane to engulf liquid or a large particle and give rise to an internal compartment called the macropinosomes or phagosome, respectively. Dictyostelium discoideum provides a powerful system to understand the molecular mechanism of these two fundamental cellular processes that impact human health and disease. Recent developments in fluorescence microscopy allow direct visualization of intracellular signaling events with high temporal and spatial resolution. Here, we describe methods to visualize temporospatial activation or localization of key signaling components that are crucial for macropinocytosis and phagocytosis using confocal fluorescence microscopy.


Assuntos
Dictyostelium/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas ras/metabolismo , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Microscopia Confocal , Microscopia de Fluorescência , Fagocitose , Pinocitose , Proteínas de Protozoários/metabolismo , Transdução de Sinais
13.
Methods Mol Biol ; 2304: 207-220, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028719

RESUMO

Eukaryotic phagocytes locate microorganisms via chemotaxis and consume them through phagocytosis. The social amoeba Dictyostelium discoideum is a stereotypical phagocyte and a well-established model to study both processes. Recent studies show that a G-protein-coupled receptor (fAR1) mediate a signaling network to control reorganization of the actin cytoskeleton leading both the directional cell movement and the engulfment of bacteria. Many live cell imaging methods have been developed and applied to monitor these signaling events. In this chapter, we will introduce how to measure GPCR-mediated signaling events for cell migration and phagocytosis in Dictyostelium.


Assuntos
Citoesqueleto de Actina/metabolismo , Dictyostelium/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Quimiotaxia , Ácido Fólico/metabolismo , Lipopolissacarídeos/metabolismo , Microscopia de Fluorescência , Fagocitose , Proteínas de Protozoários/metabolismo , Transdução de Sinais , Imagem com Lapso de Tempo
14.
J Cell Biol ; 220(7)2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-33978708

RESUMO

Polarity is essential for diverse functions in many cell types. Establishing polarity requires targeting a network of specific signaling and cytoskeleton molecules to different subregions of the cell, yet the full complement of polarity regulators and how their activities are integrated over space and time to form morphologically and functionally distinct domains remain to be uncovered. Here, by using the model system Dictyostelium and exploiting the characteristic chemoattractant-stimulated translocation of polarly distributed molecules, we developed a proteomic screening approach, through which we identified a leucine-rich repeat domain-containing protein we named Leep1 as a novel polarity regulator. We combined imaging, biochemical, and phenotypic analyses to demonstrate that Leep1 localizes selectively at the leading edge of cells by binding to PIP3, where it modulates pseudopod and macropinocytic cup dynamics by negatively regulating the Scar/WAVE complex. The spatiotemporal coordination of PIP3 signaling, Leep1, and the Scar/WAVE complex provides a cellular mechanism for organizing protrusive structures at the leading edge.


Assuntos
Actinas/economia , Polaridade Celular/genética , Pinocitose/genética , Proteínas de Protozoários/genética , Actinas/genética , Movimento Celular/genética , Quimiotaxia/genética , Citoplasma/genética , Dictyostelium/genética , Pseudópodes/genética , Transdução de Sinais/genética
15.
Elife ; 102021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34042588

RESUMO

Filopodia are thin, actin-based structures that cells use to interact with their environments. Filopodia initiation requires a suite of conserved proteins but the mechanism remains poorly understood. The actin polymerase VASP and a MyTH-FERM (MF) myosin, DdMyo7 in amoeba, are essential for filopodia initiation. DdMyo7 is localized to dynamic regions of the actin-rich cortex. Analysis of VASP mutants and treatment of cells with anti-actin drugs shows that myosin recruitment and activation in Dictyostelium requires localized VASP-dependent actin polymerization. Targeting of DdMyo7 to the cortex alone is not sufficient for filopodia initiation; VASP activity is also required. The actin regulator locally produces a cortical actin network that activates myosin and together they shape the actin network to promote extension of parallel bundles of actin during filopodia formation. This work reveals how filopodia initiation requires close collaboration between an actin-binding protein, the state of the actin cytoskeleton and MF myosin activity.


Assuntos
Actinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Dictyostelium/enzimologia , Proteínas dos Microfilamentos/metabolismo , Miosinas/metabolismo , Fosfoproteínas/metabolismo , Proteínas de Protozoários/metabolismo , Pseudópodes/enzimologia , Actinas/genética , Moléculas de Adesão Celular/genética , Dictyostelium/genética , Proteínas dos Microfilamentos/genética , Movimento , Miosinas/genética , Fosfoproteínas/genética , Proteínas de Protozoários/genética , Pseudópodes/genética , Fatores de Tempo
16.
Methods Mol Biol ; 2274: 317-336, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050483

RESUMO

GPCR signaling is the most prevailing molecular mechanism for detecting ambient signals in eukaryotes. Chemotactic cells use GPCR signaling to process chemical cues for directional migration over a broad concentration range and with high sensitivity. Dictyostelium discoideum is a classical model, in which the molecular mechanism underlying eukaryotic chemotaxis has been well studied. Here, we describe protocols to evaluate the spatiotemporal chemotactic responses of Dictyostelium discoideum by different microscopic observations combined with biochemical assays. First, two different chemotaxis assays are presented to measure the dynamic concentration ranges for different cell strains or chemotactic parameters. Next, live-cell imaging and biochemical assays are provided to detect the activities of GPCR and its partner heterotrimeric G proteins upon chemoattractant stimulation. Finally, a method for detecting how a cell deciphers chemical gradients is described.


Assuntos
Fatores Quimiotáticos/farmacologia , Quimiotaxia/efeitos dos fármacos , Dictyostelium/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , AMP Cíclico/metabolismo , Dictyostelium/efeitos dos fármacos , Imunoprecipitação , Transdução de Sinais , Análise Espaço-Temporal
17.
Methods Mol Biol ; 2306: 123-137, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33954944

RESUMO

Ceramides are a special class of sphingolipids and play a central role in sphingolipid metabolism, and have diverse structures. In this book chapter, tandem quadrupole mass spectrometric approaches applying multiple linked scannings including various constant neutral loss scan (NLS) and precursor ion scan (PIS), the unique applicable feature of a triple-stage quadrupole (TSQ) instrument for analysis of ceramides desorbed as [M-H]- and [M+Li]+ ions are described. These multiple dimensional tandem mass spectrometric approaches are fully adapted to the conventional shotgun lipidomics workflow with minimal or without prior chromatographic separation to profile ceramide molecules, and thus detection of a whole class of ceramide or various specific ceramide subclasses in crude lipid extract can be achieved. With addition of internal standard(s), semi-quantitation of ceramide in the lipid extract of biological origin is possible. Examples have shown promise in ceramide profiling of several whole lipid extracts from porcine brain, the model Dictyostelium Discoideum cells for cancer study, and skin.


Assuntos
Ceramidas/análise , Dictyostelium/química , Lipidômica/métodos , Pele/química , Animais , Química Encefálica , Humanos , Espectrometria de Massas por Ionização por Electrospray , Suínos , Espectrometria de Massas em Tandem
18.
Genes (Basel) ; 12(4)2021 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-33801615

RESUMO

Multicellularity evolved repeatedly in the history of life, but how it unfolded varies greatly between different lineages. Dictyostelid social amoebas offer a good system to study the evolution of multicellular complexity, with a well-resolved phylogeny and molecular genetic tools being available. We compare the life cycles of the Dictyostelids with closely related amoebozoans to show that complex life cycles were already present in the unicellular common ancestor of Dictyostelids. We propose frost resistance as an early driver of multicellular evolution in Dictyostelids and show that the cell signalling pathways for differentiating spore and stalk cells evolved from that for encystation. The stalk cell differentiation program was further modified, possibly through gene duplication, to evolve a new cell type, cup cells, in Group 4 Dictyostelids. Studies in various multicellular organisms, including Dictyostelids, volvocine algae, and metazoans, suggest as a common principle in the evolution of multicellular complexity that unicellular regulatory programs for adapting to environmental change serve as "proto-cell types" for subsequent evolution of multicellular organisms. Later, new cell types could further evolve by duplicating and diversifying the "proto-cell type" gene regulatory networks.


Assuntos
Amoeba/fisiologia , Dictyostelium/fisiologia , Estresse Fisiológico , Evolução Biológica , Temperatura Baixa , Evolução Molecular , Estágios do Ciclo de Vida , Filogenia , Transdução de Sinais
19.
Biomolecules ; 11(3)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33807046

RESUMO

Neddylation is a post-translational modification that is essential for a variety of cellular processes and is linked to many human diseases including cancer, neurodegeneration, and autoimmune disorders. Neddylation involves the conjugation of the ubiquitin-like modifier neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) to target proteins, and has been studied extensively in various eukaryotes including fungi, plants, and metazoans. Here, we examine the biological processes influenced by neddylation in the social amoeba, Dictyostelium discoideum, using a well-established inhibitor of neddylation, MLN4924 (pevonedistat). NEDD8, and the target of MLN4924 inhibition, NEDD8-activating enzyme E1 (NAE1), are highly conserved in D. discoideum (Nedd8 and Nae1, respectively). Treatment of D. discoideum cells with MLN4924 increased the amount of free Nedd8, suggesting that MLN4924 inhibited neddylation. During growth, MLN4924 suppressed cell proliferation and folic acid-mediated chemotaxis. During multicellular development, MLN4924 inhibited cyclic adenosine monophosphate (cAMP)-mediated chemotaxis, delayed aggregation, and suppressed fruiting body formation. Together, these findings indicate that neddylation plays an important role in regulating cellular and developmental events during the D. discoideum life cycle and that this organism can be used as a model system to better understand the essential roles of neddylation in eukaryotes, and consequently, its involvement in human disease.


Assuntos
Ciclopentanos/química , Ciclopentanos/farmacologia , Dictyostelium/efeitos dos fármacos , Proteína NEDD8/metabolismo , Pirimidinas/química , Pirimidinas/farmacologia , Quimiotaxia/efeitos dos fármacos , Processamento de Proteína Pós-Traducional
20.
Mol Biol Evol ; 38(8): 3247-3266, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-33871580

RESUMO

Alternative synonymous codons are often used at unequal frequencies. Classically, studies of such codon usage bias (CUB) attempted to separate the impact of neutral from selective forces by assuming that deviations from a predicted neutral equilibrium capture selection. However, GC-biased gene conversion (gBGC) can also cause deviation from a neutral null. Alternatively, selection has been inferred from CUB in highly expressed genes, but the accuracy of this approach has not been extensively tested, and gBGC can interfere with such extrapolations (e.g., if expression and gene conversion rates covary). It is therefore critical to examine deviations from a mutational null in a species with no gBGC. To achieve this goal, we implement such an analysis in the highly AT rich genome of Dictyostelium discoideum, where we find no evidence of gBGC. We infer neutral CUB under mutational equilibrium to quantify "adaptive codon preference," a nontautologous genome wide quantitative measure of the relative selection strength driving CUB. We observe signatures of purifying selection consistent with selection favoring adaptive codon preference. Preferred codons are not GC rich, underscoring the independence from gBGC. Expression-associated "preference" largely matches adaptive codon preference but does not wholly capture the influence of selection shaping patterns across all genes, suggesting selective constraints associated specifically with high expression. We observe patterns consistent with effects on mRNA translation and stability shaping adaptive codon preference. Thus, our approach to quantifying adaptive codon preference provides a framework for inferring the sources of selection that shape CUB across different contexts within the genome.


Assuntos
Uso do Códon , Dictyostelium/genética , Seleção Genética , Adaptação Biológica , Composição de Bases , Biossíntese de Proteínas , RNA de Transferência/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...