RESUMEN
Macropinocytosis is a broadly conserved endocytic process discovered nearly 100 years ago, yet still poorly understood. It is prominent in cancer cell feeding, immune surveillance, uptake of RNA vaccines and as an invasion route for pathogens. Macropinocytic cells extend large cups or flaps from their plasma membrane to engulf droplets of medium and trap them in micron-sized vesicles. Here they are digested and the products absorbed. A major problem - discussed here - is to understand how cups are shaped and closed. Recently, lattice light-sheet microscopy has given a detailed description of this process in Dictyostelium amoebae, leading to the 'stalled-wave' model for cup formation and closure. This is based on membrane domains of PIP3 and active Ras and Rac that occupy the inner face of macropinocytic cups and are readily visible with suitable reporters. These domains attract activators of dendritic actin polymerization to their periphery, creating a ring of protrusive F-actin around themselves, thus shaping the walls of the cup. As domains grow, they drive a wave of actin polymerization across the plasma membrane that expands the cup. When domains stall, continued actin polymerization under the membrane, combined with increasing membrane tension in the cup, drives closure at lip or base. Modelling supports the feasibility of this scheme. No specialist coat proteins or contractile activities are required to shape and close cups: rings of actin polymerization formed around PIP3 domains that expand and stall seem sufficient. This scheme may be widely applicable and begs many biochemical questions.
Asunto(s)
Membrana Celular , Dictyostelium , Pinocitosis , Pinocitosis/fisiología , Membrana Celular/metabolismo , Humanos , Actinas/metabolismo , Animales , Modelos BiológicosRESUMEN
Macropinocytosis is a relatively unexplored form of large-scale endocytosis driven by the actin cytoskeleton. Dictyostelium amoebae form macropinosomes from cups extended from the plasma membrane, then digest their contents and absorb the nutrients in the endo-lysosomal system. They use macropinocytosis for feeding, maintaining a high rate of fluid uptake that makes assay and experimentation easy. Mutants collected over the years identify cytoskeletal and signalling proteins required for macropinocytosis. Cups are organized around plasma membrane domains of intense PIP3, Ras and Rac signalling, proper formation of which also depends on the RasGAPs NF1 and RGBARG, PTEN, the PIP3-regulated protein kinases Akt and SGK and their activators PDK1 and TORC2, Rho proteins, plus other components yet to be identified. This PIP3 domain directs dendritic actin polymerization to the extending lip of macropinocytic cups by recruiting a ring of the SCAR/WAVE complex around itself and thus activating the Arp2/3 complex. The dynamics of PIP3 domains are proposed to shape macropinocytic cups from start to finish. The role of the Ras-PI3-kinase module in organizing feeding structures in unicellular organisms most likely predates its adoption into growth factor signalling, suggesting an evolutionary origin for growth factor signalling.
Asunto(s)
Amoeba , Dictyostelium , Citoesqueleto de Actina/metabolismo , Amoeba/metabolismo , Dictyostelium/genética , Dictyostelium/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , PinocitosisRESUMEN
Blebs and pseudopods can both power cell migration, with blebs often favored in tissues, where cells encounter increased mechanical resistance. To investigate how migrating cells detect and respond to mechanical forces, we used a "cell squasher" to apply uniaxial pressure to Dictyostelium cells chemotaxing under soft agarose. As little as 100 Pa causes a rapid (<10 s), sustained shift to movement with blebs rather than pseudopods. Cells are flattened under load and lose volume; the actin cytoskeleton is reorganized, with myosin II recruited to the cortex, which may pressurize the cytoplasm for blebbing. The transition to bleb-driven motility requires extracellular calcium and is accompanied by increased cytosolic calcium. It is largely abrogated in cells lacking the Piezo stretch-operated channel; under load, these cells persist in using pseudopods and chemotax poorly. We propose that migrating cells sense pressure through Piezo, which mediates calcium influx, directing movement with blebs instead of pseudopods.
Asunto(s)
Dictyostelium/citología , Dictyostelium/metabolismo , Canales Iónicos/metabolismo , Proteínas Protozoarias/metabolismo , Seudópodos/metabolismo , Fenómenos Biomecánicos , Movimiento Celular , Citoplasma/química , Citoplasma/genética , Citoplasma/metabolismo , Dictyostelium/química , Dictyostelium/genética , Canales Iónicos/genética , Mecanotransducción Celular , Miosina Tipo II/genética , Miosina Tipo II/metabolismo , Presión , Proteínas Protozoarias/genética , Seudópodos/genéticaRESUMEN
The soil is a rich ecosystem where many ecological interactions are mediated by small molecules, and in which amoebae are low-level predators and also prey. The social amoeba Dictyostelium discoideum has a high genomic potential for producing polyketides to mediate its ecological interactions, including the unique 'Steely' enzymes, consisting of a fusion between a fatty acid synthase and a chalcone synthase. We report here that D. discoideum further increases its polyketide potential by using the StlB Steely enzyme, and a downstream chlorinating enzyme, to make both a chlorinated signal molecule, DIF-1, during its multi-cellular development, and a set of abundant polyketides in terminally differentiated stalk cells. We identify one of these as a chlorinated dibenzofuran with potent anti-bacterial activity. To do this, StlB switches expression from prespore to stalk cells in late development and is cleaved to release the chalcone synthase domain. Expression of this domain alone in StlB null cells allows synthesis of the stalk-associated, chlorinated polyketides. Thus, by altered expression and processing of StlB, cells make first a signal molecule, and then abundant secondary metabolites, which we speculate help to protect the mature spores from bacterial infection.
Asunto(s)
Dictyostelium , Policétidos , Dibenzofuranos Policlorados/metabolismo , Dictyostelium/genética , Ecosistema , Ácido Graso Sintasas/metabolismo , Sintasas Poliquetidas/metabolismo , Policétidos/metabolismo , SueloRESUMEN
Macropinocytosis is an actin-driven process of large-scale and non-specific fluid uptake used for feeding by some cancer cells and the macropinocytosis model organism Dictyostelium discoideum In Dictyostelium, macropinocytic cups are organized by 'macropinocytic patches' in the plasma membrane. These contain activated Ras, Rac and phospholipid PIP3, and direct actin polymerization to their periphery. We show that a Dictyostelium Akt (PkbA) and an SGK (PkbR1) protein kinase act downstream of PIP3 and, together, are nearly essential for fluid uptake. This pathway enables the formation of larger macropinocytic patches and macropinosomes, thereby dramatically increasing fluid uptake. Through phosphoproteomics, we identify a RhoGAP, GacG, as a PkbA and PkbR1 target, and show that it is required for efficient macropinocytosis and expansion of macropinocytic patches. The function of Akt and SGK in cell feeding through control of macropinosome size has implications for cancer cell biology.
Asunto(s)
Dictyostelium/enzimología , Pinocitosis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Protozoarias/metabolismo , Dictyostelium/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Protozoarias/genéticaRESUMEN
Macropinocytosis is a conserved endocytic process used by Dictyostelium amoebae for feeding on liquid medium. To further Dictyostelium as a model for macropinocytosis, we developed a high-throughput flow cytometry assay to measure macropinocytosis, and used it to identify inhibitors and investigate the physiological regulation of macropinocytosis. Dictyostelium has two feeding states: phagocytic and macropinocytic. When cells are switched from phagocytic growth on bacteria to liquid media, the rate of macropinocytosis slowly increases, due to increased size and frequency of macropinosomes. Upregulation is triggered by a minimal medium containing three amino acids plus glucose and likely depends on macropinocytosis itself. The presence of bacteria suppresses macropinocytosis while their product, folate, partially suppresses upregulation of macropinocytosis. Starvation, which initiates development, does not of itself suppress macropinocytosis: this can continue in isolated cells, but is shut down by a conditioned-medium factor or activation of PKA signalling. Thus macropinocytosis is a facultative ability of Dictyostelium cells, regulated by environmental conditions that are identified here.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , Dictyostelium/fisiología , Pinocitosis , Aminoácidos/metabolismo , Dictyostelium/genética , Glucosa/metabolismo , Fagocitosis , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Chemotaxis--the guided movement of cells in chemical gradients--probably first emerged in our single-celled ancestors and even today is recognizably similar in neutrophils and amoebae. Chemotaxis enables immune cells to reach sites of infection, allows wounds to heal and is crucial for forming embryonic patterns. Furthermore, the manipulation of chemotaxis may help to alleviate disease states, including the metastasis of cancer cells. This review discusses recent results concerning how cells orientate in chemotactic gradients and the role of phosphatidylinositol-3,4,5-trisphosphate, what produces the force for projecting pseudopodia and a new role for the endocytic cycle in movement.
Asunto(s)
Quimiotaxis/fisiología , Animales , Quimiotaxis de Leucocito/fisiología , Dictyostelium/citología , Dictyostelium/fisiología , HumanosRESUMEN
In a role distinct from and perhaps more ancient than that in signal transduction, PIP3 and Ras help to spatially organize the actin cytoskeleton into macropinocytic cups. These large endocytic structures are extended by actin polymerization from the cell surface and have at their core an intense patch of active Ras and PIP3, around which actin polymerizes, creating cup-shaped projections. We hypothesize that active Ras and PIP3 self-amplify within macropinocytic cups, in a way that depends on the structural integrity of the cup. Signalling that triggers macropinocytosis may therefore be amplified downstream in a way that depends on macropinocytosis. This argument provides a context for recent findings that signalling to Akt (an effector of PIP3) is sensitive to cytoskeletal and macropinocytic inhibitors.
Asunto(s)
Citoesqueleto de Actina/genética , Pinocitosis/genética , Proteínas Proto-Oncogénicas c-akt/genética , Citoesqueleto de Actina/química , Membrana Celular/genética , Dictyostelium/genética , Humanos , Proteínas Proto-Oncogénicas c-akt/química , Transducción de Señal/genéticaRESUMEN
Inositol phospholipids are critical regulators of membrane biology throughout eukaryotes. The general principle by which they perform these roles is conserved across species and involves binding of differentially phosphorylated inositol head groups to specific protein domains. This interaction serves to both recruit and regulate the activity of several different classes of protein which act on membrane surfaces. In mammalian cells, these phosphorylated inositol head groups are predominantly borne by a C38:4 diacylglycerol backbone. We show here that the inositol phospholipids of Dictyostelium are different, being highly enriched in an unusual C34:1e lipid backbone, 1-hexadecyl-2-(11Z-octadecenoyl)-sn-glycero-3-phospho-(1'-myo-inositol), in which the sn-1 position contains an ether-linked C16:0 chain; they are thus plasmanylinositols. These plasmanylinositols respond acutely to stimulation of cells with chemoattractants, and their levels are regulated by PIPKs, PI3Ks and PTEN. In mammals and now in Dictyostelium, the hydrocarbon chains of inositol phospholipids are a highly selected subset of those available to other phospholipids, suggesting that different molecular selectors are at play in these organisms but serve a common, evolutionarily conserved purpose.
Asunto(s)
Dictyostelium/fisiología , Fosfatidilinositoles/metabolismo , Éteres Fosfolípidos/metabolismo , Transducción de Señal/fisiología , Animales , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Macropinocytosis is a means by which eukaryotic cells ingest extracellular liquid and dissolved molecules. It is widely conserved amongst cells that can take on amoeboid form and, therefore, appears to be an ancient feature that can be traced back to an early stage of evolution. Recent advances have highlighted how this endocytic process can be subverted during pathology - certain cancer cells use macropinocytosis to feed on extracellular protein, and many viruses and bacteria use it to enter host cells. Prion and prion-like proteins can also spread and propagate from cell to cell through macropinocytosis. Progress is being made towards using macropinocytosis therapeutically, either to deliver drugs to or cause cell death by inducing catastrophically rapid fluid uptake. Mechanistically, the Ras signalling pathway plays a prominent and conserved activating role in amoebae and in mammals; mutant amoebae with abnormally high Ras activity resemble tumour cells in their increased capacity for growth using nutrients ingested through macropinocytosis. This Commentary takes a functional and evolutionary perspective to highlight progress in understanding and use of macropinocytosis, which is an ancient feeding process used by single-celled phagotrophs that has now been put to varied uses by metazoan cells and is abused in disease states, including infection and cancer.
Asunto(s)
Pinocitosis , Animales , Muerte Celular , Enfermedades Transmisibles/inmunología , Enfermedades Transmisibles/patología , Humanos , Inmunidad , Sustancias Macromoleculares/metabolismo , Priones/metabolismoRESUMEN
As time passes, mutations accumulate in the genomes of all living organisms. These changes promote genetic diversity, but also precipitate ageing and the initiation of cancer. Food is a common source of mutagens, but little is known about how nutritional factors cause lasting genetic changes in the consuming organism. Here, we describe an unusual genetic interaction between DNA repair in the unicellular amoeba Dictyostelium discoideum and its natural bacterial food source. We found that Dictyostelium deficient in the DNA repair nuclease Xpf (xpf-) display a severe and specific growth defect when feeding on bacteria. Despite being proficient in the phagocytosis and digestion of bacteria, over time, xpf- Dictyostelium feeding on bacteria cease to grow and in many instances die. The Xpf nuclease activity is required for sustained growth using a bacterial food source. Furthermore, the ingestion of this food source leads to a striking accumulation of mutations in the genome of xpf- Dictyostelium This work therefore establishes Dictyostelium as a model genetic system to dissect nutritional genotoxicity, providing insight into how phagocytosis can induce mutagenesis and compromise survival fitness.
Asunto(s)
Dictyostelium/metabolismo , Mutagénesis , Fagocitosis , Proteínas Protozoarias/metabolismo , Secuencia de Aminoácidos , Reparación del ADN/genética , Dictyostelium/citología , Dictyostelium/crecimiento & desarrollo , Fagocitosis/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genéticaRESUMEN
Two motors can drive extension of the leading edge of motile cells: actin polymerization and myosin-driven contraction of the cortex, producing fluid pressure and the formation of blebs. Dictyostelium cells can move with both blebs and actin-driven pseudopods at the same time, and blebs, like pseudopods, can be orientated by chemotactic gradients. Here we ask how bleb sites are selected and how the two forms of projection cooperate. We show that membrane curvature is an important, yet overlooked, factor. Dictyostelium cells were observed moving under agarose, which efficiently induces blebbing, and the dynamics of membrane deformations were analyzed. Blebs preferentially originate from negatively curved regions, generated on the flanks of either extending pseudopods or blebs themselves. This is true of cells at different developmental stages, chemotaxing to either folate or cyclic AMP and moving with both blebs and pseudopods or with blebs only. A physical model of blebbing suggests that detachment of the cell membrane is facilitated in concave areas of the cell, where membrane tension produces an outward directed force, as opposed to pulling inward in convex regions. Our findings assign a role to membrane tension in spatially coupling blebs and pseudopods, thus contributing to clustering protrusions to the cell front.
Asunto(s)
Extensiones de la Superficie Celular/fisiología , Quimiotaxis/fisiología , Seudópodos/fisiología , Actinas/metabolismo , Animales , Fenómenos Biofísicos , Polaridad Celular/fisiología , AMP Cíclico/metabolismo , Dictyostelium/fisiología , Ácido Fólico/metabolismo , Fundulidae , Modelos Biológicos , Proteínas Protozoarias/metabolismoRESUMEN
Class-1 PI3-kinases are major regulators of the actin cytoskeleton, whose precise contributions to chemotaxis, phagocytosis and macropinocytosis remain unresolved. We used systematic genetic ablation to examine this question in growing Dictyostelium cells. Mass spectroscopy shows that a quintuple mutant lacking the entire genomic complement of class-1 PI3-kinases retains only 10% of wild-type PtdIns(3,4,5)P3 levels. Chemotaxis to folate and phagocytosis of bacteria proceed normally in the quintuple mutant but macropinocytosis is abolished. In this context PI3-kinases show specialized functions, only one of which is directly linked to gross PtdIns(3,4,5)P3 levels: macropinosomes originate in patches of PtdIns(3,4,5)P3, with associated F-actin-rich ruffles, both of which depend on PI3-kinase 1/2 (PI3K1/2) but not PI3K4, whereas conversion of ruffles into vesicles requires PI3K4. A biosensor derived from the Ras-binding domain of PI3K1 suggests that Ras is activated throughout vesicle formation. Binding assays show that RasG and RasS interact most strongly with PI3K1/2 and PI3K4, and single mutants of either Ras have severe macropinocytosis defects. Thus, the fundamental function of PI3-kinases in growing Dictyostelium cells is in macropinocytosis where they have two distinct functions, supported by at least two separate Ras proteins.
Asunto(s)
Actinas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Pinocitosis/genética , Actinas/genética , Quimiotaxis , Humanos , Fagocitosis , Fosfatidilinositol 3-Quinasas/genética , Transducción de SeñalRESUMEN
Shwachman-Diamond syndrome (SDS), a recessive leukemia predisposition disorder characterized by bone marrow failure, exocrine pancreatic insufficiency, skeletal abnormalities and poor growth, is caused by mutations in the highly conserved SBDS gene. Here, we test the hypothesis that defective ribosome biogenesis underlies the pathogenesis of SDS. We create conditional mutants in the essential SBDS ortholog of the ancient eukaryote Dictyostelium discoideum using temperature-sensitive, self-splicing inteins, showing that mutant cells fail to grow at the restrictive temperature because ribosomal subunit joining is markedly impaired. Remarkably, wild type human SBDS complements the growth and ribosome assembly defects in mutant Dictyostelium cells, but disease-associated human SBDS variants are defective. SBDS directly interacts with the GTPase elongation factor-like 1 (EFL1) on nascent 60S subunits in vivo and together they catalyze eviction of the ribosome antiassociation factor eukaryotic initiation factor 6 (eIF6), a prerequisite for the translational activation of ribosomes. Importantly, lymphoblasts from SDS patients harbor a striking defect in ribosomal subunit joining whose magnitude is inversely proportional to the level of SBDS protein. These findings in Dictyostelium and SDS patient cells provide compelling support for the hypothesis that SDS is a ribosomopathy caused by corruption of an essential cytoplasmic step in 60S subunit maturation.
Asunto(s)
Enfermedades de la Médula Ósea/genética , Enfermedades de la Médula Ósea/patología , Insuficiencia Pancreática Exocrina/genética , Insuficiencia Pancreática Exocrina/patología , Lipomatosis/genética , Lipomatosis/patología , Proteínas/genética , Ribosomas/patología , Enfermedades de la Médula Ósea/metabolismo , Línea Celular , Dictyostelium/genética , Dictyostelium/metabolismo , Insuficiencia Pancreática Exocrina/metabolismo , GTP Fosfohidrolasas/metabolismo , Humanos , Lipomatosis/metabolismo , Mutación , Factores de Iniciación de Péptidos/metabolismo , Proteínas/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Síndrome de Shwachman-DiamondRESUMEN
Differentiation-inducing factor 1 (DIF-1) is a polyketide-derived morphogen which drives stalk cell formation in the developmental cycle of Dictyostelium discoideum. Previous experiments demonstrated that the biosynthetic pathway proceeds via dichlorination of the precursor molecule THPH, but the enzyme responsible for this transformation has eluded characterization. Our recent studies on prokaryotic flavin-dependent halogenases and insights from the sequenced Dd genome led us to a candidate gene for this transformation. In this work, we present in vivo and in vitro evidence that chlA from Dd encodes a flavin-dependent halogenase capable of catalyzing both chlorinations in the biosynthesis of DIF-1. The results provide in vitro characterization of a eukaryotic oxygen-dependent halogenase and demonstrate a broad reach in biology for this molecular tailoring strategy, notably its involvement in the differentiation program of a social amoeba.
Asunto(s)
Dictyostelium/metabolismo , Hexanonas/metabolismo , Oxidorreductasas/metabolismo , Secuencia de Bases , Cartilla de ADN/genética , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Flavinas/metabolismo , Genes Protozoarios , Halógenos/metabolismo , Hexanonas/química , Mutagénesis Insercional , Oxidorreductasas/genética , FenotipoRESUMEN
Macropinocytosis is a conserved endocytic process by which cells engulf droplets of medium into micron-sized vesicles. We use light-sheet microscopy to define an underlying set of principles by which macropinocytic cups are shaped and closed in Dictyostelium amoebae. Cups form around domains of PIP3 stretching almost to their lip and are supported by a specialized F-actin scaffold from lip to base. They are shaped by a ring of actin polymerization created by recruiting Scar/WAVE and Arp2/3 around PIP3 domains, but how cups evolve over time to close and form a vesicle is unknown. Custom 3D analysis shows that PIP3 domains expand from small origins, capturing new membrane into the cup, and crucially, that cups close when domain expansion stalls. We show that cups can close in two ways: either at the lip, by inwardly directed actin polymerization, or the base, by stretching and delamination of the membrane. This provides the basis for a conceptual mechanism whereby closure is brought about by a combination of stalled cup expansion, continued actin polymerization at the lip, and membrane tension. We test this through the use of a biophysical model, which can recapitulate both forms of cup closure and explain how 3D cup structures evolve over time to mediate engulfment.
Asunto(s)
Actinas , Dictyostelium , Estructuras de la Membrana Celular , Citoesqueleto de Actina , EndocitosisRESUMEN
We investigated the link between cell movement and plasma membrane recycling using a fast-acting, temperature-sensitive mutant of the Dictyostelium SecA exocytic protein. Strikingly, most mutant cells become almost paralysed within minutes at the restrictive temperature. However, they can still sense cyclic-AMP (cAMP) gradients and polymerise actin up-gradient, but form only abortive pseudopodia, which cannot expand. They also relay a cAMP signal normally, suggesting that cAMP is released by a non-exocytic mechanism. To investigate why SecA is required for motility, we examined membrane trafficking in the mutant. Plasma membrane circulation is rapidly inhibited at the restrictive temperature and the cells acquire a prominent vesicle. Organelle-specific markers show that this is an undischarged contractile vacuole, and we found the cells are correspondingly osmo-sensitive. Electron microscopy shows that many smaller vesicles, probably originating from the plasma membrane, also accumulate at the restrictive temperature. Consistent with this, the surface area of mutant cells shrinks. We suggest that SecA mutant cells cannot move at the restrictive temperature because their block in exocytosis results in a net uptake of plasma membrane, reducing its area, and so restricting pseudopodial expansion. This demonstrates the importance of proper surface area regulation in cell movement.
Asunto(s)
Movimiento Celular , Dictyostelium/fisiología , Proteínas Mutantes/metabolismo , Ósmosis , Proteínas Protozoarias/metabolismo , Actinas/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Movimiento Celular/genética , AMP Cíclico/metabolismo , Exocitosis/genética , Microscopía Electrónica , Proteínas Munc18/genética , Proteínas Munc18/metabolismo , Proteínas Mutantes/genética , Transporte de Proteínas/genética , Proteínas Protozoarias/genética , Seudópodos/genética , TemperaturaRESUMEN
The mature fruiting body of Dictyostelium consists of stalk and spore cells but its construction, and the migration of the preceding slug stage, requires a number of specialized sub-types of prestalk cell whose nature and function are not well understood. The prototypic prestalk-specific gene, ecmA, is inducible by the polyketide DIF-1 in a monolayer assay and requires the DimB and MybE transcription factors for full inducibility. We perform genome-wide microarray analyses, on parental, mybE- and dimB- cells, and identify many additional genes that depend on MybE and DimB for their DIF-1 inducibility. Surprisingly, an even larger number of genes are only DIF inducible in mybE- cells, some genes are only inducible in DimB- cells and some are inducible when either transcription factor is absent. Thus in assay conditions where MybE and DimB function as inducers of ecmA these genes fall under negative control by the same two transcription factors. We have studied in detail rtaA, one of the MybE and DimB repressed genes. One especially enigmatic group of prestalk cells is the anterior-like cells (ALCs), which exist intermingled with prespore cells in the slug. A promoter fusion reporter gene, rtaA:gal(u), is expressed in a subset of the ALCs that is distinct from the ALC population detected by a reporter construct containing ecmA and ecmB promoter fragments. At culmination, when the ALC sort out from the prespore cells and differentiate to form three ancillary stalk cell structures: the upper cup, the lower cup and the outer basal disk, the rtaA:gal(u) expressing cells preferentially populate the upper cup region. This fact, and their virtual absence from the anterior and posterior regions of the slug, identifies them as a new prestalk sub-type: the pstU cells. PstU cell differentiation is, as expected, increased in a dimB- mutant during normal development but, surprisingly, they differentiate normally in a mutant lacking DIF. Thus genetic removal of MybE or DimB reveals an alternate DIF-1 activation pathway, for pstU differentiation, that functions under monolayer assay conditions but that is not essential during multicellular development.
Asunto(s)
Dictyostelium/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Proteínas Protozoarias/genética , Diferenciación Celular , Dictyostelium/citología , Mutación , Regiones Promotoras Genéticas , Proteínas Protozoarias/metabolismoRESUMEN
Macropinocytosis is a form of endocytosis performed by ruffles and cups of the plasma membrane. These close to entrap droplets of medium into micron-sized vesicles, which are trafficked through the endocytic system, their contents digested and useful products absorbed. Macropinocytosis is constitutive in certain immune cells and stimulated in many other cells by growth factors. It occurs across the animal kingdom and in amoebae, implying a deep evolutionary history. Its scientific history goes back 100 years, but increasingly work is focused on its medical importance in the immune system, cancer cell feeding, and as a backdoor into cells for viruses and drugs. Macropinocytosis is driven by the actin cytoskeleton whose dynamics can be appreciated with lattice light sheet microscopy: this reveals a surprising variety of routes for forming macropinosomes. In Dictyostelium amoebae, macropinocytic cups are organized around domains of PIP3 and active Ras and Rac in the plasma membrane. These attract activators of the Arp2/3 complex to their periphery, creating rings of actin polymerization that shape the cups. The size of PIP3 domains is controlled by RasGAPs, such as NF1, and the lipid phosphatase, PTEN. It is likely that domain dynamics determine the shape, evolution and closing of macropinocytic structures.
Asunto(s)
Amoeba , Dictyostelium , Citoesqueleto de Actina/metabolismo , Animales , Biología , Dictyostelium/metabolismo , Endocitosis , PinocitosisRESUMEN
Chemotaxing neutrophils and Dictyostelium amoebae produce in their plasma membranes the signaling lipid PI(3,4,5)P3 (PIP3) in gradients, which are orientated with the external chemotactic gradient and have been proposed to act as an internal compass, guiding movement of the cell. Evidence for and against this idea exists, but in all cases it depends on the use of inhibitors or gene knockouts, which may only incompletely abolish the PIP3 gradient. We have created a multiple gene-knockout strain in Dictyostelium lacking all five type-1 phosphoinositide 3-kinases encoded in the genome and the PTEN phosphatase and have thus removed all known ways for chemoattractant to produce PIP3 gradients in the plasma membrane. The resulting sextuple mutant is able to chemotax to cyclic-AMP with near wild-type efficiency and to trigger actin polymerization without apparent defect. There is, however, a consistent defect in movement speed in chemotaxis and especially in random movement. This work shows that polarization of membrane PIP3 is not necessary for accurate chemotaxis, but it can affect cell speed. A signaling pathway from receptor to cytoskeleton able to guide cells independently of polarized PIP3 and type-1 phosphoinositide 3-kinases must exist.