RESUMEN
Cell-cell interaction mediated by secreted and adhesive signaling molecules forms the basis of the coordinated cell movements (i.e., collective cell migration) observed in developing embryos, regenerating tissues, immune cells, and metastatic cancer. Decoding the underlying input/output rules at the single-cell level, however, remains a challenge due to the vast complexity in the extracellular environments that support such cellular behaviors. The amoebozoa Dictyostelium discoideum uses GPCR-mediated chemotaxis and cell-cell contact signals mediated by adhesion proteins with immunoglobulin-like folds to form a collectively migrating slug. Coordinated migration and repositioning of the cells in this relatively simple morphogenetic system are driven strictly by regulation of actin cytoskeleton by these signaling factors. Its unique position in the eukaryotic tree of life outside metazoa points to basic logics of tissue self-organization that are common across taxa. Here, we describe a method to reconstitute intercellular contact signals and the resulting cell polarization using purified adhesion proteins. In addition, a protocol using a microfluidic chamber is laid out where one can study how the cell-cell contact signal and chemoattractant signals, when simultaneously presented, are interpreted. Quantitative image analysis for obtaining cell morphology features is also provided. A similar approach should be applicable to study other collectively migrating cells.
Asunto(s)
Comunicación Celular , Movimiento Celular , Quimiotaxis , Dictyostelium , Dictyostelium/fisiología , Dictyostelium/citología , Adhesión Celular , Transducción de Señal , Polaridad CelularRESUMEN
Collective cell migration occurs when the orientation of cell polarity is aligned with each other in a group of cells. Such collective polarization depends on a reciprocal process between cell intrinsic mechanisms such as cell-cell adhesion and extracellular guidance mechanism such as wound healing and chemotaxis. As part of its development life cycle, individual single cells of Dictyostelium discoideum exhibit chemotaxis toward cAMP, which is secreted from a certain population of cells. During the formation of multicellular body by chemotaxis-dependent cell aggregation, D. discoideum is also known to relay on multiple cell-cell adhesion mechanisms. In particular, tail-following behavior at the contact site, called contact following of locomotion (CFL), plays a pivotal role on the formation of the multicellular body. However, whether and how CFL alone can lead to a formation of collective behavior was not well understood. KI cell is a mutant of D. discoideum that lacks all chemotactic activity. Yet, it can exhibit the CFL activity and show nontrivial collective cell migration. This mutant provides an excellent model system to analyze the mechanism of the CFL and the macroscopic phenomena brought by the CFL. This chapter describes protocols for using KI cell to understand the biophysics and cell biology behind the collective cell migration induced by CFL.
Asunto(s)
Movimiento Celular , Quimiotaxis , Dictyostelium , Dictyostelium/genética , Dictyostelium/fisiología , Dictyostelium/citología , Quimiotaxis/genética , Movimiento Celular/genética , Mutación , AMP Cíclico/metabolismo , Polaridad Celular/genética , Adhesión Celular , Modelos BiológicosRESUMEN
Living cells have the ability to detect electric fields and respond to them with directed migratory movements. Many proteomic approaches have been adopted in the past to identify the molecular mechanism behind this cellular phenomenon. However, how the cells sense the electric stimulus and transduce it into directed cell migration is still under discussion. Many eukaryotic cells react to applied electric stimulation, including Dictyostelium discoideum cells. We use them as model system for studying cell migration in electric fields, also known as electrotaxis. Here we report the protocols that we developed for our experiments. Our experimental outcomes helped us to characterize: (i) the memory that cells have in a varying electric field, which we defined as temporal electric persistence; and (ii) the accelerating motion of cells along their paths over the electric exposure time. We also report on the analysis of the role that conditioned medium factor (CMF), a protein secreted by cells when they begin to starve, plays in the mechanism of electric sensing. The results of this study can contribute to the understanding of the electrical sensing of cells and its transduction into directed cell migration.
Asunto(s)
Movimiento Celular , Dictyostelium , Dictyostelium/fisiología , Dictyostelium/metabolismo , Dictyostelium/citología , Electricidad , Estimulación Eléctrica , Taxia/fisiología , Medios de Cultivo CondicionadosRESUMEN
Normal-sized cells of Dictyostelium build up a front-tail polarity when they respond to a gradient of chemoattractant. To challenge the polarity-generating system, cells are fused to study the chemotactic response of oversized cells that extend multiple fronts toward the source of attractant. An aspect that can be explored in these cells is the relationship of spontaneously generated actin waves to actin reorganization in response to chemoattractant.
Asunto(s)
Quimiotaxis , Dictyostelium , Dictyostelium/fisiología , Dictyostelium/citología , Factores Quimiotácticos/farmacología , Factores Quimiotácticos/metabolismo , Actinas/metabolismo , Fusión Celular/métodos , Células Gigantes/citología , Células Gigantes/metabolismo , Polaridad CelularRESUMEN
Amoeboid cells such as the protist Dictyostelium, human neutrophils, and the fungus B.d. chytrid move by extending pseudopods. The trajectories of cell movement depend on the size, rhythm, and direction of long series of pseudopods. These pseudopod properties are regulated by internal factors such as memory of previous directions and by external factors such as gradients of chemoattractants or electric currents. Here a simple method is described that defines the X, Y time coordinates of a pseudopod at the start and the end of the extension phase. The connection between the start and end of an extending pseudopod defines a vector, which is the input of different levels of analysis that defines cell movement. The primary information of the vector is its spatial length (pseudopod size), temporal length (extension time), extension rate (size divided by time), and direction. The second layer of information describes the sequence of two (or more) pseudopods: the direction of the second pseudopod relative to the direction of the first pseudopod, the start of the second pseudopod relative to the extension phase of the first pseudopod (the second starts while the first is still extending or after the first has stopped), and the alternating right/left extension of pseudopods. The third layer of information is provided by specific and detailed statistical analysis of these data and addresses question such as: is pseudopod extension in buffer in random direction or has the system internal directional memory, and how do shallow external electrical or chemical gradients bias the intrinsic pseudopod extension. The method is described for Dictyostelium, but has been used successfully for fast-moving neutrophils, slow-moving stem cells, and the fungus B.d. chytrid.
Asunto(s)
Quimiotaxis , Dictyostelium , Quimiotaxis/fisiología , Dictyostelium/fisiología , Dictyostelium/citología , Seudópodos/fisiología , Movimiento Celular/fisiología , Humanos , Tampones (Química) , Neutrófilos/citología , Neutrófilos/fisiologíaRESUMEN
Amoeboid cell motility is fundamental for a multitude of biological processes such as embryogenesis, immune responses, wound healing, and cancer metastasis. It is characterized by specific cell shape changes: the extension and retraction of membrane protrusions, known as pseudopodia. A common approach to investigate the mechanisms underlying this type of cell motility is to study phenotypic differences in the locomotion of mutant cell lines. To characterize such differences, methods are required to quantify the contour dynamics of migrating cells. AmoePy is a Python-based software package that provides tools for cell segmentation, contour detection as well as analyzing and simulating contour dynamics. First, a digital representation of the cell contour as a chain of nodes is extracted from each frame of a time-lapse microscopy recording of a moving cell. Then, the dynamics of these nodes-referred to as virtual markers-are tracked as the cell contour evolves over time. From these data, various quantities can be calculated that characterize the contour dynamics, such as the displacement of the virtual markers or the local stretching rate of the marker chain. Their dynamics is typically visualized in space-time plots, the so-called kymographs, where the temporal evolution is displayed for the different locations along the cell contour. Using AmoePy, you can straightforwardly create kymograph plots and videos from stacks of experimental bright-field or fluorescent images of motile cells. A hands-on guide on how to install and use AmoePy is provided in this chapter.
Asunto(s)
Movimiento Celular , Programas Informáticos , Procesamiento de Imagen Asistido por Computador/métodos , Imagen de Lapso de Tiempo/métodos , Quimografía/métodos , Dictyostelium/citología , Dictyostelium/fisiología , Dictyostelium/crecimiento & desarrollo , SeudópodosRESUMEN
Cell-to-cell communication is fundamental to the organization and functionality of multicellular organisms. Intercellular signals orchestrate a variety of cellular responses, including gene expression and protein function changes, and contribute to the integrated functions of individual tissues. Dictyostelium discoideum is a model organism for cell-to-cell interactions mediated by chemical signals and multicellular formation mechanisms. Upon starvation, D. discoideum cells exhibit coordinated cell aggregation via cyclic adenosine 3',5'-monophosphate (cAMP) gradients and chemotaxis, which facilitates the unicellular-to-multicellular transition. During this process, the calcium signaling synchronizes with the cAMP signaling. The resulting multicellular body exhibits organized collective migration and ultimately forms a fruiting body. Various signaling molecules, such as ion signals, regulate the spatiotemporal differentiation patterns within multicellular bodies. Understanding cell-to-cell and ion signaling in Dictyostelium provides insight into general multicellular formation and differentiation processes. Exploring cell-to-cell and ion signaling enhances our understanding of the fundamental biological processes related to cell communication, coordination, and differentiation, with wide-ranging implications for developmental biology, evolutionary biology, biomedical research, and synthetic biology. In this review, I discuss the role of ion signaling in cell motility and development in D. discoideum.
Asunto(s)
Movimiento Celular , AMP Cíclico , Dictyostelium , Transducción de Señal , Dictyostelium/metabolismo , Dictyostelium/crecimiento & desarrollo , Dictyostelium/genética , Dictyostelium/citología , AMP Cíclico/metabolismo , Quimiotaxis , Comunicación Celular , Iones/metabolismo , Diferenciación Celular , Señalización del CalcioRESUMEN
The social amoeba Dictyostelium discoideum is a versatile model for understanding many different cellular processes involving cell motility including chemotaxis, phagocytosis, and cytokinesis. Cytokinesis, in particular, is a model cell-shaped change process in which a cell separates into two daughter cells. D. discoideum has been used extensively to identify players in cytokinesis and understand how they comprise the mechanosensory and biochemical pathways of cytokinesis. In this chapter, we describe how we use cDNA library complementation with D. discoideum to discover potential regulators of cytokinesis. Once identified, these regulators are further analyzed through live cell imaging, immunofluorescence imaging, fluorescence correlation and cross-correlation spectroscopy, micropipette aspiration, and fluorescence recovery after photobleaching. Collectively, these methods aid in detailing the mechanisms and signaling pathways that comprise cell division.
Asunto(s)
Citocinesis , Dictyostelium , Dictyostelium/metabolismo , Dictyostelium/genética , Dictyostelium/citología , Biblioteca de Genes , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Transducción de Señal , Recuperación de Fluorescencia tras Fotoblanqueo/métodosRESUMEN
Over the last decade, the use of microfabricated substrates has proven pivotal for studying the effect of substrate topography on cell deformation and migration. Microfabrication techniques allow one to construct a transparent substrate with topographic features with high designability and reproducibility and thus well suited to experiments that microscopically address how spatial and directional bias are brought about in the cytoskeletal machineries and hence cell motility. While much of the progress in this avenue of study has so far been made in adhesive cells of epithelial and mesenchymal nature, whether related phenomena exist in less adhesive fast migrating cells is relatively unknown. In this chapter, we describe a method that makes use of micrometer-scale ridges to study fast-migrating Dictyostelium cells where it was recently shown that membrane evagination associated with macropinocytic cup formation plays a pivotal role in the topography sensing. The method requires only basic photolithography, and thus the step-by-step protocol should be a good entry point for cell biologists looking to incorporate similar microfabrication approaches.
Asunto(s)
Movimiento Celular , Dictyostelium , Microtecnología , Dictyostelium/citología , Dictyostelium/fisiología , Microtecnología/métodos , Adhesión CelularRESUMEN
Macropinocytosis mediates the non-selective bulk uptake of extracellular fluid, enabling cells to survey the environment and obtain nutrients. A conserved set of signaling proteins orchestrates the actin dynamics that lead to membrane ruffling and macropinosome formation across various eukaryotic organisms. At the center of this signaling network are Ras GTPases, whose activation potently stimulates macropinocytosis. However, how Ras signaling is initiated and spatiotemporally regulated during macropinocytosis is not well understood. By using the model system Dictyostelium and a proteomics-based approach to identify regulators of macropinocytosis, we uncovered Leep2, consisting of Leep2A and Leep2B, as a RasGAP complex. The Leep2 complex specifically localizes to emerging macropinocytic cups and nascent macropinosomes, where it modulates macropinosome formation by regulating the activities of three Ras family small GTPases. Deletion or overexpression of the complex, as well as disruption or sustained activation of the target Ras GTPases, impairs macropinocytic activity. Our data reveal the critical role of fine-tuning Ras activity in directing macropinosome formation.
Asunto(s)
Dictyostelium , Pinocitosis , Proteínas Activadoras de ras GTPasa , Dictyostelium/citología , Dictyostelium/metabolismo , Proteínas Protozoarias/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas ras/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND INFORMATION: Two pore channels (TPCs) are voltage-gated ion channel superfamily members that release Ca2+ from acidic intracellular stores and are ubiquitously present in both animals and plants. Starvation initiates multicellular development in Dictyostelium discoideum. Increased intracellular calcium levels bias Dictyostelium cells towards the stalk pathway and thus we decided to analyze the role of TPC2 in development, differentiation, and autophagy. RESULTS: We showed TPC2 protein localizes in lysosome-like acidic vesicles and the in situ data showed stalk cell biasness. Deletion of tpc2 showed defective and delayed development with formation of multi-tipped structures attached to a common base, while tpc2OE cells showed faster development with numerous small-sized aggregates and wiry fruiting bodies. The tpc2OE cells showed higher intracellular cAMP levels as compared to the tpc2- cells while pinocytosis was found to be higher in the tpc2- cells. Also, TPC2 regulates cell-substrate adhesion and cellular morphology. Under nutrient starvation, deletion of tpc2 reduced autophagic flux as compared to Ax2. During chimera formation, tpc2- cells showed a bias towards the prestalk/stalk region while tpc2OE cells showed a bias towards the prespore/spore region. tpc2 deficient strain exhibits aberrant cell-type patterning and loss of distinct boundary between the prestalk/prespore regions. CONCLUSION: TPC2 is required for effective development and differentiation in Dictyostelium and supports autophagic cell death and cell-type patterning. SIGNIFICANCE: Decreased calcium due to deletion of tpc2 inhibit autophagic flux.
Asunto(s)
Autofagia , Dictyostelium , Proteínas Protozoarias , Dictyostelium/genética , Dictyostelium/metabolismo , Dictyostelium/citología , Dictyostelium/crecimiento & desarrollo , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Eliminación de Gen , Canales de Calcio/metabolismo , Canales de Calcio/genética , Calcio/metabolismo , Diferenciación CelularRESUMEN
Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) is a signaling lipid on the plasma membrane that plays a fundamental role in cell signaling with a strong impact on cell physiology and diseases. It is responsible for the protruding edge formation, cell polarization, macropinocytosis, and other membrane remodeling dynamics in cells. It has been shown that the membrane confinement and curvature affects the wave formation of PIP3 and F-actin. But, even in the absence of F-actin, a complex self-organization of the spatiotemporal PIP3 waves is observed. In recent findings, we have shown that these waves can be guided and pinned on strongly bended Dictyostelium membranes caused by molecular crowding and curvature-limited diffusion. Based on these experimental findings, we investigate the spatiotemporal PIP3 wave dynamics on realistic three-dimensional cell-like membranes to explore the effect of curvature-limited diffusion, as observed experimentally. We use an established stochastic reaction-diffusion model with enzymatic Michaelis-Menten-type reactions that mimics the dynamics of Dictyostelium cells. As these cells mimic the three-dimensional shape and size observed experimentally, we found that the PIP3 wave directionality can be explained by a Hopf-like and a reverse periodic-doubling bifurcation for uniform diffusion and curvature-limited diffusion properties. Finally, we compare the results with recent experimental findings and discuss the discrepancy between the biological and numerical results.
Asunto(s)
Membrana Celular , Dictyostelium , Modelos Biológicos , Fosfatos de Fosfatidilinositol , Membrana Celular/metabolismo , Dictyostelium/citología , Dictyostelium/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , DifusiónRESUMEN
It has been experimentally reported that chemotactic cells exhibit cellular memory, that is, a tendency to maintain the migration direction despite changes in the chemoattractant gradient. In this study, we analyzed a phenomenological model assuming the presence of cellular inertia, as well as a response time in motility, resulting in the reproduction of the cellular memory observed in the previous experiments. According to the analysis, the cellular motion is described by the superposition of multiple oscillative functions induced by the multiplication of the oscillative polarity and motility. The cellular intertia generates cellular memory by regulating phase differences between those oscillative functions. By applying the theory to the experimental data, the cellular inertia was estimated at [Formula: see text] min. In addition, physiological parameters, such as response time in motility and intracellular processing speed, were also evaluated. The agreement between the experiemental data and theory suggests the possibility of the presence of the response time in motility, which has never been biologically verified and should be explored in the future.
Asunto(s)
Fenómenos Fisiológicos Celulares , Quimiotaxis/fisiología , Modelos Biológicos , Algoritmos , Animales , Dictyostelium/citología , HumanosRESUMEN
Ketogenic diets, used in epilepsy treatment, are considered to work through reduced glucose and ketone generation to regulate a range of cellular process including autophagy induction. Recent studies into the medium-chain triglyceride (MCT) ketogenic diet have suggested that medium-chain fatty acids (MCFAs) provided in the diet, decanoic acid and octanoic acid, cause specific therapeutic effects independent of glucose reduction, although a role in autophagy has not been investigated. Both autophagy and MCFAs have been widely studied in Dictyostelium, with findings providing important advances in the study of autophagy-related pathologies such as neurodegenerative diseases. Here, we utilize this model to analyze a role for MCFAs in regulating autophagy. We show that treatment with decanoic acid but not octanoic acid induces autophagosome formation and modulates autophagic flux in high glucose conditions. To investigate this effect, decanoic acid, but not octanoic acid, was found to induce the expression of autophagy-inducing proteins (Atg1 and Atg8), providing a mechanism for this effect. Finally, we demonstrate a range of related fatty acid derivatives with seizure control activity, 4BCCA, 4EOA, and Epilim (valproic acid), also function to induce autophagosome formation in this model. Thus, our data suggest that decanoic acid and related compounds may provide a less-restrictive therapeutic approach to activate autophagy.
Asunto(s)
Autofagia , Ácidos Decanoicos/farmacología , Dictyostelium/citología , Autofagosomas/efectos de los fármacos , Autofagosomas/metabolismo , Autofagia/efectos de los fármacos , Dictyostelium/efectos de los fármacos , Dictyostelium/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
The social amoeba Dictyostelium discoideum provides an excellent model for research across a broad range of disciplines within biology. The organism diverged from the plant, yeast, fungi and animal kingdoms around 1 billion years ago but retains common aspects found in these kingdoms. Dictyostelium has a low level of genetic complexity and provides a range of molecular, cellular, biochemical and developmental biology experimental techniques, enabling multidisciplinary studies to be carried out in a wide range of areas, leading to research breakthroughs. Numerous laboratories within the United Kingdom employ Dictyostelium as their core research model. This review introduces Dictyostelium and then highlights research from several leading British research laboratories, covering their distinct areas of research, the benefits of using the model, and the breakthroughs that have arisen due to the use of Dictyostelium as a tractable model system.
Asunto(s)
Biología , Dictyostelium/fisiología , Modelos Biológicos , Investigación , Animales , Dictyostelium/citología , Descubrimiento de Drogas , Procesamiento Proteico-Postraduccional , Reino UnidoRESUMEN
Defects in mitochondrial dynamics, fission, fusion, and motility have been implicated in the pathogenesis of multiple neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Huntington's disease, and Charcot-Marie-Tooth disease. Another key feature of neurodegeneration is the increase in reactive oxygen species (ROS). Previous work has shown that the cytoskeleton, in particular the microtubules, and ROS generated by rotenone significantly regulate mitochondrial dynamics in Dictyostelium discoideum. The goal of this project is to study the effects of ROS on mitochondrial dynamics within our model organism D. discoideum to further understand the underlying issues that are the root of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. We chose three likely ROS inducers, cumene hydroperoxide, hydroxylamine hydrochloride, and Antimycin A. Our work demonstrates that alteration of the microtubule cytoskeleton is not required to alter dynamics in response to ROS and there is no easy way to predict how mitochondrial dynamics will be altered based on which ROS generator is used. This research contributes to the better understanding of the cellular mechanisms that induce the pathogenesis of incurable neurodegenerative diseases with the hope that it will translate into developing new and more effective treatments for patients afflicted by them.
Asunto(s)
Citoesqueleto/metabolismo , Dictyostelium/metabolismo , Microtúbulos/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Especies Reactivas de Oxígeno/metabolismo , Enfermedad de Alzheimer/metabolismo , Antimicina A/farmacología , Derivados del Benceno/farmacología , Enfermedad de Charcot-Marie-Tooth/metabolismo , Citoesqueleto/efectos de los fármacos , Dictyostelium/citología , Dictyostelium/efectos de los fármacos , Humanos , Enfermedad de Huntington/metabolismo , Hidroxilamina/farmacología , Microtúbulos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Modelos Biológicos , Enfermedad de Parkinson/metabolismoRESUMEN
Navigation of fast migrating cells such as amoeba Dictyostelium and immune cells are tightly associated with their morphologies that range from steady polarized forms that support high directionality to those more complex and variable when making frequent turns. Model simulations are essential for quantitative understanding of these features and their origins, however systematic comparisons with real data are underdeveloped. Here, by employing deep-learning-based feature extraction combined with phase-field modeling framework, we show that a low dimensional feature space for 2D migrating cell morphologies obtained from the shape stereotype of keratocytes, Dictyostelium and neutrophils can be fully mapped by an interlinked signaling network of cell-polarization and protrusion dynamics. Our analysis links the data-driven shape analysis to the underlying causalities by identifying key parameters critical for migratory morphologies both normal and aberrant under genetic and pharmacological perturbations. The results underscore the importance of deciphering self-organizing states and their interplay when characterizing morphological phenotypes.
Asunto(s)
Movimiento Celular/fisiología , Aprendizaje Profundo , Modelos Biológicos , Animales , Polaridad Celular/fisiología , Forma de la Célula/fisiología , Extensiones de la Superficie Celular/fisiología , Células Cultivadas , Cíclidos , Biología Computacional , Simulación por Computador , Dictyostelium/citología , Dictyostelium/fisiología , Fibroblastos/citología , Fibroblastos/fisiología , Células HL-60 , HumanosRESUMEN
BACKGROUND: The evolution of multicellularity is a critical event that remains incompletely understood. We use the social amoeba, Dictyostelium discoideum, one of the rare organisms that readily transits back and forth between both unicellular and multicellular stages, to examine the role of epigenetics in regulating multicellularity. RESULTS: While transitioning to multicellular states, patterns of H3K4 methylation and H3K27 acetylation significantly change. By combining transcriptomics, epigenomics, chromatin accessibility, and orthologous gene analyses with other unicellular and multicellular organisms, we identify 52 conserved genes, which are specifically accessible and expressed during multicellular states. We validated that four of these genes, including the H3K27 deacetylase hdaD, are necessary and that an SMC-like gene, smcl1, is sufficient for multicellularity in Dictyostelium. CONCLUSIONS: These results highlight the importance of epigenetics in reorganizing chromatin architecture to facilitate multicellularity in Dictyostelium discoideum and raise exciting possibilities about the role of epigenetics in the evolution of multicellularity more broadly.
Asunto(s)
Dictyostelium/citología , Dictyostelium/genética , Epigénesis Genética , Acetilación , Animales , Caenorhabditis elegans/citología , Cromatina/metabolismo , Perfilación de la Expresión Génica , Histonas/metabolismo , Metilación , Schizosaccharomyces/citología , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Dictyostelium/efectos de los fármacos , Dictyostelium/genética , Mutación , Fosfatidilserinas/farmacología , Adhesión Celular/efectos de los fármacos , Membrana Celular/metabolismo , Dictyostelium/citología , Proteínas Protozoarias/genéticaRESUMEN
The ability of cells to polarize and move toward external stimuli plays a crucial role in development, as well as in normal and pathological physiology. Migrating cells maintain dynamic complementary distributions of Ras activity and of the phospholipid phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). Here, we show that lagging-edge component PI(3,4)P2 also localizes to retracting leading-edge protrusions and nascent macropinosomes, even in the absence of phosphatidylinositol 3,4,5-trisphosphate (PIP3). Once internalized, macropinosomes break up into smaller PI(3,4)P2-enriched vesicles, which fuse with the plasma membrane at the rear of the cell. Subsequently, the phosphoinositide diffuses toward the front of the cell, where it is degraded. Computational modeling confirms that this cycle gives rise to stable back-to-front gradient. These results uncover a surprising "reverse-fountain flow" of PI(3,4)P2 that regulates polarity.