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Mechanism of Superoxide Mediated Regulation of Particle Uptake and Exocytosis by a GPI-anchored Superoxide Dismutase C in DictyosteliumPulido, Maria 27 June 2014 (has links)
Dictyostelium discoideum is a simple model organism that can be used to study endocytic pathways such as phagocytosis and macropinocytosis because of its homology to cells of the mammalian innate immune system, namely macrophages and neutrophils. Consequently, Dictyostelium can also be used to study the process of exocytosis. In our laboratory, we generated Dictyostelium cells lacking superoxide dismutase SodC. Our data suggest that cells that lack SodC are defective in macropinocytosis and exocytosis when compared to wild type cells.
In this study I describe a regulatory mechanism of macropinocytosis by SodC via regulation of RasG, which in turn controls PI3K activation and thus macropinocytosis. Our results show that proper metabolism of superoxide is critical for efficient particle uptake, for the proper trafficking of internalized particles, and a timely exocytosis of fluid uptake in Dictyostelium cells.
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Bleb-driven chemotaxis in Dictyostelium discoideumZatulovskiy, Evgeny January 2013 (has links)
Migrating cells have two basic ways of extending their leading edge: by dendritic actin polymerization beneath the membrane, or by fluid pressure, which produces blebs. Most cells are believed to move using actin-driven projections, but in more physiological conditions, blebbing motility is also apparent. It has been shown that certain cells even can switch between these two modes of motility, although it is not known how this switch is triggered. Besides, it is unclear whether blebbing can be regulated by chemotactic stimuli, and generally, how blebbing is controlled in the cell. In this study I employed a popular model organism – Dictyostelium discoideum – to investigate the role of blebbing in chemotaxis. Here I confirm that in standard conditions Dictyostelium cells move by a combination of F-actin-driven protrusions and blebs. Blebbing is characterized by the rapid projection of hemispherical patches of plasma membrane at 2-4 times the speed of an actin-driven projection, and leaves transient scars of F-actin marking the original cortex in the base of blebs. I demonstrate that Dictyostelium cells can adjust their mode of movement according to the conditions: in a resistive environment they switch almost entirely to “bleb mode”. I show that in chemotaxing cells, blebs are mainly restricted to the leading edge, and they often lead the way when a cell is forced to re-orientate. Bleb location appears to be controlled directly by chemotactic gradients. To investigate how chemoattractant induces blebbing, I have screened signal transduction mutants for altered blebbing. I have found that blebbing is unaffected in many chemotactic mutants, but unexpectedly depends on PI3-kinases and two downstream PIP3-binding proteins of unknown function – PhdA and CRAC. I conclude that Dictyostelium cells move using a hybrid motor in which hydrostatic pressure-driven bleb formation is as important as F-actin-driven membrane extension, and that cells can change the balance between modes as required. I propose that blebbing motility of Dictyostelium cells is a direct response to mechanical resistance of environment. More generally, bleb-driven motility may be a ‘”high-force” mode of movement that is suited to penetrating tissues. Blebs are chemotactic and their induction may involve branches of the chemotactic signal transduction pathway distinct from F-actin regulation.
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Developmental regulation of alkaline phosphatase in Dictyostelium discoideumMohandas, Devaki Velayudhan January 1983 (has links)
The membrane bound alkaline phosphatase activity' in vegetative cells of Dictydstelium discdideum was found to exhibit a 8 to 10 fold increase in specific activity when incubated at 50°C. This activation was reversed on transfer of the preparation to 0°C. Similar activation of the vegetative enzyme was achieved by dialysing the crude membrane preparation, suggesting the removal of a low molecular weight inhibitor. Alkaline phosphatase solubilized from the membranes using Triton X-100 was similarly activated by 50°C treatment or dialysis and the 50°C activation was reversed by incubation at 0°C. Both the dialysed vegetative membrane and the dialysed Triton X-100 extract were inhibited by the addition of concentrated dialysate. This inhibition could be relieved by subsequent dialysis.
A 620 fold purified alkaline phosphatase preparation was obtained from crude vegetative membranes by affinity and ion exchange chromatography after solubilization of the enzyme using Triton X-100-5'-nucleotidase activity copurified along with the alkaline phosphatase activity in all the fractionation steps employed.
The membrane bound 5'-nucleotidase activity was not activated either by incubation at 50°C or by dialysis and the activity was far less stable than the alkaline phosphatase. However, the Triton X-100
extracted 5'-nucleotidase was activated by dialysis to the same extent as the alkaline phosphatase and both activities in the partially purified preparation were equally inhibited by the dialysates from vegetative membranes. These results suggest that both alkaline phosphatase and 5'-nucleotidase are due to a single protein but the interaction of the two substrates, AMP and pNPP, with the enzyme are different and are markedly influenced by conformational changes induced by the inhibitor.
Unlike the vegetative enzyme, the alkaline phosphatase activity in the culminating membrane was not markedly activated by incubation at 50°C or by dialysis. Since the vegetative enzyme was activated by both treatments to levels similar to those found in culminating cells, it was proposed that the developmental increase in alkaline phosphatase in D.discoideum was due to the unmasking of already existing enzyme by the removal of inhibitor.
However, the alkaline phosphatase activity of culminating cells differed from that of vegetative cells in chromatography on DEAE-Sephacel and conA-Sepharose and it was less stable in low concentrations of Tris-Cl and in SDS. In contrast, the culminating enzyme was more stable in high concentrations of Tris-Cl. These results suggest that the vegetative enzyme is modified during development. This modification appears to be slight, since the vegetative and culminating enzymes migrate identically in SDS polyacrylamide electrophoretic gels and the enzymes from the two
developmental stages were similar in pH optima, in inhibition by phosphate and in inhibition by concentrated dialysate. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate
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The developmental expression of the Dictyostelium discoideum ras gene and preliminary detection of a second ras-homologous sequence in its genomeGray, Virginia Elaine January 1987 (has links)
The expression of a mammalian ras gene analog was previously found by Reymond et al. to be developmentally regulated in Dictyostelium discoideum using Northern analysis of strain AX-3 RNA (1984, Cell 39;141) and by Pawson et al. using specific immunoprecipitation of in vivo synthesized proteins from strain V12M2 (1985, Mol. Cell Biol. 5;33). Due to differences in the results of the two studies, it was decided to further examine ras expression by applying both protein and RNA techniques to a single strain of D.discoideum, V12M2. RNA samples from strain V12M2 cells at different stages of development were analyzed using Northern blotting. The same RNAs were translated in vitro, and the ras proteins synthesized were immunoprecipitated and analysed by polyacrylamide gel electrophoresis.
In agreement with the findings of Reymond et al. (1984, Cell 39;141), Northern analysis with the cDNA ras probe revealed that the highest levels of the 1.2 and 0.9 kb ras mRNAs were present in the total RNA of V12M2 cells at the pseudoplasmodial stage of development, and very little ras mRNA was present in early developing cells.
In contrast to the Northern analysis the greatest amount of ras protein was in vitro translated from the RNA of vegetative and 2 hour cells. Hence this work confirms in a single strain of Dictyostelium that the greatest amount of ras protein is synthesized at those developmental stages that contained the lowest levels of mRNA detectable by the cDNA probe. Possible reasons for this phenomena are discussed. In vitro RNA translation was also used to study the relationship between the two ras proteins of 23 and 24 kd. The proteins did not appear to be derived from one another by degradation or by post-translational modification. This result suggested that the two ras proteins of strain V12M2 must be derived from two different mRNAs.
High stringency Southern blots of AX-3 DNA showed the expected restriction fragments detected by Reymond et al. (1984, Cell 39;141) . Low stringency blots showed three faint additional restriction fragments in Eco RI digests of AX-3 DNA. No additional restriction fragments were generated by an Eco RI-Bg1 II digest, but two of the three faint bands were smaller. This suggested that at least two of the Eco RI ras fragments are non-contiguous, and hence two to three ras genes may be present in addition to the one characterized by Reymond et al. (1984, Cell 39;141). All Northern and Southern bolts were probed with antisense RNA probes in order to gain greater sensitivity of detection as described by Cox et al. (1984, Dev. Biol. 101;485). / Science, Faculty of / Microbiology and Immunology, Department of / Graduate
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Plasma membrane lipid composition of Dictyostelium Discoideum during early development in aqueous suspensionWithers, Howard Keith January 1979 (has links)
Cell-cell contact must be made and maintained for normal development and eventual differentiation of D. discoideum to occur. Certain plasma membrane components are known to alter in activity or abundance during the organism's developmental cycle although no quantitative measurement of plasma membrane neutral lipid and phospholipid content has been reported to date.
Optimal conditions for the extraction, separation and assay of lipid components were derived and tested by quantification of the neutral lipid and phospholipid components of intact cells of strain Ax-2. Development was initiated in D. discoideum populations suspended in aqueous buffer and plasma membrane fractions were purified from both exponentially growing and aggregation-phase cells by a modified procedure which minimized phospholipid degradation
during the plasma membrane isolation. Neutral lipid and phospholipid compositions of the plasma membrane fractions PM1 and PM2 from exponentially growing cells and from those in early aggregation phase were determined.
Exponential phase cells' plasma membranes contained large proportions of phosphatidylethanolamine, phosphatidylcholine and lysophosphatidylethanolamine. Lysophosphatidylcholine was absent. A significant quantity of phosphatidylinositol was detected and cardiolipin, phosphatidylglycerol, phosphatidic acid and lyso-phosphatidic acid were each present in small amounts. The presence of phosphatidylethanolamine plasmalogen was suspected but not proven. No acylglycerol components were detected, the major
neutral lipid fraction being that of free sterol which largely comprised stigmast-22-en-3β-ol; sterol ester was present in extremely small quantities. An unidentified neutral lipid component of plasma membranes was detected by its characteristic absorption and fluorescence upon irradiation at ultraviolet wavelengths. After sixteen hours aggregation the phosphatidylcholine content of the plasma membranes was greatly reduced, a significant proportion of the phosphatidylethanolamine appeared to have been converted to lysophosphatidylethanolamine, and phosphatidylglycerol, phosphatidic acid and lysophosphatidic acid were all in greater abundance than in growing cells' membranes. The free sterol component remained relatively constant but sterol ester had increased dramatically (7 to 10-fold) and the fatty acid composition of the plasma membrane phospholipids was more saturated, primarily because of the accumulation
of palmitate and stearate and a reduction of the octadeca-dienoic fatty acid components. / Science, Faculty of / Microbiology and Immunology, Department of / Unknown
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Characterization of the function and mechanism of an orphan 3'-5' polymeraseimplicated in noncoding RNA processing.Dodbele, Samantha January 2019 (has links)
No description available.
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Endogenous cyclic AMP-dependent phosphorylation in vitro of cytosolic proteins from Dictyostelium discoideumFrame, Lynn Teresa January 1983 (has links)
Endogenous phosphorylation was measured in soluble fractions at four stages of Dictyostelium discoideum development. A peak of activity occurred at the slug stage, coincident with the appearance of cyclic AMP-dependent protein kinase. After partial purification by DE-52 cellulose and Sephacryl S-300 chromatography, cyclic AMP dependency of six cytosolic proteins was observed, with apparent subunit molecular weights of > 200,000, 110,000, 107,000, 91,000, 75,000, and 69,000. Phosphorylated bands at 107,000 and 91,000 MW were found to be specific for a partially-purified catalytic subunit prepared from the Dictyostelium holoenzyme form. / M.S.
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The relationship between two forms of glycogen phosphorylase in Dictyostelium discoideumSucic, Joseph F. January 1988 (has links)
The cellular slime mold, Dictyortelium dixcoideum, provides an ideal model system to study eukaryotic cell differentiation. In D. discoideum glycogen degradation provides precursors for the synthesis of developmentally regulated structural end products. The enzyme responsible for glycogen degradation, glycogen phosphorylase, exists in active and inactive forms. The activity of the ’a' form (the active form) is independent of 5′adenosine monophosphate (5′AMP) while the activity of the ’b' form (the inactive form) is 5′AMP dependent, The two forms are developmentally regulated. Polyclortal antibodies raised to the purified forms of this enzyme show low cross reactivity. The anti-’a' antiserum reacts with a 104 kd protein that is associated with phosphorylase ’a' activity; the anti-’b' antiserum reacts with a 92 kd protein that is associated with phosphorylase ’b' activity and cross reacts weakly with the 104 kd protein. Cyclic AMP perturbation of intact cells caused induction of both phosphorylase ’a' activity and the appearance of the 104 kd protein. lmmunotitration data suggest that the ’a’ form accumulates due to de novo protein synthesis, although this result must be interpreted with caution. In vitro translation experiments indicate that separate mRNA species exist for the two forms of phosphorylase. The mRNA for the 'b’ form is present throughout development while that of the ’a' form appears late in development. / Master of Science
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A study of the regulation of glycogen metabolism in Dictyostelium discoideumBrickey, Debra A. January 1988 (has links)
This work discusses the regulation of glycogen metabolism in Dictyostelium discoideum during its developmental cycle. Specifically, the possible cAMP dependent regulation of glycogen phosphorylase and glycogen synthase was examined. In other systems, cAMP can regulate at the level of the gene (procaryotes, CAP protein) or at the level of covalent, reversible modification of the enzyme activity (eucaryotes, cAMP-dependent protein kinase-cAMPdPK). In Dictyostelium, glycogen phosphorylase and glycogen synthase have each been found to occur in two forms; one regulated allosterically and the other independent of allosteric regulation.
The regulation of the two forms of glycogen phosphorylase was examined in single-cell suspensions to which cAMP or one of several cAMP analogs were added to mimic differentiative conditions. The allosterically regulated form of glycogen phosphorylase, phosphorylase b, decreased in the presence of cAMP while a corresponding increase in phosphorylase a, the non-allosterically regulated form of glycogen phosphorylase, was observed over an 8 hr period in the same cultures. In the presence of cAMP analogs, a similar time course of regulation for the two forms of glycogen phosphorylase occurred but only 2’deoxy-cAMP gave an effect comparable to cAMP. Under these same conditions, northern blot analysis of three developmentally regulated mRNAs--PL3, D11, and D3--revealed that normal gene regulation was occurring. Under conditions where elevation of intracellular cAMP was inhibited, neither regulation of phosphorylase enzyme activity nor of the 3 genes was observed. This indicated that under these conditions intracellular elevation of cAMP was necessary for the observed effects on enzyme and gene activity. This requirement for intracellular cAMP may indicate the involvement of a cAMPdPK.
The properties of a phosphorylase b kinase found in amoebal extracts are described. The kinase activity coeluted with the phosphorylase b activity on a DE·52 anion exchange column. Under the conditions described conversion of the phosphorylase b activity to the phosphorylase a activity was observed. However, an increase in molecular weight to 104 kd (as seen for purified phosphorylase a) was not observed.
The characterization of a partially purified glycogen synthase I and its developmental regulation are described. Also described are in vitro attempts to convert the I form to the allosterically regulated, D form, under conditions conducive to phosphorylation. / Ph. D.
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Identificação de genes codificadores de serina/treonina fosfatases em Dictyostelium discoideum e caracterização funcional da proteína fosfatase do tipo 4 (PP4) / Identification of genes coding for serine/threonine phosphatases in Dictyostelium discoideum and functional characterization of type 4 protein phosphatase (PP4)Fiorini, Leonardo Costa 27 May 2003 (has links)
As serina/treonina fosfatases (PPs) são enzimas responsáveis pela desfosforilação de resíduos de fosfoserina e/ou fosfotreonina e estão subdivididas em duas famílias gênicas designadas PPP e PPM. A família PPP está dividida em cinco subfamílias, que compreendem as PPs do tipo 1 (PP1), 2A (PP2A), 2B (PP2B), 5 (PP5) e 7 (PP7), de acordo com a similaridade entre as sequências de aminoácidos das subunidades ou domínios destas enzimas. Novas PPs estão sendo descobertas em diferentes organismos e classificadas nestas famílias ou subfamílias com base na análise comparada de suas sequências. Uma delas é a proteína fosfatase do tipo 4 (PP4), descoberta originalmente em coelhos e cujas funções biológicas vêm sendo progressivamente elucidadas. Neste estudo tivemos como objetivos a identificação e caracterização de uma nova serina/treonina fosfatase de Dictyostelium discoideum. Para isto, rastreamos uma biblioteca de cDNA e um cDNA completo que codifica a subunidade catalítica da fosfatase do tipo 4 (PP4c) foi isolado e seqüenciado. Verificamos que o gene da PP4c é essencial e está presente em cópia única localizada no cromossomo 2, originando um mRNA expresso ao longo de todo o ciclo de vida de D. discoideum. Imunodetecção da PP4c realizada com anticorpo específico indicou que os níveis desta proteína também são constitutivos. Observamos que a superexpressão da PP4c sob o controle de um promotor constitutivo não causa alterações fenotípicas detectáveis. A análise da localização celular da PP4c expressa em células de D. discoideum como proteína de fusão com GFP (Green Fluorescent Protein) revelou que a enzima está localizada no citossol, contrastando com outros organismos, onde ela se encontra enriquecida no centrossomo. Por fim, descrevemos neste trabalho, além da organização genômica da PP4, a estrutura dos genes de todas as serina/treonina fosfatases da família PPP encontradas no cromossomo 2 e apresentamos dados das relações entre estas enzimas de D. discoideum. / The serine/threonine protein phosphatases (PPs) are enzymes responsible for dephosphorylation of phosphoserine and/or phosphothreonine residues and are divided in two gene families designated as PPP and PPM. The PPP family is divided in five subfamilies, which comprise type 1 (PP1), 2A (PP2A), 2B (PP2B), 5 (PP5) and 7 (PP7) phosphatases. This subdivision is based on aminoacid sequence similarity or enzyme domains. Novel PPs have been discovered in different organisms and classified in these families or subfamilies based on comparative sequence analysis. One of these is type 4 protein phosphatase (PP4), originally discovered in rabbit, which functions have been progressively uncovered. In this study our goal is to identify and characterize a novel serine/threonine phosphatase in Dictyostelium discoideum. We first screened a cDNA library and isolated a complete cDNA encoding the catalytic subunit of protein phosphatase 4 (PP4c), confirmed by manual DNA sequencing. The PP4c is an essential, single-copy gene located in chromosome 2, which encondes a mRNA constitutively expressed throughout D. discoideum life cycle. Immunodetection of PP4c performed with specific antibodies indicated corresponding protein levels. Overexpression of PP4c under a strong promoter caused no detectable phenotype. Subcellular localization of PP4c expressed as a GFP-fusion protein revealed its cytosolic location, in contrast to other organisms, where it has been reported to be enriched in centrosomes. We also describe here the genetic organization of PP4c, the genetic structure of all serine/threonine protein phosphatases belonging to the PPP family found in chromosome 2, and a phylogenetic analysis indicating relationships among these enzymes in D. discoideum and other selected organisms.
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