Search PubMed⌕ Search

Biomedical subjects

M B Coukell

Publications and source records attributed to M B Coukell.

At least 19 recordsLinked to original sources
PubMed1999

Involvement of the Ca2+-ATPase PAT1 and the contractile vacuole in calcium regulation in Dictyostelium discoideum.

In Dictyostelium discoideum, the Ca2+-ATPase, PAT1, is localized to membranes of the contractile vacuole and its expression is upregulated substantially when the cells are grown in Ca2+-rich medium. In this study, we have analyzed the cellular/molecular mechanisms regulating PAT1 expression and examined the role of PAT1 and the contractile vacuole in Ca2+ regulation. During both growth and development, Dictyostelium cells respond to low millimolar concentrations of extracellular Ca2+ and upregulate PAT1 in a few hours. This process is dependent on protein synthesis and the serine/threonine phosphatase, calcineurin. Immunofluorescence analysis indicates that the upregulated PAT1 is associated mainly with the contractile vacuole, but it is also on the plasma membrane. This latter finding suggests that the contractile vacuole fuses with the plasma membrane to eliminate excess intracellular Ca2+. In support of this idea, it was observed that conditions which impair contractile vacuolar function reduce the rate of Ca2+ secretion. It was also found that cells deficient in PAT1, due to the expression of antisense patA RNA or to the presence of calcineurin antagonists, grow normally in low Ca2+ medium but poorly or not at all in high Ca2+ medium. Together, these results suggest that PAT1 and the contractile vacuole are components of a Ca2+ sequestration and excretion pathway, which functions to help maintain Ca2+ homeostasis, especially under conditions of Ca2+ stress.

Animals↗
PubMed1996

Calcium influences the stability and conformation of rotavirus SA11 glycoprotein VP7 expressed in Dictyostelium discoideum.

We have previously reported expression of the rotavirus outer capsid glycoprotein, VP7, in the relatively new expression host, Dictyostelium discoideum. To optimise yields of recombinant VP7, we examined the role of Ca2+ since stability of both VP7 and mature rotavirus during a rotavirus infection are calcium-dependent. Low micromolar levels of free extracellular Ca2+ were required to maximise yields of VP7 in D. discoideum whilst levels of VP7 were reduced following depletion of intracellular Ca2+ reserves using A23187 and EGTA. Immunoblot analysis suggested that VP7 was being degraded in an intracellular compartment. Immunoprecipitation with a conformation-dependent neutralising antibody confirmed that EGTA-induced Ca2+ chelation alters the conformation of VP7. These results suggest that stability of VP7 is dependent on maintaining adequate levels of intracellular Ca2+ and that conformational changes in VP7 which occur following depletion of Ca2+ reserves induce rapid proteolysis of the protein. Since these results establish conditions for expressing optimal levels of VP7 in the correct conformation they have important implications for the development of a subunit vaccine based on recombinant VP7.

Animals↗
PubMed1996

Antisense RNA inhibition of the putative vacuolar H(+)-ATPase proteolipid of Dictyostelium reduces intracellular Ca2+ transport and cell viability.

Transport of Ca2+ via a P-type pump into the contractile vacuole of Dictyostelium discoideum appears to be facilitated by vacuolar proton (V-H+) ATPase activity. To investigate the involvement of the V-H(+)-ATPase in this process using molecular techniques, we cloned a cDNA (vatP) encoding the putative proteolipid subunit of this enzyme. The deduced protein product of this cDNA is composed of 196 amino acids with a calculated M(r) of 20,148 and the primary structure exhibits high amino acid sequence identity with V-H(+)-ATPase proteolipids from other organisms. vatP is a single-copy gene and it produces one approximately 900 nt transcript at relatively constant levels during growth and development. Attempts to disrupt the endogenous gene using vatP cDNA were unsuccessful. But, expression of vatP antisense RNA reduced the levels of vatP message and V-H(+)-ATPase activity by 50% or more. These antisense strains grew and developed slowly, especially under acidic conditions, and the cells seemed to have difficulty forming acidic vesicles. During prolonged cultivation, all of the antisense strains either reverted to a wild-type phenotype or died. Thus in Dictyostelium, unlike yeast, the V-H(+)-ATPase seems to be indispensable for cell viability. When different antisense strains were analyzed for Ca2+ uptake by the contractile vacuole, they all accumulated less Ca2+ than control transformants. These results are consistent with earlier pharmacological studies which suggested that the V-H(+)-ATPase functions in intracellular Ca2+ transport in this organism.

Amino Acid Sequence↗
PubMed1995

Molecular cloning of an intracellular P-type ATPase from Dictyostelium that is up-regulated in calcium-adapted cells.

Results from a number of laboratories suggest that intracellular Ca2+ is involved in the regulation of Dictyostelium discoideum growth and development. To learn more about the regulation and function of intracellular Ca2+ in this organism, we have cloned and sequenced cDNAs that encode a putative P-type Ca2+ ATPase designated patA. The deduced protein product of this gene (PAT1) has a calculated molecular mass of 120,718 daltons. It exhibits about 46% amino acid identity with Ca2+ ATPases of the plasma membrane Ca2+ ATPase family and lower identity with sarco(endo)plasmic reticulum Ca2+ ATPase family members and monovalent cation pumps. However, PAT1 lacks the highly conserved calmodulin-binding domain present in the C-terminal region of most plasma membrane Ca2+ ATPase-type enzymes. When Dictyostelium amoebae are adapted to grow in the presence of 80 mM CaCl2, both the patA message and protein product are up-regulated substantially. These cells also exhibit an increase in the rate and magnitude of intracellular P-type Ca2+ uptake activity. Immunofluorescence analysis indicates that PAT1 colocalizes with bound calmodulin to intracellular membranes, probably components of the contractile vacuole complex. The presence of PAT1 on the contractile vacuole suggests that in Dictyostelium this organelle might function in Ca2+ homeostasis as well as in water regulation.

Acclimatization↗
PubMed1992

Association of a cGMP-binding activity with nuclei isolated from amoebae of Dictyostelium discoideum and Polysphondylium violaceum.

When vegetative and early slug stage amoebae of Dictyostelium discoideum or Polysphondylium violaceum were lysed by filter breakage in a nuclear isolation buffer not containing detergents, substantial levels of a cGMP-binding activity with slow-dissociation kinetics were detected. After fractionation by centrifugation, 50% or more of this binding activity was associated with isolated nuclei. In addition, with Polysphondylium cells, the fraction of stable, nuclear-associated binding activity appeared to increase during cell aggregation. These results support the idea that cGMP might function in the nucleus during early development.

Animals↗
PubMed1992

Involvement of intracellular calcium in protein secretion in Dictyostelium discoideum.

We reported previously that Ca2+ depletion of Dictyostelium discoideum cells severely inhibits extracellular cyclic nucleotide phosphodiesterase (PD) synthesis at a post-transcriptional step. In this study, further experiments were performed to learn more about the nature of this phenomenon. Examination of the polysomal distribution of PD transcripts in control cells and in cells depleted of Ca2+ by incubation with EGTA and A23187 (EA) suggested that inhibition of PD production does not involve translational control. Kinetic analysis of this inhibitory process revealed that soluble, intracellular PD activity, synthesized from either the 2.4 or 1.9 kb PD mRNA, decreased very rapidly upon addition of EA. Furthermore, this decrease in activity was accompanied by the preferential loss of PD-related polypeptides, indicating a proteolytic event. EA-induced PD degradation required cellular energy and concomitant protein synthesis but was unaffected by most of the lysosomotropic agents tested. Therefore, PD proteolysis might not occur in the lysosome. In cell fractionation experiments, the EA-sensitive, intracellular PD activity comigrated with a rough ER marker in Percoll/KCl gradients. In addition to its effect on the PD, EA were also observed to inhibit production and rapidly lower the intracellular levels of another secreted glycoprotein, the PD inhibitor. Together, these results suggest that depletion of some intracellular Ca2+ store(s) in Dictyostelium, possibly the ER, disrupts the normal function of the secretory pathway, resulting in selective degradation of certain proteins.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1991

A Ca2+ transport system associated with the plasma membrane of Dictyostelium discoideum is activated by different chemoattractant receptors.

Amebae of Dictyostelium exhibit a transient uptake of extracellular Ca2+ approximately 5 s after activation of surface folate or cAMP receptors (Bumann, J., B. Wurster, and D. Malchow. 1984. J. Cell Biol. 98:173-178). To further characterize these Ca2+ entry systems, we analyzed 45Ca2+ uptake by resting and activated amebae. Like the surface chemoreceptors, folate- and cAMP-induced Ca2+ uptake responses were developmentally regulated; the former response was evident in vegetative but not aggregation-competent cells, whereas the latter response displayed the opposite pattern of expression. In contrast, other characteristics of these Ca2(+)-uptake pathways were remarkably similar. Both systems (a) exhibited comparable kinetic properties, (b) displayed a high specificity for Ca2+, and (c) were inhibited effectively by Ruthenium Red, sodium azide, and carbonylcyanide m-chlorophenyl-hydrazone. These results, together with the finding that vegetative cells transformed with a plasmid expressing the surface cAMP receptor exhibit a cAMP-induced Ca2+ uptake, suggest that different chemoreceptors activate a single Ca2+ entry pathway. Additional pharmacological and ion competition studies indicated that receptor-mediated Ca2+ entry probably does not involve a Na+/Ca2+ exchanger or voltage-activated channels. Chemoattractant binding appears to generate intracellular signals that induce activation and adaption of the Ca2(+)-uptake response. Analysis of putative signaling mutants suggests that Ca2+ entry is not regulated by the guanine nucleotide-binding (G) protein subunits G alpha 1 or G alpha 2, or by G protein-mediated changes in intracellular cAMP or guanosine 3,'5'-cyclic monophosphate (cGMP).

Biological Transport↗
PubMed1991

Biochemical and genetic characterization of a rapid-development strain in Dictyostelium discoideum.

We have examined the rates of development of six wild-type and rapid-development strains of Dictyostelium discoideum. Strains NC4 and HU1231, a derivative of V12, have very similar developmental profiles. In comparison to these two strains, amoebae of the rapid-development strain HT100 reach the aggregation stage about 6 h earlier. Also, intracellular cAMP levels in HT100 increase precociously during early development. Postaggregative morphogenesis of HT100 proceeds at the same rate as NC4 and HU1231. In addition we have assessed the progress of development by RNA hybridization blotting. The appearance in HT100 of mRNAs associated with aggregation is advanced by 6 h while the appearance of early gene products is not affected appreciably. These data suggest that HT100 differs from strains NC4 and HU1231 primarily in the process of aggregation. We have partially characterized the genetic background of strain HT100. Mating and cell fusion analyses suggest that HT100 is a derivative of V12. We constructed a growth-temperature-sensitive derivative of HT100, and fused it with HU1231 cells. The resulting diploids develop at the same rate as HT100, suggesting that the rapid-development phenotype in HT100 is dominant.

Animals↗
PubMed1990

Effects of DNA and synthetic oligodeoxyribonucleotides on the binding properties of a cGMP-binding protein from Dictyostelium discoideum.

Previously, we identified a cGMP-binding protein (cGBP) in Dictyostelium discoideum that can exist in two forms: a fast-dissociating (F-type) activity and a slow-dissociating (S-type) activity. Moreover, the F-type activity was converted effectively to S-type by the addition of nucleic acids, especially DNA (Parissenti, A.M. and Coukell, M. B. (1989) J. Cell Sci. 92, 291-301). In this study, we examined the effects of heterologous DNA and various synthetic homo-oligodeoxyribonucleotides on the cGMP-binding properties of partially purified F-type activity. Equilibrium and kinetic binding experiments revealed that DNA increased the affinity of the protein for cGMP without altering the number of binding sites. However, the presence of DNA decreased only slightly the apparent Kd of the protein for cGMP because the nucleic acid also reduced the rate of cGMP association. Addition of oligo(dGMP)8 or oligo(dCMP)8 to the protein increased both total cGMP binding and the conversion of F-type activity to S-type; in contrast, oligo(dAMP)8 or oligo(dTMP)8, at the same concentration, had no effect. Oligodeoxycytidylic acids with chain lengths less than about eight nucleotides were also ineffective or inhibitory. Analysis of cGMP binding to intact, filipin-permeabilized cells revealed a binding activity with association and dissociation rates comparable to isolated S-type activity. This observation suggests that in vivo the cGBP might exist in its S-form.

Carrier Proteins↗
PubMed1989

Identification of a high-affinity Ca2+ pump associated with endocytotic vesicles in Dictyostelium discoideum.

In the cellular slime mold Dictyostelium discoideum, changes in free cytosolic Ca2+ are thought to regulate certain processes during cell aggregation and differentiation. To understand the mechanisms controlling free Ca2+ levels in this organism, we previously isolated and characterized an ATP/Mg2+-dependent, high-affinity Ca2+ pump which appeared to be a component of "inside-out" plasma membrane vesicles [J. L. Milne and M. B. Coukell (1988) Biochem. J. 249. 223-230]. In this report, we demonstrate that a high-affinity Ca2+ pump, with properties virtually identical to the isolated pump, can be detected in filipin- or digitonin-permeabilized cells of Dictyostelium. Moreover, Ca2+-pumping vesicles, which migrate on Percoll/KCl gradients like the vesicles identified earlier, can be isolated from the permeabilized cells. Results of additional experiments suggest that this intracellular Ca2+ transporter is associated with a high-capacity non-IP3-releasable Ca2+ store which is generated by endocytosis. A possible role for this store in maintaining Ca2+ homeostasis in Dictyostelium is discussed.

Animals↗
PubMed1988

Isolation and characterization of a plasma membrane calcium pump from Dictyostelium discoideum.

During the aggregation and differentiation of amoebae of Dictyostelium discoideum, changes in free cytosolic Ca2+ appear to regulate a number of physiological processes. To understand the mechanisms regulating free intracellular Ca2+ in this organism, we have isolated and characterized an ATP/Mg2+-dependent, high-affinity Ca2+ pump. When homogenates of 2 h starved cells were fractionated on Percoll/KCl gradients, one peak of high-affinity Ca2+-pumping activity was detected. This activity was resolved from enzyme markers of the mitochondrion and the rough endoplasmic reticulum but it cosedimented with the plasma membrane marker, alkaline phosphatase. Further studies suggested that the pump was associated with 'inside-out' plasma membrane vesicles. Like plasma membrane Ca2+-transport ATPases from other systems, this isolated Ca2+ pump: (1) was Mg2+-dependent, (2) displayed a high specificity for ATP as an energy source, (3) exhibited a high affinity for free Ca2+ with a Km of 0.3 microM, and (4) was very sensitive to inhibition by vanadate (IC50 2 microM) but was unaffected by mitochondrial inhibitors, ouabain and Ca2+-channel blockers. Unlike plasma membrane Ca2+ pumps from most other systems, this enzyme appeared not to be regulated by calmodulin. During development, non-mitochondrial, vanadate-sensitive, high-affinity Ca2+-pumping activity in crude lysates remained relatively constant for at least 15 h. These observations suggest that this plasma membrane Ca2+ pump probably functions in Dictyostelium to maintain Ca2+ homeostasis by extruding free cytosolic Ca2+ from the cells.

Adenosine Triphosphatases↗
PubMed1988

Effects of suboptimal levels of extracellular calcium on the regulation of the cyclic AMP phosphodiesterase-inhibitor system and membrane differentiation in Dictyostelium discoideum.

When starved wild-type amoebae of Dictyostelium discoideum were washed and incubated in 1 mM-EGTA, their ability to induce soluble cyclic AMP phosphodiesterase (PD) activity in response to either millimolar cyclic AMP or a series of nanomolar cyclic AMP pulses was reduced by 55-75%. Supplementation of EGTA-treated cells with exogenous Ca2+ stimulated PD induction in a dose-dependent fashion (EC50 = 100-200 nM free extracellular Ca2+), and enzyme production was maximal at about 1 microM free Ca2+. Ca2+ depletion also strongly impaired production of the phosphodiesterase inhibitor (PDI). In contrast, other than delaying their appearance by about 1 h, EGTA had little effect on the induction by cyclic AMP pulses of cell surface markers such as contact sites A and membrane-bound PD activity. Similar changes in both the soluble and membrane activities were observed with strain NP368, a mutant that overproduces cyclic GMP when stimulated by cyclic AMP. Thus, Ca2+ depletion does not appear to inhibit PD and PDI production by reducing intracellular cyclic GMP. To determine whether Ca2+ depletion alters signal transduction, two mutants that produce the soluble PD activities constitutively were examined. Suboptimal concentrations of free extracellular Ca2+ were found to inhibit PD production in these cells to the same degree and with the same concentration dependence as low Ca2+ inhibited PD induction by cyclic AMP in wild-type cells. These results suggest that Ca2+ depletion by EGTA probably inhibits PD and PDI production indirectly by perturbing an intracellular Ca2+ pool(s) rather than by altering a surface cyclic AMP-receptor-mediated process.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Effects of calcium antagonists on cyclic AMP phosphodiesterase induction in Dictyostelium discoideum.

Previous studies have suggested that cyclic GMP and/or Ca2+ might function as second messengers in the induction by exogenous cyclic AMP of the cyclic AMP phosphodiesterase (PD) in Dictyostelium discoideum. To assess further the role of Ca2+ in PD induction we examined the effect on this process of a number of putative Ca2+-channel blockers. At relatively low micromolar concentrations, TMB-8, nicardipine, nifedipine, diltiazem and verapamil all altered cell shape and inhibited PD induction in a similar dose-dependent fashion. Concentrations of these drugs that abolished PD induction had no effect on cell viability; however, higher concentrations reduced viability and caused cell lysis. All effects of these compounds on the cells were antagonized at least partially by 5-10 mM-Ca2+. Other cations tested were considerably less effective. Like the organic inhibitors, La3+ also altered cell shape, inhibited PD induction and reduced cell viability at elevated concentrations, but its effect on the cells appeared to be more complex. Inhibition of PD induction by the organic antagonists could not be attributed solely to an impaired uptake of extracellular Ca2+, a reduction of ATP pools in the cells or a direct effect on calmodulin. Concentrations of TMB-8 that inhibited PD induction had little effect on the cyclic GMP response. Therefore, this compound did not inhibit PD induction indirectly by blocking cyclic GMP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Characterization of revertants of stmF mutants of Dictyostelium discoideum: evidence that stmF is the structural gene of the cGMP-specific phosphodiesterase.

stmF mutants of Dictyostelium discoideum produce long, banded aggregation streams on growth plates and exhibit altered cGMP metabolism. To learn more about the role of cGMP in chemotaxis and the nature of the defect in these mutants, 15 nonstreaming (Stm+) revertants of two stmF mutants were isolated and characterized. Fourteen of the revertants continued to show the elevated cAMP-induced cGMP response and very low cGMP-specific phosphodiesterase (cGPD) activity characteristic of their stmF parents. Parasexual genetic analysis revealed that many of these Stm+ revertants carried phenotypic suppressors unlinked to stmF. One Stm+ revertant, strain HC344, exhibited a low, prolonged cGMP response and relatively high cGPD activity throughout development. To determine whether the elevated cGPD activity in this revertant resulted from increased enzyme production or enhanced enzyme activity, cGPDs were partially purified from the wild-type strain, the stmF parent and revertant HC344, and properties of the enzymes were compared. cGPDs from the stmF mutant and the revertant showed similar differences from the wild-type enzyme in kinetic properties, thermal stability, and sensitivity to certain inhibitors. These results suggest that stmF is the structural gene of the cGPD. In addition, the unusual cGMP response in revertant HC344 appeared to be due to increased production of an altered cGPD.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1985

Genetic locus (stmF) associated with cyclic GMP phosphodiesterase activity in Dictyostelium discoideum maps in linkage group II.

Previous attempts to map the stmF locus in Dictyostelium discoideum, by using only clone morphology as a marker, have led to equivocal results. Since strains carrying mutations at the stmF locus possess very low cyclic GMP phosphodiesterase activity, we have remapped this locus using both morphological and biochemical markers. Our results indicate that mutations producing a stable "streamer" phenotype and reduced cyclic GMP phosphodiesterase activity are located in linkage group II, probably centromere distal to acrA.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1984

Developmental regulation and properties of the cGMP-specific phosphodiesterase in Dictyostelium discoideum.

A simple assay has been developed to measure cGMP-specific phosphodiesterase (cGPD) activity in crude soluble extracts of amoebae of Dictyostelium discoideum. When amoebae of different wild-type strains were starved on buffered agar, all strains exhibited an 8- to 12-fold increase in cGMP-specific hydrolyzing activity during development, with the major increase occurring at aggregation. cGMP-specific activity was found in both prestalk and prespore cells. To determine if the elevated cGMP-specific hydrolyzing activity observed during late development was associated with the same enzyme present in vegetative cells, cGMP-specific activities were partially purified from cells at different developmental stages and characterized. Activity in vegetative cells was fractionated by gel filtration into three components with molecular weights of approximately 172,000, 115,000 and 56,000. In contrast, cells starved 4 hr in suspension or 18 hr on agar possessed only the 172,000 or 115,000 Mr forms, respectively. The low-molecular-weight enzyme differed from the two larger forms in kinetic properties and in sensitivity to sulfhydryl reagents. Nevertheless, the three activities probably represent different forms of the same enzyme because mutants defective at the stmF locus lacked appreciable cGMP-specific hydrolyzing activity throughout development. These results indicate that D. discoideum produces a single cGPD which is strongly developmentally regulated. These findings further suggest that intracellular cGMP might be involved in regulating postaggregative as well as preaggregative development.

3',5'-Cyclic-AMP Phosphodiesterases↗