Biomedical subjects

Carole A Parent

Publications and source records attributed to Carole A Parent.

At least 19 recordsLinked to original sources

PubMed2006

S-adenosylhomocysteine hydrolase is localized at the front of chemotaxing cells, suggesting a role for transmethylation during migration.

Chemotaxis of bacteria requires regulated methylation of chemoreceptors. However, despite considerable effort in the 1980s, transmethylation has never been established as a component of eukaryotic cell chemotaxis. S-adenosylhomocysteine (SAH), the product formed when the methyl group of the universal donor S-adenosylmethionine (SAM) is transferred to an acceptor molecule, is a potent inhibitor of all transmethylation reactions. In eukaryotic cells, this inhibition is relieved by hydrolysis of SAH to adenosine and homocysteine catalyzed by SAH hydrolase (SAHH). We now report that SAHH, which is diffuse in the cytoplasm of nonmotile Dictyostelium amoebae and human neutrophils, concentrates with F-actin in pseudopods at the front of motile, chemotaxing cells, but is not present in filopodia or at the very leading edge. Tubercidin, an inhibitor of SAHH, inhibits both chemotaxis and chemotaxis-dependent cell streaming of Dictyostelium, and chemotaxis of neutrophils at concentrations that have little effect on cell viability. Tubercidin does not inhibit starvation-induced expression of the cAMP receptor, cAR1, or G protein-mediated stimulation of adenylyl cyclase activity and actin polymerization in Dictyostelium. Tubercidin has no effect on either capping of Con A receptors or phagocytosis in Dictyostelium. These results add SAHH to the list of proteins that redistribute in response to chemotactic signals in Dictyostelium and neutrophils and strongly suggest a role for transmethylation in chemotaxis of eukaryotic cells.

Actins↗

PubMed2006

A chemoattractant-mediated Gi-coupled pathway activates adenylyl cyclase in human neutrophils.

Neutrophils and Dictyostelium use conserved signal transduction pathways to decipher chemoattractant gradients and migrate directionally. In both cell types, addition of chemoattractants stimulates the production of cAMP, which has been suggested to regulate chemotaxis. We set out to define the mechanism by which chemoattractants increase cAMP levels in human neutrophils. We show that chemoattractants elicit a rapid and transient activation of adenylyl cyclase (AC). This activation is sensitive to pertussis toxin treatment but independent of phosphoinositide-3 kinase activity and an intact cytoskeleton. Remarkably, and in sharp contrast to Galpha(s)-mediated activation, chemoattractant-induced AC activation is lost in cell lysates. Of the nine, differentially regulated transmembrane AC isoforms in the human genome, we find that isoforms III, IV, VII, and IX are expressed in human neutrophils. We conclude that the signal transduction cascade used by chemoattractants to activate AC is conserved in Dictyostelium and human neutrophils and is markedly different from the canonical Galpha(s)-meditated pathway.

Adenylyl Cyclases↗

PubMed2006

A secondary disruption of the dmpA gene encoding a large membrane protein allows aggregation defective Dictyostelium rasC- cells to form multicellular structures.

The disruption of the gene encoding the Dictyostelium Ras subfamily protein, RasC, results in a strain that does not aggregate and has defects in both cAMP signal relay and cAMP chemotaxis. Disruption of a second gene in the rasC(-) strain by Restriction Enzyme Mediated Integration produced cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene (dmpA) encoded a novel membrane protein that was designated Dmp1. Although the rasC(-)/dmpA(-) cells progressed through early development, they did not form aggregation streams on a plastic surface under submerged starvation conditions. Phosphorylation of PKB in response to cAMP, which is significantly reduced in rasC(-) cells, remained low in the rasC(-)/dmpA(-) cells. However, in spite of this low PKB phosphorylation, the rasC(-)/dmpA(-) cells underwent efficient chemotaxis to cAMP in a spatial gradient. Cyclic AMP accumulation, which was greatly reduced in the rasC(-) cells, was restored in the rasC(-)/dmpA(-) strain, but cAMP relay in these cells was not apparent. These data indicate that although the rasC(-)/dmpA(-) cells were capable of associating to form multicellular structures, normal aggregative cell signaling was clearly not restored. Disruption of the dmpA gene in a wild-type background resulted in cells that exhibited a slight defect in aggregation and a more substantial defect in late development. These results indicate that, in addition to the role played by Dmp1 in aggregation, it is also involved in late development.

Amino Acid Sequence↗

PubMed2006

Signal relay during the life cycle of Dictyostelium.

A fundamental property of multicellular organisms is signal relay, the process by which information is transmitted from one cell to another. The integration of external information, such as nutritional status or developmental cues, is critical to the function of organisms. In addition, the spatial organizations of multicellular organisms require intricate signal relay mechanisms. Signal relay is remarkably exhibited during the life cycle of the social amoebae Dictyostelium discoideum, a eukaryote that retains a simple way of life, yet it has greatly contributed to our knowledge of the mechanisms cells use to communicate and integrate information. This chapter focuses on the molecules and mechanisms that Dictyostelium employs during its life cycle to relay temporal and spatial cues that are required for survival.

Adenine↗

PubMed2006

Chemotaxis: moving forward and holding on to the past.

The ability of cells to sense external chemical cues and respond by directionally migrating towards them is a fundamental process called chemotaxis. This phenomenon is essential for many biological responses in the human body, including the invasion of neutrophils to sites of inflammation. Remarkably, many of the molecular mechanisms involved in controlling neutrophils chemotaxis arose millions of years ago in the simple eukaryotic organism Dictyostelium discoideum. Both neutrophils and Dictyostelium use G protein-coupled signaling cascades to mediate chemotactic responses, which are responsible for transducing external cues into highly organized cytoskeletal rearrangements that ultimately lead to directed migration. By using the genetically and biochemically tractable organism Dictyostelium as a model system, it has been possible to decipher many of the signal transduction events that are involved in chemotaxis.

Actins↗

PubMed2005

Phosphoinositide 3-kinase activity controls the chemoattractant-mediated activation and adaptation of adenylyl cyclase.

The binding of chemoattractants to cognate G protein-coupled receptors activates a variety of signaling cascades that provide spatial and temporal cues required for chemotaxis. When subjected to uniform stimulation, these responses are transient, showing an initial peak of activation followed by a period of adaptation, in which activity subsides even in the presence of stimulus. A tightly regulated balance between receptor-mediated stimulatory and inhibitory pathways controls the kinetics of activation and subsequent adaptation. In Dictyostelium, the adenylyl cyclase expressed during aggregation (ACA), which synthesizes the chemoattractant cAMP, is essential to relay the signal to neighboring cells. Here, we report that cells lacking phosphoinositide 3-kinase (PI3K) activity are deficient in signal relay. In LY294002-treated cells, this defect is because of a loss of ACA activation. In contrast, in cells lacking PI3K1 and PI3K2, the signal relay defect is because of a loss of ACA adaptation. We propose that the residual low level of 3-phosphoinositides in pi3k(1-/2-) cells is sufficient to generate the initial peak of ACA activity, yet is insufficient to sustain the inhibitory phase required for its adaptation. Thus, PI3K activity is poised to regulate both ACA activation and adaptation, thereby providing a link to ensure the proper balance of counteracting signals required to maintain optimal chemoresponsiveness.

Adenylyl Cyclases↗

PubMed2005

TOR complex 2 integrates cell movement during chemotaxis and signal relay in Dictyostelium.

Dictyostelium cells form a multicellular organism through the aggregation of independent cells. This process requires both chemotaxis and signal relay in which the chemoattractant cAMP activates adenylyl cyclase through the G protein-coupled cAMP receptor cAR1. cAMP is produced and secreted and it activates receptors on neighboring cells, thereby relaying the chemoattractant signal to distant cells. Using coimmunoprecipitation and mass spectrometric analyses, we have identified a TOR-containing complex in Dictyostelium that is related to the TORC2 complex of Saccharomyces cerevisiae and regulates both chemotaxis and signal relay. We demonstrate that mutations in Dictyostelium LST8, RIP3, and Pia, orthologues of the yeast TORC2 components LST8, AVO1, and AVO3, exhibit a common set of phenotypes including reduced cell polarity, chemotaxis speed and directionality, phosphorylation of Akt/PKB and the related PKBR1, and activation of adenylyl cyclase. Further, we provide evidence for a role of Ras in the regulation of TORC2. We propose that, through the regulation of chemotaxis and signal relay, TORC2 plays an essential role in controlling aggregation by coordinating the two essential arms of the developmental pathway that leads to multicellularity in Dictyostelium.

Amino Acid Sequence↗

PubMed2005

Selenocysteine tRNA identification in the model organisms Dictyostelium discoideum and Tetrahymena thermophila.

Characterizing Sec tRNAs that decode UGA provides one of the most direct and easiest means of determining whether an organism possesses the ability to insert selenocysteine (Sec) into protein. Herein, we used a combination of two techniques, computational to identify Sec tRNA genes and RT-PCR to sequence the gene products, to unequivocally demonstrate that two widely studied, model protozoans, Dictyostelium discoideum and Tetrahymena thermophila, encode Sec tRNA in their genomes. The advantage of using both procedures is that computationally we could easily detect potential Sec tRNA genes and then confirm by sequencing that the Sec tRNA was present in the tRNA population, and thus the identified gene was not a pseudogene. Sec tRNAs from both organisms decode UGA. T. thermophila Sec tRNA, like all other sequenced Sec tRNAs, is 90 nucleotides in length, while that from D. discoideum is 91 nucleotides long making it the longest eukaryotic sequenced to date. Evolutionary analyses of known Sec tRNAs reveal the two forms identified herein are the most divergent eukaryotic Sec tRNAs thus far sequenced.

Animals↗

PubMed2005

The PI3K-mediated activation of CRAC independently regulates adenylyl cyclase activation and chemotaxis.

The ability of a cell to detect an external chemical signal and initiate a program of directed migration along a gradient comprises the fundamental process called chemotaxis. Investigations in Dictyostelium discoideum and neutrophils have established that pleckstrin homology (PH) domain-containing proteins that bind to the PI3K products PI(3,4)P2 and PI(3,4,5)P3, such as CRAC (cytosolic regulator of adenylyl cyclase) and Akt/PKB, translocate specifically to the leading edge of chemotaxing cells. CRAC is essential for the chemoattractant-mediated activation of the adenylyl cyclase ACA, which converts ATP into cAMP, the primary chemoattractant for D. discoideum. The mechanisms by which CRAC activates ACA remain to be determined. We now show that in addition to its essential role in the activation of ACA, CRAC is involved in regulating chemotaxis. Through mutagenesis, we show that these two functions are independently regulated downstream of PI3K. A CRAC mutant that has lost the capacity to bind PI3K products does not support chemotaxis and shows minimal ACA activation. Finally, overexpression of CRAC and various CRAC mutants show strong effects on ACA activation with little effect on chemotaxis. These findings establish that chemoattractant-mediated activation of PI3K is important for the CRAC-dependent regulation of both chemotaxis and adenylyl cyclase activation.

Adenylyl Cyclases↗

PubMed2004

Teaching resources. Spatial and temporal dynamics of signaling components involved in the control of chemotaxis in Dictyostelium discoideum.

This animation shows the redistribution of several proteins during the chemotactic response of the single-celled organism Dictyostelium discoideum to the chemoattractant cAMP. Much like neutrophils, this organism is able to sense and move toward very shallow gradients of chemoattractant. Mutant strains carrying mutations in genes encoding various signaling components are available and, thus, D. discoideum serves as a model organism for the study of the cellular mechanisms that control chemotaxis and amplification of a signaling gradient across a single cell. The animation would be useful in depicting the dynamic nature of signal transduction, the ability of cells to polarize to allow for directional movement, and the spatial and temporal changes in the activity of specific proteins that accompany chemotactic responses.

Animals↗

PubMed2004

A G alpha-dependent pathway that antagonizes multiple chemoattractant responses that regulate directional cell movement.

Chemotactic cells, including neutrophils and Dictyostelium discoideum, orient and move directionally in very shallow chemical gradients. As cells polarize, distinct structural and signaling components become spatially constrained to the leading edge or rear of the cell. It has been suggested that complex feedback loops that function downstream of receptor signaling integrate activating and inhibiting pathways to establish cell polarity within such gradients. Much effort has focused on defining activating pathways, whereas inhibitory networks have remained largely unexplored. We have identified a novel signaling function in Dictyostelium involving a Galpha subunit (Galpha9) that antagonizes broad chemotactic response. Mechanistically, Galpha9 functions rapidly following receptor stimulation to negatively regulate PI3K/PTEN, adenylyl cyclase, and guanylyl cyclase pathways. The coordinated activation of these pathways is required to establish the asymmetric mobilization of actin and myosin that typifies polarity and ultimately directs chemotaxis. Most dramatically, cells lacking Galpha9 have extended PI(3,4,5)P(3), cAMP, and cGMP responses and are hyperpolarized. In contrast, cells expressing constitutively activated Galpha9 exhibit a reciprocal phenotype. Their second message pathways are attenuated, and they have lost the ability to suppress lateral pseudopod formation. Potentially, functionally similar Galpha-mediated inhibitory signaling may exist in other eukaryotic cells to regulate chemoattractant response.

Actins↗

PubMed2004

Making all the right moves: chemotaxis in neutrophils and Dictyostelium.

Neutrophils and Dictyostelium discoideum share the ability to migrate directionally in response to external chemoattractant gradients. The binding of chemoattractants to specific receptors that are coupled to heterotrimeric G proteins leads to a wide range of biochemical responses that become highly localized as cells polarize and migrate by chemotaxis. The signaling mechanisms that lead to the predominant polymerization of F-actin at the front of cells for propulsion and to myosin II assembly at the sides to suppress lateral pseudopod formation and at the back for retraction are now beginning to emerge.

Actins↗

PubMed2004

Adenylyl cyclase expression and regulation during the differentiation of Dictyostelium discoideum.

Cyclic AMP metabolism is essential for the survival of the social amoebae Dictyostelium discoideum. Three distinct adenylyl cyclases are expressed and required for the normal development of this simple eukaryote. The adenylyl cyclase expressed during aggregation, ACA, is related to the mammalian and Drosophila G protein-coupled enzymes and is responsible for the synthesis of cAMP that is required for cell-cell signaling in early development. ACB harbors histidine kinase and response-regulator domains and is required for terminal differentiation. Finally, the adenylyl cyclase expressed during germination, ACG, acts as an osmosensor and is involved in controlling spore germination. Together, these enzymes generate the various levels of cAMP that are required for D. discoideum to transition from uni- to multi-cellularity. This review will highlight the properties of these enzymes and describe the signaling cascades that lead to their activation.

Adenylyl Cyclases↗

PubMed2003

The signal to move: D. discoideum go orienteering.

Cells migrating directionally toward a chemoattractant source display a highly polarized cytoskeletal organization, with F-actin localized predominantly at the anterior and myosin II at the lateral and posterior regions. Dictyostelium discoideum has proven a useful system for elucidating signaling pathways that regulate this chemotactic response. During development, extracellular adenosine 3', 5' monophosphate (cAMP) functions as a primary signal to activate cell surface cAMP receptors (cARs). These receptors transduce different signals depending on whether or not they are coupled to heterotrimeric guanine nucleotide-binding proteins (G proteins) (see the STKE Connections Maps). Multiple G protein-stimulated pathways interact to establish polarity in chemotaxing D. discoideum cells by localizing F-actin at their leading edge and by regulating the phosphorylation state and assembly of myosin II. Many of the molecular interactions described are fundamental to the regulation of chemotaxis in other eukaryotic cells.

Actins↗

PubMed2003

Adenylyl cyclase localization regulates streaming during chemotaxis.

We studied the role of the adenylyl cyclase ACA in Dictyostelium discoideum chemotaxis and streaming. In this process, cells orient themselves in a head to tail fashion as they are migrating to form aggregates. We show that cells lacking ACA are capable of moving up a chemoattractant gradient, but are unable to stream. Imaging of ACA-YFP reveals plasma membrane labeling highly enriched at the uropod of polarized cells. This localization requires the actin cytoskeleton but is independent of the regulator CRAC and the effector PKA. A constitutively active mutant of ACA shows dramatically reduced uropod enrichment and has severe streaming defects. We propose that the asymmetric distribution of ACA provides a compartment from which cAMP is secreted to locally act as a chemoattractant, thereby providing a unique mechanism to amplify chemical gradients. This could represent a general mechanism that cells use to amplify chemotactic responses.

Actins↗

PubMed2003

Receptor-mediated regulation of PI3Ks confines PI(3,4,5)P3 to the leading edge of chemotaxing cells.

Recent studies have demonstrated that PH domains specific for PI(3,4,5)P3 accumulate at the leading edge of a number of migrating cells and that PI3Ks and PTEN associate with the membrane at the front and back, respectively, of chemotaxing Dictyostelium discoideum cells. However, the dependence of chemoattractant induced changes in PI(3,4,5)P3 on PI3K and PTEN activities have not been defined. We find that bulk PI(3,4,5)P3 levels increase transiently upon chemoattractant stimulation, and the changes are greater and more prolonged in pten- cells. PI3K activation increases within 5 s of chemoattractant addition and then declines to a low level of activity identically in wild-type and pten- cells. Reconstitution of the PI3K activation profile can be achieved by mixing membranes from stimulated pi3k1-/pi3k2- cells with cytosolic PI3Ks from unstimulated cells. These studies show that significant control of chemotaxis occurs upstream of the PI3Ks and that regulation of the PI3Ks and PTEN cooperate to shape the temporal and spatial localization of PI(3,4,5)P3.

Animals↗

PubMed2002

Tail chimeras of Dictyostelium myosin II support cytokinesis and other myosin II activities but not full development.

Dictyostelium lacking myosin II cannot grow in suspension culture, develop beyond the mound stage or cap concanavalin A receptors and chemotaxis is impaired. Recently, we showed that the actin-activated MgATPase activity of myosin chimeras in which the tail domain of Dictyostelium myosin II heavy chain is replaced by the tail domain of either Acanthamoeba or chicken smooth muscle myosin II is unregulated and about 20 times higher than wild-type myosin. The Acanthamoeba chimera forms short bipolar filaments similar to, but shorter than, filaments of Dictyostelium myosin and the smooth muscle chimera forms much larger side-polar filaments. We now find that the Acanthamoeba chimera expressed in myosin null cells localizes to the periphery of vegetative amoeba similarly to wild-type myosin but the smooth muscle chimera is heavily concentrated in a single cortical patch. Despite their different tail sequences and filament structures and different localization of the smooth muscle chimera in interphase cells, both chimeras support growth in suspension culture and concanavalin A capping and colocalize with the ConA cap but the Acanthamoeba chimera subsequently disperses more slowly than wild-type myosin and the smooth muscle chimera apparently not at all. Both chimeras also partially rescue chemotaxis. However, neither supports full development. Thus, neither regulation of myosin activity, nor regulation of myosin polymerization nor bipolar filaments is required for many functions of Dictyostelium myosin II and there may be no specific sequence required for localization of myosin to the cleavage furrow.

Acanthamoeba↗

PubMed2002

Visualizing PI3 kinase-mediated cell-cell signaling during Dictyostelium development.

BACKGROUND: Starving amoebae of Dictyostelium discoideum communicate by relaying extracellular cAMP signals, which direct chemotactic movement, resulting in the aggregation of thousands of cells into multicellular aggregates. Both cAMP relay and chemotaxis require the activation of PI3 kinase signaling. The spatiotemporal dynamics of PI3 kinase signaling can be followed in individual cells via the cAMP-induced membrane recruitment of a GFP-tagged PH domain-containing protein, CRAC, which is required for the activation of adenylylcyclase. RESULTS: We show that polarized periodic CRAC-GFP translocation occurs during the aggregation and mound stages of development in response to periodic cAMP signals. The duration of CRAC translocation to the membrane is determined by the duration of the rising phase of the cAMP signal. The system shows rapid adaptation and responds to the rate of change of the extracellular cAMP concentration. When the cells are in close contact, it takes 10 s for the signal to propagate from one cell to the next. In slugs, all cells show a permanent polarized PI3 kinase signaling in their leading edge, which is dependent on cell-cell contact. CONCLUSIONS: Measuring the redistribution of GFP-tagged CRAC has enabled us to study the dynamics of PI3 kinase-mediated cell-cell communication at the individual cell level in the multicellular stages of Dictyostelium development. This approach should also be useful to study the interactions between cell-cell signaling, cell polarization, and movement in the development of other organisms.

Animals↗