Derrick Brazill

A Second UDP-glucose Pyrophosphorylase Is Required for Differentiation and Development in Dictyostelium discoideum

Uridine diphosphoglucose pyrophosphorylase (UDPGP) is a developmentally regulated enzyme in Dictyostelium discoideum, which is involved in trehalose, cellulose, and glycogen synthesis. Two independent UDPGP proteins are believed to be responsible for this activity. To determine the relative contributions of each protein, the genes encoding them were disrupted individually. Cells lacking the udpgp1 gene exhibit normal growth and development and make normal levels of cellulose. In agreement with these phenotypes, udpgp1 − cells still have UDPGP activity, although at a reduced level. This supports the importance of the second UDPGP gene. This newly identified gene,ugpB, encodes an active UDPGP as determined by complementation in Escherichia coli. When this gene is disrupted, cells undergo aberrant differentiation and development ending with small, gnarled fruiting bodies. These cells also have decreased spore viability and decreased levels of glycogen, whose production requires UDPGP activity. These phenotypes suggest that UgpB constitutes the major UDPGP activity produced during development. Sequence analysis of the two UDPGP genes shows that UgpB has higher homology to other eukaryotic UDPGPs than does UDPGP1. This includes the presence of 5 conserved lysine residues. Udpgp1 only has 1 of these lysines.

Cell density sensing and size determination

The social amoeba Dictyostelium discoideum is one of the leading model systems used to study how cells count themselves to determine the number and/or density of cells. In this review, we describe work on three different cell‐density sensing systems used by Dictyostelium. The first involves a negative feedback loop in which two secreted signals inhibit cell proliferation during the growth phase. As the cell density increases, the concentrations of the secreted factors concomitantly increase, allowing the cells to sense their density. The two signals act as message authenticators for each other, and the existence of two different signals that require each other for activity may explain why previous efforts to identify autocrine proliferation‐inhibiting signals in higher eukaryotes have generally failed. The second system involves a signal made by growing cells that is secreted only when they starve. This then allows cells to sense the density of just the starving cells, and is an example of a mechanism that allows cells in a tissue to sense the density of one specific cell type. The third cell density counting system involves cells in aggregation streams secreting a signal that limits the size of fruiting bodies. Computer simulations predicted, and experiments then showed, that the factor increases random cell motility and decreases cell–cell adhesion to cause streams to break up if there are too many cells in the stream. Together, studies on Dictyostelium cell density counting systems will help elucidate how higher eukaryotes regulate the size and composition of tissues.

A G protein-coupled receptor with a lipid kinase domain is involved in cell-density sensing.

One mechanism multicellular structures use for controlling cell number [1, 2] involves the secretion and sensing of a factor, such as leptin [3] or myostatin [4], in mammals. Dictyostelium cells secrete autocrine factors for sensing cell density prior to aggregation and multicellular development [5, 6] such as CMF (conditioned-medium factor), which enables starving cells to respond to cAMP pulses [7-9]. Its actions are mediated by two receptors. CMFR1 activates a G protein-independent signaling pathway regulating gene expression [10]. An unknown Galpha1-dependent receptor activates phospholipase C (PLC), which regulates the lifetime of Galpha2-GTP [11-13]. Here, we describe RpkA, an unusual seven-transmembrane receptor that is fused to a C-terminal PIP5 kinase domain and that localizes in membranes of a late endosomal compartment. Loss of RpkA resulted in formation of persistent loose aggregates and altered expression of cAMP-regulated genes. The developmental defect can be rescued by full-length RpkA and the transmembrane domain only. The PIP5 kinase domain is dispensable for the developmental role of RpkA. rpkA- cells secrete and bind CMF but are unable to induce downstream responses. Inactivation of Galpha1, a negative regulator of CMF signaling, rescued the developmental defect of the rpkA- cells, suggesting that RpkA actions are mediated by Galpha1.

PldB, a putative phospholipase D homologue in Dictyostelium discoideum mediates quorum sensing during development.

ABSTRACT Quorum sensing, also known as cell-density sensing in the unicellular eukaryote Dictyostelium discoideum, is required for efficient entry into the differentiation and development segment of its life cycle. Quorum sensing is accomplished by simultaneously secreting and sensing the glycoprotein Conditioned Medium Factor, or CMF. When the density of starving cells is high, CMF levels are high, which leads to aggregation followed by development. Here, we describe the role of pldB, a gene coding for a putative phospholipase D (PLD) homologue, in quorum sensing. We find that in submerged culture, adding butanol, an inhibitor of PLD-catalyzed phosphatidic acid production, allows cells to bypass the requirement for CMF mediated quorum sensing and aggregate at low cell density. Deletion of pldB mimics the presence of butanol, allowing cells to aggregate at low cell density. pldB− cells also initiate and finish aggregation rapidly. Analysis of early developmental gene expression in pldB− cells reveals that the cyclic AMP receptor cAR1 is expressed at higher levels earlier than in wild-type cells, which could explain the rapid aggregation phenotype. As would be predicted, cells overexpressing pldB are unable to aggregate even at high cell density. Adding CMF to these pldB− overexpressing cells does not rescue aggregation. Both of these phenotypes are cell autonomous, as mixing a small number of pldB− cells with wild-type cells does not cause the wild-type cells to behave like pldB− cells.

Serum amyloid P inhibits granulocyte adhesion

BackgroundThe extravasation of granulocytes (such as neutrophils) at a site of inflammation is a key aspect of the innate immune system. Signals from the site of inflammation upregulate granulocyte adhesion to the endothelium to initiate extravasation, and also enhance granulocyte adhesion to extracellular matrix proteins to facilitate granulocyte movement through the inflamed tissue. During the resolution of inflammation, other signals inhibit granulocyte adhesion to slow and ultimately stop granulocyte influx into the tissue. In a variety of inflammatory diseases such as acute respiratory distress syndrome, an excess infiltration of granulocytes into a tissue causes undesired collateral damage, and being able to reduce granulocyte adhesion and influx could reduce this damage.ResultsWe found that serum amyloid P (SAP), a constitutive protein component of the blood, inhibits granulocyte spreading and granulocyte adhesion to extracellular matrix components. This indicates that in addition to granulocyte adhesion inhibitors that are secreted during the resolution of inflammation, a granulocyte adhesion inhibitor is present at all times in the blood. Although SAP affects adhesion, it does not affect the granulocyte adhesion molecules CD11b, CD62L, CD18, or CD44. SAP also has no effect on the production of hydrogen peroxide by resting or stimulated granulocytes, or N-formyl-methionine-leucine-phenylalanine (fMLP)-induced granulocyte migration. In mice treated with intratracheal bleomycin to induce granulocyte accumulation in the lungs, SAP injections reduced the number of granulocytes in the lungs.ConclusionsWe found that SAP, a constitutive component of blood, is a granulocyte adhesion inhibitor. We hypothesize that SAP allows granulocytes to sense whether they are in the blood or in a tissue.

A cell-density sensing factor regulates the lifetime of a chemoattractant-induced Gα-GTP conformation

© 1997 Federation of European Biochemical Societies.

Phospholipase D controls Dictyostelium development by regulating G protein signaling.

Dictyostelium discoideum cells normally exist as individual amoebae, but will enter a period of multicellular development upon starvation. The initial stages of development involve the aggregation of individual cells, using cAMP as a chemoattractant. Chemotaxis is initiated when cAMP binds to its receptor, cAR1, and activates the associated G protein, Gα2βγ. However, chemotaxis will not occur unless there is a high density of starving cells present, as measured by high levels of the secreted quorum sensing molecule, CMF. We previously demonstrated that cells lacking PldB bypass the need for CMF and can aggregate at low cell density, whereas cells overexpressing pldB do not aggregate even at high cell density. Here, we found that PldB controlled both cAMP chemotaxis and cell sorting. PldB was also required by CMF to regulate G protein signaling. Specifically, CMF used PldB, to regulate the dissociation of Gα2 from Gβγ. Using fluorescence resonance energy transfer (FRET), we found that along with cAMP, CMF increased the dissociation of the G protein. In fact, CMF augmented the dissociation induced by cAMP. This augmentation was lost in cells lacking PldB. PldB appears to mediate the CMF signal through the production of phosphatidic acid, as exogenously added phosphatidic acid phenocopies overexpression of pldB. These results suggest that phospholipase D activity is required for CMF to alter the kinetics of cAMP-induced G protein signaling.

Dictyostelium discoideum paxillin regulates actin-based processes.

Paxillin is a key player in integrating the actin cytoskeleton with adhesion, and thus is essential to numerous cellular processes, including proliferation, differentiation, and migration in animal cells. PaxB, the Dictyostelium discoideum orthologue of paxillin, has been shown to be important for adhesion and development, much like its mammalian counterpart. Here, we use the overproduction of PaxB to gain better insight into its role in regulating the actin cytoskeleton and adhesion. We find that PaxB-overexpressing (PaxBOE) cells can aggregate and form mounds normally, but are blocked in subsequent development. This arrest can be rescued by addition of wild-type cells, indicating a non-cell autonomous role for PaxB. PaxBOE cells also have alterations in several actin-based processes, including adhesion, endocytosis, motility, and chemotaxis. PaxBOE cells exhibit an EDTA-sensitive increase in cell-cell cohesion, suggesting that PaxB-mediated adhesion is Ca(2+) or Mg(2+) dependent. Interestingly, cells overexpressing paxB are less adhesive to the substratum. In addition, PaxBOE cells display decreased motility under starved conditions, decreased endocytosis, and are unable to efficiently chemotax up a folate gradient. Taken together, the data suggest that proper expression of PaxB is vital for the regulation of development and actin-dependent processes.

PakD, a Putative p21-Activated Protein Kinase in Dictyostelium discoideum, Regulates Actin

ABSTRACT Proper regulation of the actin cytoskeleton is essential for cell function and ultimately for survival. Tight control of actin dynamics is required for many cellular processes, including differentiation, proliferation, adhesion, chemotaxis, endocytosis, exocytosis, and multicellular development. Here we describe a putative p21-activated protein kinase, PakD, that regulates the actin cytoskeleton in Dictyostelium discoideum. We found that cells lacking pakD are unable to aggregate and thus unable to develop. Compared to the wild type, cells lacking PakD have decreased membrane extensions, suggesting defective regulation of the actin cytoskeleton. pakD− cells show poor chemotaxis toward cyclic AMP (cAMP) but normal chemotaxis toward folate, suggesting that PakD mediates some but not all chemotaxis responses. pakD− cells have decreased polarity when placed in a cAMP gradient, indicating that the chemotactic defects of the pakD− cells may be due to an impaired cytoskeletal response to cAMP. In addition, while wild-type cells polymerize actin in response to global stimulation by cAMP, pakD− cells exhibit F-actin depolymerization under the same conditions. Taken together, the results suggest that PakD is part of a pathway coordinating F-actin organization during development.

Paxillin phosphorylation and complexing with Erk and FAK are regulated by PLD activity in MDA-MB-231 cells.

MDA-MB-231 cells are highly aggressive human breast adenocarcinoma cells that depend on PLD activity for survival. In response to the stress of serum withdrawal, there is increased motility and invasiveness of these cells that is associated with a rapid increase in PLD activity. In addition, PLD activity is elevated in response to most mitogenic signals. Similar to PLD, paxillin, a focal adhesion adaptor protein, and Erk, mitogen-activated protein kinase, play vital roles in cell motility through regulation of focal adhesion dynamics. Here, we addressed whether there is a functional correlation between paxillin and PLD that may influence cancer cell motility. We investigated the role of PLD activity on paxillin regulation, Erk activation and formation of a paxillin-Erk and paxillin-FAK association. Inhibition of PLD activity led to an increase in paxillin tyrosine phosphorylation, a decrease in Erk activation, as measured by phosphorylation, and enhanced association of paxillin with Erk. In addition, we found that paxillin tyrosine phosphorylation depends upon Erk activity and may be a consequence of an increased association with FAK. Taken together, these results suggest that Erk activity is governed by PLD activity and regulates the tyrosine phosphorylation of paxillin, potentially explaining its role in cell motility. This study indicated that PLD, Erk, paxillin and FAK participate in the same signaling pathway in this breast cancer cell line.