Chapter 17 Transmembrane Signaling in Dictyostelium

Devreotes, Peter N.; Fontana, Donna R.; Klein, Peter S.; Sherring, Jane; Theibert, Anne B.

doi:10.1016/s0091-679x(08)61653-2

Cited by 87 publications

(69 citation statements)

References 63 publications

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“…Cells were differentiated to the chemotaxis-competent stage as described (27,28). Briefly, log-phase cells (ϳ2 ϫ 10 6 cells/ml) were harvested by low-speed centrifugation, washed, and re-suspended in developmental buffer (5 mM Na 2 HPO 4 , 5 mM KH 2 PO 4 , pH 6.2, 2 mM Mg 2 SO 4 , 0.2 mM CaCl 2 ) at 2 ϫ 10 7 cells/ml (25), and developed in suspension at 100 rpm for 5-6 h with pulses of 50 l of 4 M cAMP into 5 ml of cells every 6 min.…”

Section: Methodsmentioning

confidence: 99%

Expression of Y53A-Actin in Dictyostelium Disrupts the Cytoskeleton and Inhibits Intracellular and Intercellular Chemotactic Signaling

Shu

Liu

Kriebel

et al. 2010

Journal of Biological Chemistry

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We showed previously that phosphorylation of Tyr 53 , or its mutation to Ala, inhibits actin polymerization in vitro with formation of aggregates of short filaments, and that expression of Y53A-actin in Dictyostelium blocks differentiation and development at the mound stage (Liu, X., Shu, S. We now show that expression of Y53A-actin, which does not affect cell growth, phagocytosis, or pinocytosis, inhibits the formation of head-to-tail cell streams during cAMP-induced aggregation, although individual amoebae chemotax normally. We show that expression of Y53A-actin causes a 50% reduction of cell surface cAMP receptors, and inhibits cAMPinduced increases in adenylyl cyclase A activity, phosphorylation of ERK2, and actin polymerization. Trafficking of vesicles containing adenylyl cyclase A to the rear of the cell and secretion of the ACA vesicles are also inhibited. The actin cytoskeleton of cells expressing Y53A-actin is characterized by numerous short filaments, and bundled and aggregated filaments similar to the structures formed by copolymerization of purified Y53A-actin and wild-type actin in vitro. This disorganized actin cytoskeleton may be responsible for the inhibition of intracellular and intercellular cAMP signaling in cells expressing F-Y53A-actin.

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Section: Methodsmentioning

confidence: 99%

Expression of Y53A-Actin in Dictyostelium Disrupts the Cytoskeleton and Inhibits Intracellular and Intercellular Chemotactic Signaling

Shu

Liu

Kriebel

et al. 2010

Journal of Biological Chemistry

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“…The cells were pulsed with 75 nM cAMP (final concentration) at 6-min intervals (43), and samples were taken at 1-h intervals for SDS/PAGE.…”

Section: Methodsmentioning

confidence: 99%

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

Shu

Mahadeo

Liu

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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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 starvationinduced expression of the cAMP receptor, cAR1, or G proteinmediated 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.chemotaxis ͉ Dictyostelium ͉ neutrophils ͉ tubercidin S -adenosylhomocysteine (SAH), the product of the transfer of the methyl group from S-adenosylmethionine (SAM) to DNA, RNA, phospholipids and many small molecules ( Fig. 1), is a strong inhibitor of transmethylation (1). The inhibition is relieved in prokaryotes by hydrolysis of SAH to adenine and ribosylhomocysteine (2), and in eukaryotes by hydrolysis of SAH to adenosine and homocysteine, catalyzed by SAH hydrolase (SAHH) (3). Because hydrolysis of SAH is reversible, and more favored in the synthetic direction, efficient transmethylation from SAM in eukaryotes also requires removal of adenosine and homocysteine (Fig. 1).Stimulated by the discovery in the mid-1970s that bacterial chemotaxis is initiated by SAM-dependent transient carboxyl-O-methylation of glutamic acid residues in chemotactic ligand receptor proteins (4, 5), the possibility that transmethylation reactions have a role in the chemotactic response of Dictyostelium and leukocytes was investigated in the early 1980s. Investigators asked whether chemotactic factors increase methylation of proteins, phospholipids, and/or nucleic acids, and whether inhibition of methylation by inhibiting SAHH or adenosine deaminase inhibits chemotaxis. The results of these studies were inconclusive and often contradictory (see Discussion).In the contemporary view of chemotaxis of Dictyostelium and leukocytes, chemoattractants mediate their effects by binding to transmembrane receptors coupled to heterotrimer...

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“…Amoebae aggregation was assayed on 35-mm nonnutrient agar as described previously (Devreotes et al, 1987). Chemotaxis assays and cAMP-binding assays were carried out according to the protocols by Xiao et al (1997).…”

Section: Functional Assaysmentioning

confidence: 99%

Gγ inDictyostelium: Its Role in Localization of Gβγ to the Membrane Is Required for Chemotaxis in Shallow Gradients

Zhang

Lan

Devreotes

2001

MBoC

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Monitoring Editor: Joan S. Brugge G-protein-mediated signal transduction pathways play an essential role in the developmental program of the simple eukaryotic organism Dictyostelium discoideum. Database searches have yielded 11 G␣-subunits, a single G␤-subunit, but no G␥-subunits. We report here the purification, cDNA isolation, and functional analysis of a G␥-subunit. Like G␤, the G␥ appears to be unique and hybridization studies show that G␥ and G␤ are expressed in parallel during development. Species-wide sequence comparisons of G␥-subunits and ␥-like domains of RGS proteins reveal short stretches of highly conserved residues as well as the common CXXL motif at the COOHterminal of G␥s that target G␤␥s to plasma membrane. Overexpression of a CSVL-deleted G␥ (G␥⌬) in wild-type cells shifts G␤␥ to the cytosol and selectively impairs certain G-proteinmediated signal transduction pathways. These cells are able to respond to increments in the stimulus, but are unable to sense chemoattractant gradients. They neither move directionally nor recruit PH-domains to their leading edge. Thus, a full complement of membrane-tethered G␤␥ is required for sensing shallow gradients, but is not essential for responses to increments in extracellular stimuli.

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Chapter 17 Transmembrane Signaling in Dictyostelium

Cited by 87 publications

References 63 publications

Expression of Y53A-Actin in Dictyostelium Disrupts the Cytoskeleton and Inhibits Intracellular and Intercellular Chemotactic Signaling

Expression of Y53A-Actin in Dictyostelium Disrupts the Cytoskeleton and Inhibits Intracellular and Intercellular Chemotactic Signaling

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

Gγ inDictyostelium: Its Role in Localization of Gβγ to the Membrane Is Required for Chemotaxis in Shallow Gradients

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