A vesicle surface tyrosine kinase regulates phagosome maturation

Phagocytosis is crucial for host defense against microbial pathogens and for obtaining nutrients in Dictyostelium discoideum. Phagocytosed particles are delivered via a complex route from phagosomes to lysosomes for degradation, but the molecular mechanisms involved in the phagosome maturation process are not well understood. Here, we identify a novel vesicle-associated receptor tyrosine kinase-like protein, VSK3, in D. discoideum. We demonstrate how VSK3 is involved in phagosome maturation. VSK3 resides on the membrane of late endosomes/lysosomes with its C-terminal kinase domain facing the cytoplasm. Inactivation of VSK3 by gene disruption reduces the rate of phagocytosis in cells, which is rescued by re-expression of VSK3. We found that the in vivo function of VSK3 depends on the presence of the kinase domain and vesicle localization. Furthermore, VSK3 is not essential for engulfment, but instead, is required for the fusion of phagosomes with late endosomes/lysosomes. Our findings suggest that localized tyrosine kinase signaling on the surface of endosome/lysosomes represents a control mechanism for phagosome maturation

Distinct External Signals Trigger Sequential Release of Apical Organelles during Erythrocyte Invasion by Malaria Parasites

The invasion of erythrocytes by Plasmodium merozoites requires specific interactions between host receptors and parasite ligands. Parasite proteins that bind erythrocyte receptors during invasion are localized in apical organelles called micronemes and rhoptries. The regulated secretion of microneme and rhoptry proteins to the merozoite surface to enable receptor binding is a critical step in the invasion process. The sequence of these secretion events and the external signals that trigger release are not known. We have used time-lapse video microscopy to study changes in intracellular calcium levels in Plasmodium falciparum merozoites during erythrocyte invasion. In addition, we have developed flow cytometry based methods to measure relative levels of cytosolic calcium and study surface expression of apical organelle proteins in P. falciparum merozoites in response to different external signals. We demonstrate that exposure of P. falciparum merozoites to low potassium ion concentrations as found in blood plasma leads to a rise in cytosolic calcium levels through a phospholipase C mediated pathway. Rise in cytosolic calcium triggers secretion of microneme proteins such as the 175 kD erythrocyte binding antigen (EBA175) and apical membrane antigen-1 (AMA-1) to the merozoite surface. Subsequently, interaction of EBA175 with glycophorin A (glyA), its receptor on erythrocytes, restores basal cytosolic calcium levels and triggers release of rhoptry proteins. Our results identify for the first time the external signals responsible for the sequential release of microneme and rhoptry proteins during erythrocyte invasion and provide a starting point for the dissection of signal transduction pathways involved in regulated exocytosis of these key apical organelles. Signaling pathway components involved in apical organelle discharge may serve as novel targets for drug development since inhibition of microneme and rhoptry secretion can block invasion and limit blood-stage parasite growth

Gα-Mediated Inhibition of Developmental Signal Response

AbstractBackground: Seven-transmembrane receptor (7-TMR)-G protein networks are molecular sensors of extracellular signals in all eukarya. These pathways cycle through activated (sensitized) and inhibited (desensitized) states, and, while many of the molecular components for signal activation have been described, inhibitory mechanisms are not well characterized. In Dictyostelium, 7-TM cAMP receptors direct chemotaxis and development but also regulate the periodic synthesis of their own ligand, the chemoattractant/morphogen cAMP. We now demonstrate through loss-of-function/gain-of-function studies that the novel heterotrimeric Gα9 protein subunit regulates an inhibitory pathway during early Dictyostelium development for the cAMP signal response.Results:gα9 null cells form more cAMP signaling centers, are more resistant to compounds that inhibit cAMP signaling, and complete aggregation sooner and at lower cell densities than wild-type cells. These phentoypes are consistent with the loss of an inhibitory signaling pathway during development of gα9 null cells. Cells expressing constitutively activated Gα9 are defective in cAMP signaling center formation and development at low cell density and display an increased sensitivity to cAMP signal inhibition that is characteristic of enhanced suppression of the cAMP signal response. Finally, we demonstrate that gα9 null cells, which have been codeveloped with a majority of wild-type cells, primarily establish cAMP signaling centers and are able to non-autonomously direct wild-type cells to adopt a gα9 null-like phenotype.Conclusions: We suggest that Gα9 functions in an inhibitory-feedback pathway that regulates cAMP signaling center formation and propagation. Gα9 may be part of the mechanism that regulates lateral signal inhibition or that modulates receptor desensitization

A Gα-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 Gα subunit (Gα9) that antagonizes broad chemotactic response. Mechanistically, Gα9 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 Gα9 have extended PI(3,4,5)P(3), cAMP, and cGMP responses and are hyperpolarized. In contrast, cells expressing constitutively activated Gα9 exhibit a reciprocal phenotype. Their second message pathways are attenuated, and they have lost the ability to suppress lateral pseudopod formation. Potentially, functionally similar Gα-mediated inhibitory signaling may exist in other eukaryotic cells to regulate chemoattractant response

Imaging Protein‐Protein Interactions by Förster Resonance Energy Transfer (FRET) Microscopy in Live Cells

This unit describes an acceptor-sensitized emission FRET method using a confocal microscope for image acquisition. In contrast to acceptor photobleaching, which is an end-point assay that destroys the acceptor fluorophore, the sensitized emission method is amenable for FRET measurements in live cells and can be used to measure changes in FRET efficiency over time. The purpose of this unit is to provide a basic starting point for understanding and performing the sensitized emission method with a simple teaching tool for live-cell imaging. References that discuss the vagaries of acquiring and analyzing FRET between individually tagged molecules are provided

Imaging protein-protein interactions by Förster resonance energy transfer (FRET) microscopy in live cells

This unit describes an acceptor-sensitized emission FRET method using a confocal microscope for image acquisition. In contrast to acceptor photobleaching, which is an end-point assay that destroys the acceptor fluorophore, the sensitized emission method is amenable for FRET measurements in live cells and can be used to measure changes in FRET efficiency over time. The purpose of this unit is to provide a basic starting point for understanding and performing the sensitized emission method with a simple teaching tool for live-cell imaging. References that discuss the vagaries of acquiring and analyzing FRET between individually tagged molecules are provided

The Elmo family forms an ancient group of actin-regulating proteins

The Elmo protein family members are important mediators of small G protein activity, regulating actin-mediated processes such as chemotaxis and engulfment. Until recently,1 Elmo function has not been explored in professional phagocytes such as Dictyostelium discoideum. We discuss the significance of this family with respect to pathways that regulate Rac signaling, we present a comparison of Elmo proteins between representative taxa, and discuss our findings on ElmoA, one of six Elmo proteins found in D. discoideum

Coupling mechanism of a GPCR and a heterotrimeric G protein during chemoattractant gradient sensing in Dictyostelium.

The coupling of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) with G proteins is fundamental for GPCR signaling; however, the mechanism of coupling is still debated. Moreover, how the proposed mechanisms affect the dynamics of downstream signaling remains unclear. Here, through experiments involving fluorescence recovery after photobleaching and single-molecule imaging, we directly measured the mobilities of cyclic adenosine monophosphate (cAMP) receptor 1 (cAR1), a chemoattractant receptor, and a G protein βγ subunit in live cells. We found that cAR1 diffused more slowly in the plasma membrane than did Gβγ. Upon binding of ligand to the receptor, the mobility of cAR1 was unchanged, whereas the speed of a fraction of the faster-moving Gβγ subunits decreased. Our measurements showed that cAR1 was relatively immobile and Gβγ diffused freely, suggesting that chemoattractant-bound cAR1 transiently interacted with G proteins. Using models of possible coupling mechanisms, we computed the temporal kinetics of G protein activation. Our fluorescence resonance energy transfer imaging data showed that fully activated cAR1 induced the sustained dissociation of G protein α and βγ subunits, which indicated that ligand-bound cAR1 activated G proteins continuously. Finally, simulations indicated that a high-affinity coupling of ligand-bound receptors and G proteins was essential for cAR1 to translate extracellular gradient signals into directional cellular responses. We suggest that chemoattractant receptors use a ligand-induced coupling rather than a precoupled mechanism to control the activation of G proteins during chemotaxis