Adenylyl cyclase mRNA localizes to the posterior of polarized DICTYOSTELIUM cells during chemotaxis (original) (raw)
Related papers
Adenylyl cyclase localization to the uropod of aggregating Dictyostelium cells requires RacC
Biochemical and Biophysical Research Communications, 2015
The localization of adenylyl cyclase A (ACA) to uropod of cells is required for the stream formation during Dictyostelium development. RacC is a Dictyostelium orthologue of Cdc42. We identified a streaming defect of racC − cells as they are clearly less polarized and form smaller and fragmented streams. ACA-YFP is mainly associated with intracellular vesicular structures, but not with the plasma membrane in racC − cells. racC − cells have a slightly higher number of vesicles than Ax3 cells, suggesting that the defect of ACA trafficking is not simply due to the lack of vesicle formation. While the ACA-YFP vesicles traveled with an average velocity of 9.1 µm/min in Ax3 cells, a slow and diffusional movement without direction with an average velocity of 4 µm/min was maintained in racC − cells. Images acquired by using total internal reflection fluorescence (TIRF) microscopy and fluorescence recovery after photobleaching (FRAP) analysis revealed that a significantly decreased number of ACA-YFP vesicles appeared near the cell membrane, indicating a defect in ACA-YFP vesicle trafficking. These results suggest an important role of RacC in the rapid and directional movements of ACA vesicles on microtubules to the plasma membrane, especially to the back of polarized cell.
The Cyclase-associated Protein CAP as Regulator of Cell Polarity and cAMP Signaling in Dictyostelium
Molecular Biology of the Cell, 2004
involved in regulating the adenylyl cyclase activity. We show that cell polarization, F-actin organization, and phototaxis are altered in a Dictyostelium CAP knockout mutant. Furthermore, in complementation assays we determined the roles of the individual domains in signaling and regulation of the actin cytoskeleton. We studied in detail the adenylyl cyclase activity and found that the mutant cells have normal levels of the aggregation phase-specific adenylyl cyclase and that receptor-mediated activation is intact. However, cAMP relay that is responsible for the generation of propagating cAMP waves that control the chemotactic aggregation of starving Dictyostelium cells was altered, and the cAMP-induced cGMP production was significantly reduced. The data suggest an interaction of CAP with adenylyl cyclase in Dictyostelium and an influence on signaling pathways directly as well as through its function as a regulatory component of the cytoskeleton.
Eukaryotic Cell, 2005
Cyclic AMP (cAMP) functions as the extracellular chemoattractant in the aggregation phase of Dictyostelium development. There is some question, however, concerning what role, if any, it plays intracellularly in motility and chemotaxis. To test for such a role, the behavior of null mutants of acaA, the adenylyl cyclase gene that encodes the enzyme responsible for cAMP synthesis during aggregation, was analyzed in buffer and in response to experimentally generated spatial and temporal gradients of extracellular cAMP. acaA ؊ cells were defective in suppressing lateral pseudopods in response to a spatial gradient of cAMP and to an increasing temporal gradient of cAMP. acaA ؊ cells were incapable of chemotaxis in natural waves of cAMP generated by majority control cells in mixed cultures. These results indicate that intracellular cAMP and, hence, adenylyl cyclase play an intracellular role in the chemotactic response. The behavioral defects of acaA ؊ cells were surprisingly similar to those of cells of null mutants of regA, which encodes the intracellular phosphodiesterase that hydrolyzes cAMP and, hence, functions opposite adenylyl cyclase A (ACA). This result is consistent with the hypothesis that ACA and RegA are components of a receptor-regulated intracellular circuit that controls protein kinase A activity. In this model, the suppression of lateral pseudopods in the front of a natural wave depends on a complete circuit. Hence, deletion of any component of the circuit (i.e., RegA or ACA) would result in the same chemotactic defect.
Journal of Cell Science, 2008
The directed cell migration towards a chemotactic source, chemotaxis, involves three complex and interrelated processes: directional sensing, cell polarization and motility. Directional sensing allows migrating eukaryotic cells to chemotax in extremely shallow gradients (<2% across the cell body) of the chemoattractant. Although directional sensing has been observed as spatially restricted responses along the plasma membrane, our understanding of the `compass' of the cell that controls the gradient-induced translocation of proteins during chemotactic movements is still largely lacking. Until now, the dynamical behaviour and mobility of the chemoattractant-receptor molecule has been neglected in models describing the directional sensing mechanisms. Here, we show by single-molecule microscopy an agonist-induced increase in the mobile fraction of cAMP-receptor at the leading edge of chemotacting Dictyostelium discoideum cells. The onset of receptor mobility was correlated to the...
The Journal of Cell Biology, 2001
We have identified a novel gene, Tortoise ( TorA ), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA Ϫ partially restores chemotaxis, whereas overexpression of TorA in mek1 Ϫ does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA-green fluorescent protein (GFP) is clustered near one end of mitochondria. Dele-tion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.
A temperature-sensitive adenylyl cyclase mutant of Dictyostelium
The EMBO Journal, 2000
Dictyostelium development starts with the chemotactic aggregation of up to 10 6 amoebae in response to propagating cAMP waves. cAMP is produced by the aggregation stage adenylyl cyclase (ACA) and cells lacking ACA (aca null) cannot aggregate. Temperature-sensitive mutants of ACA were selected from a population of aca null cells transformed with a library of ACA genes, a major segment of which had been ampli®ed by error-prone PCR. One mutant (tsaca2) that can complement the aggregation null phenotype of aca null cells at 22°C but not at 28°C was characterized in detail. The basal catalytic activity of the enzyme in this mutant was rapidly and reversibly inactivated at 28°C. Using this mutant strain we show that cell movement in aggregates and mounds is organized by propagating waves of cAMP. Synergy experiments between wild-type and tsaca2 cells, shifted to the restrictive temperature at various stages of development, showed that ACA plays an important role in the control of cell sorting and tip formation.
A Continuum Analysis of the Chemotactic Signal Seen byDictyostelium discoideum
Journal of Theoretical Biology, 1998
We develop a mathematical model of cell-to-cell-signalling in Dictyostelium discoideum that predicts the cAMP signal seen by individual cells in early aggregation. The model employs two cells on a plane and is designed to predict the space-time characteristics of both the extracellular cAMP signal seen by one cell when a nearby cell relays, and the intracellular cAMP response produced by the stimulus in the receiving cell. The effect of membrane bound phosphodiesterase is studied and it is shown that cells can orient effectively even in its absence. Our results give a detailed picture of how the spatio-temporal characteristics of the extracellular signal can be transduced into a timeand space-dependent intracellular gradient, and they suggest a plausible mechanism for orientation in a natural chemotactic wave.
BMC Developmental Biology, 2012
Background: Cell adhesion, an integral part of D. discoideum development, is important for morphogenesis and regulated gene expression in the multicellular context and is required to trigger cell-differentiation. G-protein linked adenylyl cyclase pathways are crucially involved and a mutant lacking the aggregation specific adenylyl cyclase ACA does not undergo multicellular development. Results: Here, we have investigated the role of cyclase-associated protein (CAP), an important regulator of cell polarity and F-actin/G-actin ratio in the acamutant. We show that ectopic expression of GFP-CAP improves cell polarization, streaming and aggregation in acacells, but it fails to completely restore development. Our studies indicate a requirement of CAP in the ACA dependent signal transduction for progression of the development of unicellular amoebae into multicellular structures. The reduced expression of the cell adhesion molecule DdCAD1 together with csA is responsible for the defects in acacells to initiate multicellular development. Early development was restored by the expression of GFP-CAP that enhanced the DdCAD1 transcript levels and to a lesser extent the csA mRNA levels.
Analysis of chemotaxis in Dictyostelium
Methods in molecular biology (Clifton, N.J.), 2012
Dictyostelium discoideum is an excellent model organism for the study of directed cell migration, since Dictyostelium cells show robust chemotactic responses to the chemoattractant cAMP. Many powerful experimental tools are applicable, including forward and reverse genetics, biochemistry, microscopy, and proteomics. Recent studies have demonstrated that many components involved in chemotaxis are functionally conserved between human neutrophils and Dictyostelium amoebae. In this chapter, we describe how to define the functions of proteins that mediate and regulate cell motility, cell polarity, and directional sensing during chemotaxis in Dictyostelium.