RasC Plays a Role in Transduction of Temporal Gradient Information in the Cyclic-AMP Wave ofDictyostelium discoideum

Abstract

To define the role that RasC plays in motility and chemotaxis, the behavior of a rasC null mutant, rasC ؊ , in buffer and in response to the individual spatial, temporal, and concentration components of a natural cyclic AMP (cAMP) wave was analyzed by using computer-assisted two-dimensional and three-dimensional motion analysis systems. These quantitative studies revealed that rasC ؊ cells translocate at the same velocity and exhibit chemotaxis up spatial gradients of cAMP with the same efficiency as control cells. However, rasC ؊ cells exhibit defects in maintaining anterior-posterior polarity along the substratum and a single anterior pseudopod when translocating in buffer in the absence of an attractant. rasC ؊ cells also exhibit defects in their responses to both the increasing and decreasing temporal gradients of cAMP in the front and the back of a wave. These defects result in the inability of rasC ؊ cells to exhibit chemotaxis in a natural wave of cAMP. The inability to respond normally to temporal gradients of cAMP results in defects in the organization of the cytoskeleton, most notably in the failure of both F actin and myosin II to exit the cortex in response to the decreasing temporal gradient of cAMP in the back of the wave. While the behavioral defect in the front of the wave is similar to that of the myoA ؊ /myoF ؊ myosin I double mutant, the behavioral and cytoskeletal defects in the back of the wave are similar to those of the S13A myosin II regulatory light-chain phosphorylation mutant. Expression array data support the premise that the behavioral defects exhibited by the rasC ؊ mutant are the immediate result of the absence of RasC function.The Ras GTPases function as molecular switches in the regulation of a variety of responses to extracellular signals (3,21,35,36). Dictyostelium discoideum, like higher eukaryotes, contains a number of Ras GTPases (6,21,22). Because of its attributes, Dictyostelium provides a unique experimental system for exploring the roles played by the Ras GTPases in cell motility and chemotaxis (6,21,49). First, because it is haploid, null mutations are readily generated and rescued (16,18). Second, because the behavior of Dictyostelium amoebae in buffer and in response to the temporal, spatial, and concentration components of the natural chemotactic wave has been characterized in detail by computer-assisted methods (33, 34), a unique contextual framework exists for identifying specific behavioral defects manifested in mutants (29) and for deducing from them the specific role played by mutated genes in motility and/or chemotaxis (4,8,10,42,45,50,51).In a previous study, it was demonstrated that rasC Ϫ cells could not progress through the early stages of development or form aggregates unless they were pulsed with the chemoattractant cyclic AMP (cAMP), indicating that RasC was necessary for signaling (20). rasC Ϫ cells artificially pulsed with cAMP were then capable of forming aggregates on filter pads. When mixed with a majority of normal cells, they could also enter aggr...

Delineation of the Roles Played by RasG and RasC in cAMP-dependent Signal Transduction during the Early Development ofDictyostelium discoideum

“…Calculation of the rates of cell motility revealed no significant difference between the JH10 and JH10/rasC Ϫ cells, but average instantaneous velocities of the JH10/rasG Ϫ cells and JH10/rasG Ϫ rasC Ϫ were lower than those for the wild type (Table 1). The instantaneous velocity values obtained for the JH10 and JH10/rasC Ϫ cells were not significantly different from the values published previously for AX2 and AX2/rasC Ϫ strains (Wessels et al, 2004). A calculation of chemotactic index revealed no reduction relative to JH10 for the JH10/rasC Ϫ cells, but the index was lower for the JH10/rasG Ϫ cells and very low for the …”

“…At t ϭ 0, a micropipette (Eppendorf Femtotip, Hamburg, Germany) filled with 100 M cAMP was placed in the field of view, and cell movements toward the micropipette tip were monitored by time-lapse videomicroscopy using an Olympus IX-70 inverted microscope (Melville, NY), a DAGE-MTI CCD-100 camera (Michigan City, IN), and Scion (Frederick, MD) Image 4.0 software. Instantaneous velocities and chemotaxis indices were determined as described previously (Wessels et al, 2004).…”

The IplA Ca++ Channel OfDictyostelium discoideumIs Necessary For Ca++, But Not cAMP Chemotaxis, And Plays A Fundamental Role In Natural Aggregation

“…This prediction is predicated on their normal responses to spatial and temporal gradients of cAMP assayed in vitro. To test this prediction, we analyzed the behavior of vitally stained minority iplA -cells seeded in majority wildtype cell aggregation territories (Wessels et al, 2000a;Wessels et al, 2000b;Wessels et al, 2004;Wessels et al, 2007;Heid et al, 2004;Stepanovic et al, 2005). Under these conditions, minority mutant cells are challenged with natural waves relayed by majority wild-type cells.…”

Interferon regulatory factor 6 regulates keratinocyte migration

“…Persistence was essentially computed by dividing the speed by direction change, the latter given in grads rather than degrees, and adding one to the denominator to prevent division by 0. Area and roundness were computed from contours of the beta-spline replacement images (Soll and Voss, 1998;Wessels et al, 2004).…”