Insight from the maximal activation of the signal transduction excitable network in Dictyostelium discoideum.

Cell migration requires the coordination of an excitable signal transduction network involving Ras and PI3K pathways with cytoskeletal activity. We show that expressing activated Ras GTPase-family proteins in cells lacking PTEN or other mutations which increase cellular protrusiveness transforms cells into a persistently activated state. Leading- and trailing-edge markers were found exclusively at the cell perimeter and the cytosol, respectively, of the dramatically flattened cells. In addition, the lifetimes of dynamic actin puncta were increased where they overlapped with actin waves, suggesting a mechanism for the coupling between these two networks. All of these phenotypes could be reversed by inhibiting signal transduction. Strikingly, maintaining cells in this state of constant activation led to a form of cell death by catastrophic fragmentation. These findings provide insight into the feedback loops that control excitability of the signal transduction network, which drives migration.

Shear force-based genetic screen reveals negative regulators of cell adhesion and protrusive activity.

The model organism Dictyostelium discoideum has greatly facilitated our understanding of the signal transduction and cytoskeletal pathways that govern cell motility. Cell-substrate adhesion is downstream of many migratory and chemotaxis signaling events. Dictyostelium cells lacking the tumor suppressor PTEN show strongly impaired migratory activity and adhere strongly to their substrates. We reasoned that other regulators of migration could be obtained through a screen for overly adhesive mutants. A screen of restriction enzyme-mediated integration mutagenized cells yielded numerous mutants with the desired phenotypes, and the insertion sites in 18 of the strains were mapped. These regulators of adhesion and motility mutants have increased adhesion and decreased motility. Characterization of seven strains demonstrated decreased directed migration, flatness, increased filamentous actin-based protrusions, and increased signal transduction network activity. Many of the genes share homology to human genes and demonstrate the diverse array of cellular networks that function in adhesion and migration.

Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes.

Chemotaxis, or directed migration of cells along a chemical gradient, is a highly coordinated process that involves gradient sensing, motility, and polarity. Most of our understanding of chemotaxis comes from studies of cells undergoing amoeboid-type migration, in particular the social amoeba Dictyostelium discoideum and leukocytes. In these amoeboid cells the molecular events leading to directed migration can be conceptually divided into four interacting networks: receptor/G protein, signal transduction, cytoskeleton, and polarity. The signal transduction network occupies a central position in this scheme as it receives direct input from the receptor/G protein network, as well as feedback from the cytoskeletal and polarity networks. Multiple overlapping modules within the signal transduction network transmit the signals to the actin cytoskeleton network leading to biased pseudopod protrusion in the direction of the gradient. The overall architecture of the networks, as well as the individual signaling modules, is remarkably conserved between Dictyostelium and mammalian leukocytes, and the similarities and differences between the two systems are the subject of this review.

Behavioral bioassays and their uses in Tetrahymena.

The swimming behaviors of Tetrahymena can be used in sensitive behavioral bioassays for estimating the effects of drugs, mutations, and other conditions on the physiological state of the cell. These assays can be used in both forward and reverse genetic approaches to help understand cellular functions from genotype to phenotype.

A knockout mutation of a constitutive GPCR in Tetrahymena decreases both G-protein activity and chemoattraction.

Although G-protein coupled receptors (GPCRs) are a common element in many chemosensory transduction pathways in eukaryotic cells, no GPCR or regulated G-protein activity has yet been shown in any ciliate. To study the possible role for a GPCR in the chemoresponses of the ciliate Tetrahymena, we have generated a number of macronuclear gene knockouts of putative GPCRs found in the Tetrahymena Genome database. One of these knockout mutants, called G6, is a complete knockout of a gene that we call GPCR6 (TTHERM_00925490). Based on sequence comparisons, the Gpcr6p protein belongs to the Rhodopsin Family of GPCRs. Notably, Gpcr6p shares highest amino acid sequence homologies to GPCRs from Paramecium and several plants. One of the phenotypes of the G6 mutant is a decreased responsiveness to the depolarizing ions Ba²âº and K⁺, suggesting a decrease in basal excitability (decrease in Ca²âº channel activity). The other major phenotype of G6 is a loss of chemoattraction to lysophosphatidic acid (LPA) and proteose peptone (PP), two known chemoattractants in Tetrahymena. Using microsomal [³5S]GTPγS binding assays, we found that wild-type (CU427) have a prominent basal G-protein activity. This activity is decreased to the same level by pertussis toxin (a G-protein inhibitor), addition of chemoattractants, or the G6 mutant. Since the basal G-protein activity is decreased by the GPCR6 knockout, it is likely that this gene codes for a constitutively active GPCR in Tetrahymena. We propose that chemoattractants like LPA and PP cause attraction in Tetrahymena by decreasing the basal G-protein stimulating activity of Gpcr6p. This leads to decreased excitability in wild-type and longer runs of smooth forward swimming (less interrupted by direction changes) towards the attractant. Therefore, these attractants may work as inverse agonists through the constitutively active Gpcr6p coupled to a pertussis-sensitive G-protein.

Chemoattraction to lysophosphatidic acid does not require a change in membrane potential in Tetrahymena thermophila.

LPA (lysophosphatidic acid), a known chemoattractant for many types of eukaryotic cells, is also a reliable chemoattractant for Tetrahymena. Since LPA receptors are GPCRs (G-protein coupled receptors) in many cell types and several putative GPCR sequences can be found in the Tetrahymena Genome Database, we are interested to determine whether similar GPCR pathways can be used for chemosensory transduction in Tetrahymena. To confirm our procedures, we tested the known chemoattractant proteose peptone (at 1.0 mg/ml), which caused hyperpolarization and increased forward swimming speed in Tetrahymena, consistent with the current model for ciliate chemoattraction. Although 10 µM LPA did not produce these same responses, it was still an effective chemoattractant. PTX (pertussis toxin) blocked attraction to both of these compounds, suggesting a possible G-protein involvement in chemoattraction. Both of these chemoattractants also decreased the basal percent of cells showing direction changes [PDC (percent directional change)] and the duration of backward swimming in 0.5 mM Ba2+ (a general excitability assay). LPA probably causes chemoattraction in Tetrahymena by decreasing the basal PDC without changing either membrane potential or swim speed. Since a pertussis-sensitive G-protein might modulate the ciliate voltage-dependent Ca2+ channels, we propose that LPA acts through an uncharacterized GPCR to lower the PDC by decreasing cellular excitability. These combined behavioural and electrophysiological analyses support the novel hypothesis that chemoattraction to some attractants, like LPA, can occur without hyperpolarization and increased swim speed in Tetrahymena.