Jane Borleis

Structurally distinct and stage-specific adenylyl cyclase genes play different roles in dictyostelium development

We have isolated two adenylyl cyclase genes, designated ACA and ACG, from Dictyostelium. The proposed structure for ACA resembles that proposed for mammalian adenylyl cyclases: two large hydrophilic domains and two sets of six transmembrane spans. ACG has a novel structure, reminiscent of the membrane-bound guanylyl cyclases. An aca- mutant, created by gene disruption, has little detectable adenylyl cyclase activity and fails to aggregate, demonstrating that cAMP is required for cell-cell communication. cAMP is not required for motility, chemotaxis, growth, and cell division, which are unaffected. Constitutive expression in aca- cells of either ACA or ACG, which is normally expressed only during germination, restores aggregation and the ability to complete the developmental program. ACA expression restores receptor and guanine nucleotide-regulated adenylyl cyclase activity, while activity in cells expressing ACG is insensitive to these regulators. Although they lack ACA, which has a transporter-like structure, the cells expressing ACG secrete cAMP constitutively.

Random mutagenesis of the cAMP chemoattractant receptor, cAR1, of Dictyostelium. Mutant classes that cause discrete shifts in agonist affinity and lock the receptor in a novel activational intermediate

The cAMP chemoattractant receptor, cAR1, of Dictyostelium transduces extracellular cAMP signals via G protein-dependent and G protein-independent mechanisms. While site-directed mutagenesis studies of G protein-coupled receptors have provided a host of information regarding the domains essential for various functions, many mechanistic and structural questions remain to be resolved. We therefore carried out polymerase chain reaction-mediated random mutagenesis over a large part of the cAR1 sequence (from TMIII through the proximal part of the cytoplasmic tail). We devised a rapid screen for loss-of-function mutations based on the essential role of cAR1 in the developmental program of Dictyostelium. Although there were an average of two amino acid substitutions per receptor, ∼90% of the mutants were able to substitute for wild-type cAR1 when expressed in receptor null cells. About 2% were loss-of-function mutants that expressed wild-type levels of receptor protein. We used biochemical screens to select about 100 of these mutants and chose eight representative mutants for extensive characterization. These fell into distinct classes. One class had a conditional defect in cAMP binding that was reversed by high salt. Another large class had decreased affinity under all conditions. Curiously, the decreases were clustered into three discrete intervals. One of the most interesting class of mutants lost all capacity for signal transduction but was phosphorylated in response to agonist binding. This latter finding suggests that there are at least two activated states of cAR1 that can be recognized by different downstream effectors.

Tumor suppressor Hippo/MST1 kinase mediates chemotaxis by regulating spreading and adhesion

Chemotaxis depends on a network of parallel pathways that coordinate cytoskeletal events to bias cell movement along a chemoattractant gradient. Using a forward genetic screen in Dictyostelium discoideum, we identified the Ste20 kinase KrsB, a homolog of tumor suppressors Hippo and MST1/2, as a negative regulator of cell spreading and substrate attachment. The excessive adhesion of krsB− cells reduced directional movement and prolonged the streaming phase of multicellular aggregation. These phenotypes depended on an intact kinase domain and phosphorylation of a conserved threonine (T176) within the activation loop. Chemoattractants triggered a rapid, transient autophosphorylation of T176 in a heterotrimeric G protein-dependent and PI3K- and TorC2-independent manner. The active phosphorylated form of KrsB acts to decrease adhesion to the substrate. Taken together these studies suggest that cycling between active and inactive forms of KrsB may provide the dynamic regulation of cell adhesion needed for proper cell migration and chemotaxis. KrsB interacts genetically with another D. discoideum Hippo/MST homolog, KrsA, but the two genes are not functionally redundant. These studies show that Hippo/MST proteins, like the tumor suppressor PTEN and oncogenes Ras and PI3K, play a key role in cell morphological events in addition to their role in regulating cell growth.

Chemical and mechanical stimuli act on common signal transduction and cytoskeletal networks

Significance Cells directionally migrate in response to a variety of external cues, including chemical, electrical, and mechanical stimuli; however, only response to chemoattractants has been characterized at the molecular level. Binding of chemoattractants to specific surface receptors triggers rapid, transient activation of many signal transduction and cytoskeletal events. We discovered that brief application of shear stress to cells likely elicits activation of all of the same events. Responses to chemoattractants and shear stress are susceptible to many of the same perturbations, although that to mechanical stimulation uniquely is blocked by disruption of the actin cytoskeleton. Our finding provides insight into the molecular mechanism of cellular response to mechanical stimuli and has important implications for integration of chemical and mechanical inputs. Signal transduction pathways activated by chemoattractants have been extensively studied, but little is known about the events mediating responses to mechanical stimuli. We discovered that acute mechanical perturbation of cells triggered transient activation of all tested components of the chemotactic signal transduction network, as well as actin polymerization. Similarly to chemoattractants, the shear flow-induced signal transduction events displayed features of excitability, including the ability to mount a full response irrespective of the length of the stimulation and a refractory period that is shared with that generated by chemoattractants. Loss of G protein subunits, inhibition of multiple signal transduction events, or disruption of calcium signaling attenuated the response to acute mechanical stimulation. Unlike the response to chemoattractants, an intact actin cytoskeleton was essential for reacting to mechanical perturbation. These results taken together suggest that chemotactic and mechanical stimuli trigger activation of a common signal transduction network that integrates external cues to regulate cytoskeletal activity and drive cell migration.

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

Significance We report a forward genetic screen to identify genes involved in cell adhesion and motility. Cells with mutations in these genes have increased adhesion, flattened morphology, and decreased motility. The mutants display increased cytoskeletal and signal transduction network activity suggesting that these genes are negative regulators. The GFP-tagged localization of these proteins shows the remarkable diversity in the regulation of these cell behaviors. Several of the identified proteins have strong homologs throughout metazoans and have relevance to human disease. Because many of the resulting mutant phenotypes are similar to those of cells lacking PTEN or expressing active Ras GTPases, these gene families are promising cancer targets in humans. Better understanding of these pathways holds the possibility for therapeutic intervention. 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.

Novel protein Callipygian defines the back of migrating cells

Significance Though the asymmetric distribution of proteins is a crucial first step in establishing polarity and guiding cell migration, the molecular mechanisms regulating many of these localizations are unknown. Our study reports on the novel protein Callipygian (CynA), which localizes to the rear of cells during symmetry breaking, thereby promoting polarity and increasing migration efficiency. Our data indicate that CynA localization is mediated by two distinct mechanisms, which may be important for segregating proteins in other polarized cell types including epithelial cells, neurons, and immune cells. Thus, our findings have implications for tissue formation during embryonic development, the migration of immune cells during wound healing and infection, and the aberrant migrations associated with arthritis, asthma, atherosclerosis, cancer metastasis, and other diseases. Asymmetric protein localization is essential for cell polarity and migration. We report a novel protein, Callipygian (CynA), which localizes to the lagging edge before other proteins and becomes more tightly restricted as cells polarize; additionally, it accumulates in the cleavage furrow during cytokinesis. CynA protein that is tightly localized, or “clustered,” to the cell rear is immobile, but when polarity is disrupted, it disperses throughout the membrane and responds to uniform chemoattractant stimulation by transiently localizing to the cytosol. These behaviors require a pleckstrin homology-domain membrane tether and a WD40 clustering domain, which can also direct other membrane proteins to the back. Fragments of CynA lacking the pleckstrin homology domain, which are normally found in the cytosol, localize to the lagging edge membrane when coexpressed with full-length protein, showing that CynA clustering is mediated by oligomerization. Cells lacking CynA have aberrant lateral protrusions, altered leading-edge morphology, and decreased directional persistence, whereas those overexpressing the protein display exaggerated features of polarity. Consistently, actin polymerization is inhibited at sites of CynA accumulation, thereby restricting protrusions to the opposite edge. We suggest that the mutual antagonism between CynA and regions of responsiveness creates a positive feedback loop that restricts CynA to the rear and contributes to the establishment of the cell axis.