Carole A. Parent

G PROTEIN SIGNALING EVENTS ARE ACTIVATED AT THE LEADING EDGE OF CHEMOTACTIC CELLS

Directional sensing by eukaryotic cells does not require polarization of chemoattractant receptors. The translocation of the PH domain-containing protein CRAC in D. discoideum to binding sites on the inner face of the plasma membrane reflects activation of the G protein-linked signaling system. Increments in chemoattractant elicit a uniform response around the cell periphery. Yet when cells are exposed to a gradient, the activation occurs selectively at the stimulated edge, even in immobilized cells. We propose that such localized activation, transmitted by the recruitment of cytosolic proteins, may be a general mechanism for gradient sensing by G protein-linked chemotactic systems including those involving chemotactic cytokines in leukocytes.

The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase

We have identified a MAP kinase kinase (DdMEK1) that is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild‐type cells. Time‐lapse video microscopy and in vitro assays indicate that the cells are able to produce cAMP waves that move through the aggregation domains. However, these cells are unable to undergo chemotaxis properly during aggregation in response to the chemoattractant cAMP or activate guanylyl cyclase, a known regulator of chemotaxis in Dictyostelium. The activation of guanylyl cyclase in response to osmotic stress is, however, normal. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect and the ability to activate guanylyl cyclase. However, this does not result in constitutive activation of guanylyl cyclase, suggesting that DdMEK1 activity is necessary, but not sufficient, for cAMP activation of guanylyl cyclase. Analysis of a temperature‐sensitive DdMEK1 mutant suggests that DdMEK1 activity is required throughout aggregation at the time of guanylyl cyclase activation, but is not essential for proper morphogenesis during the later multicellular stages. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium.

Constitutively Active Adenylyl Cyclase Mutant Requires Neither G Proteins nor Cytosolic Regulators

Receptor-mediated G protein-linked adenylyl cyclase systems are universal signal transducers. We exploited the essential role of this cascade in Dictyostelium development to screen for random mutations in the catalytic component, ACA. This enzyme is activated by G protein βγ-subunits acting in concert with a novel cytosolic regulator, CRAC. By suppression of the CRAC-null phenotype, we isolated constitutively active versions of the enzyme that require neither exogenous stimuli nor internal regulators. One mutant displayed a 15-fold increase in its Vmax. It harbors a single amino acid substitution (L394S) affecting a conserved residue located in the first cytoplasmic loop near the N-terminal hydrophobic domain of ACA. The screening procedure can be adapted for isolation of constitutive mutations in mammalian adenylyl cyclases.

A temperature‐sensitive adenylyl cyclase mutant of Dictyostelium

Dictyostelium development starts with the chemotactic aggregation of up to 106 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 amplified 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.