Research this year was focused on: (1) the biochemical and biophysical properties of actin with mutations of Tyr-53, and (2) the effects of expression of actin Tyr-53 mutants on the growth and development of Dictyostelium. Actin is one of the most highly conserved proteins. About 95% of all actins have the same amino acid in about 66% of the 375 positions, and in about 74% of the remaining positions the substitution is always the same amino acid, and often a conservative, substitution. For example, Tyr is at position 53 in all but 11 of the 323 actin sequences in the data base, and Phe replaces Tyr in all 11 exceptions, which raises the following questions. What is the critical role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently?. Interestingly, Tyr-53 is dynamically phosphorylated in Dictyostelium during the developmental cycle (50% of the actin in spores is phosphorylated), and when amoebae are subjected to stress. Actin Tyr-53 is also phosphorylated in cancer cells grown in culture. (1) Last year we found that mutation of Tyr-53 to Phe has no effect on actin's properties but that mutation of Tyr-53 to either Ala or Glu profoundly affects the biochemical and biophysical properties of Dictyostelium actin, in ways similar, but not identical, to phosphorylation of Tyr53. The Ala and Glu mutations modify the conformation of the D-loop so that actin's affinity for DNase I and pointed-end elongation are reduced, the critical concentration is increased, and the polymerized mutant actins form unstable filaments and small oligomers. In addition, the rates of nucleotide exchange by actin monomers and filaments are increased. We concluded that both Tyr and Phe (but not Ala or Glu) at position 53 maintain the functional conformations of the D-loop required for interaction with DNase I and actin polymerization, and that the conformation of the D-loop allosterically affects the conformation of the nucleotide-binding cleft. The evolutionary preference for Tyr at this position may be explained by providing the possibility of dynamic regulation of actins properties by phosphorylation. These studies were extended this year to see if the hydrophobic amino acids Trp and Leu could replace Tyr-53. These two mutations had much less, but still significant, effect on actin's properties. Y53W-actin polymerized at the same rate as WT-actin and Y53L-actin slightly slower but faster than Y53A-actin or Y53E-actin. The critical concentrations of both Y53W-actin and Y53L-actin were not significantly different than WT-actin, and both polymerized to normal filaments. The rates of nucleotide exchange by monomers of both Y53W-actin and Y53L-actin were faster than for WT-actin monomers and similar to the rates of nucleotide exchange for Y53A-actin and Y53E-actin. Thus, neither Trp nor Leu replaced Tyr-53 as well as Phe, but both were better substitutes for Tyr than Ala or Glu. (2) Last year we found that the properties of purified N-terminal FLAG-tagged WT-actin and the Y53F, Y53A and Y53E mutants were very similar to the properties of the actins without a FLAG-tag. When expressed in Dictyostelium, to the extent of 20% of total actin, the FLAG-actins co-localized with endogenous actin. Y53F-actin had no effect on cell phenotype. Y53A-actin had no effect on growth, pinocytosis or phagocytosis, but profoundly affected chemotaxis and development;although individual cells chemotaxed normally, the chemotaxing cells did not form streams, and formed smaller mounds that did not develop to normal fruiting bodies. The cells exressing Y53A-actin (Y53A-cells) had fewer cAMP-receptors, and reduced cAMP-induced ERK2 phosphorylation, cAMP-activation of adenylyl cyclase A and reduced cAMP-induced actin polymerization. In extension of these studies this year,we found that WT-cells did not follow chemotaxing Y53A-cells but did follow chemotaxing Y53F-cells. These observations, and the earlier data, are consistent with an inability of the Y53A-cells to secrete sufficient cAMP to attract neighboring cells, in addition to their diminished response to external cAMP. Electron microscopy of platinum-carbon replicas of detergent-extracted cytoskeletons of polarized, migrating cells revealed that expression of Y53A-actin substantially disrupted the actin cytoskeleton. Whereas the cytoskeletons of WT-cells and Y53F-cells consisted of a mostly homogeneous array of long actin filaments, the cytoskeleton of Y53A-cells had fewer long filaments, many short filaments, and bundles and aggregates of filaments consistent with the appearance of copolymers of purified WT-actin and Y53A-actin. There were also many large empty spaces in the cytoskeleton of Y53A-cells that were not present in either WT-cells or Y53F-cells. cAMP-secretion of chemotaxing amoebae involves the movement of vesicles containing adenylyl cyclase A from the front to the rear of the cells from which the vesicles are secreted forming a trail for neighboring cells to follow. ACA-vesicles traffic along microtubules and trafficking requires a functional actin cytoskeleton. We found that this process is dramatically inhibited in the Y53A-cells. Thus, it seems that the abnormal actin cytoskeleton of cells expressing Y53A-actin inhibits both intracellular and intercellular cAMP-signaling.
