Loss of the histidine kinase DhkD results in mobile mounds during development of Dictyostelium discoideum.

BACKGROUND: Histidine kinases are receptors for sensing cellular and environmental signals, and in response to the appropriate cue they initiate phosphorelays that regulate the activity of response regulators. The Dictyostelium discoideum genome encodes 15 histidine kinases that function to regulate several processes during the multicellular developmental program, including the slug to culmination transition, osmoregulation, and spore differentiation. While there are many histidine kinases, there is only a single response regulator, RegA. Not surprisingly given the ubiquitous involvement of cAMP in numerous processes of development in Dictyostelium, RegA is a cAMP phosphodiesterase that is activated upon receiving phosphates through a phosphorelay. Hence, all of the histidine kinases characterized to date regulate developmental processes through modulating cAMP production. Here we investigate the function of the histidine kinase DhkD. PRINCIPAL FINDINGS: The dhkD gene was disrupted, and the resulting cells when developed gave a novel phenotype. Upon aggregation, which occurred without streaming, the mounds were motile, a phenotype termed the pollywog stage. The pollywog phenotype was dependent on a functional RegA. After a period of random migration, the pollywogs attempted to form fingers but mostly generated aberrant structures with no tips. While prestalk and prespore cell differentiation occurred with normal timing, proper patterning did not occur. In contrast, wild type mounds are not motile, and the cAMP chemotactic movement of cells within the mound facilitates proper prestalk and prespore patterning, tip formation, and the vertical elongation of the mound into a finger. CONCLUSIONS: We postulate that DhkD functions to ensure the proper cAMP distribution within mounds that in turn results in patterning, tip formation and the transition of mounds to fingers. In the absence of DhkD, aberrant cell movements in response to an altered cAMP distribution result in mound migration, a lack of proper patterning, and an inability to generate normal finger morphology.

eIF2α kinases regulate development through the BzpR transcription factor in Dictyostelium discoideum.

BACKGROUND: A major mechanism of translational regulation in response to a variety of stresses is mediated by phosphorylation of eIF2α to reduce delivery of initiator tRNAs to scanning ribosomes. For some mRNAs, often encoding a bZIP transcription factor, eIF2α phosphorylation leads to enhanced translation due to delayed reinitiation at upstream open reading frames. Dictyostelium cells possess at least three eIF2α kinases that regulate various portions of the starvation-induced developmental program. Cells possessing an eIF2α that cannot be phosphorylated (BS167) show abnormalities in growth and development. We sought to identify a bZIP protein in Dictyostelium whose production is controlled by the eIF2α regulatory system. PRINCIPAL FINDINGS: Cells disrupted in the bzpR gene had similar developmental defects as BS167 cells, including small entities, stalk defects, and reduced spore viability. ß-galactosidase production was used to examine translation from mRNA containing the bzpR 5' UTR. While protein production was readily apparent and regulated temporally and spatially in wild type cells, essentially no ß-galactosidase was produced in developing BS167 cells even though the lacZ mRNA levels were the same as those in wild type cells. Also, no protein production was observed in strains lacking IfkA or IfkB eIF2α kinases. GFP fusions, with appropriate internal controls, were used to directly demonstrate that the bzpR 5' UTR, possessing 7 uORFs, suppressed translation by 12 fold. Suppression occurred even when all but one uORF was deleted, and translational suppression was removed when the ATG of the single uORF was mutated. CONCLUSIONS: The findings indicate that BzpR regulates aspects of the development program in Dictyostelium, serving as a downstream effector of eIF2α phosphorylation. Its production is temporally and spatially regulated by eIF2α phosphorylation by IfkA and IfkB and through the use of uORFs within the bzpR 5' UTR.

eIF2α kinases control chalone production in Dictyostelium discoideum.

Growing Dictyostelium cells secrete CfaD and AprA, two proteins that have been characterized as chalones. They exist within a high-molecular-weight complex that reversibly inhibits cell proliferation, but not growth, via cell surface receptors and a signaling pathway that includes G proteins. How the production of these two proteins is regulated is unknown. Dictyostelium cells possess three GCN2-type eukaryotic initiation factor 2 α subunit (eIF2α) kinases, proteins that phosphorylate the translational initiation factor eIF2α and possess a tRNA binding domain involved in their regulation. The Dictyostelium kinases have been shown to function during development in regulating several processes. We show here that expression of an unregulated, activated kinase domain greatly inhibits cell proliferation. The inhibitory effect on proliferation is not due to a general inhibition of translation. Instead, it is due to enhanced production of a secreted factor(s). Indeed, extracellular CfaD and AprA proteins, but not their mRNAs, are overproduced in cells expressing the activated kinase domain. The inhibition of proliferation is not seen when the activated kinase domain is expressed in cells lacking CfaD or AprA or in cells that contain a nonphosphorylatable eIF2α. We conclude that production of the chalones CfaD and AprA is translationally regulated by eIF2α phosphorylation. Both proteins are upregulated at the culmination of development, and this enhanced production is lacking in a strain that possesses a nonphosphorylatable eIF2α.

Subcellular localization of ammonium transporters in Dictyostelium discoideum.

BACKGROUND: With the exception of vertebrates, most organisms have plasma membrane associated ammonium transporters which primarily serve to import a source of nitrogen for nutritional purposes. Dictyostelium discoideum has three ammonium transporters, Amts A, B and C. Our present work used fluorescent fusion proteins to determine the cellular localization of the Amts and tested the hypothesis that the transporters mediate removal of ammonia generated endogenously from the elevated protein catabolism common to many protists. RESULTS: Using RFP and YFP fusion constructs driven by the actin 15 promoter, we found that the three ammonium transporters were localized on the plasma membrane and on the membranes of subcellular organelles. AmtA and AmtB were localized on the membranes of endolysosomes and phagosomes, with AmtB further localized on the membranes of contractile vacuoles. AmtC also was localized on subcellular organelles when it was stabilized by coexpression with either the AmtA or AmtB fusion transporter. The three ammonium transporters exported ammonia linearly with regard to time during the first 18 hours of the developmental program as revealed by reduced export in the null strains. The fluorescently tagged transporters rescued export when expressed in the null strains, and thus they were functional transporters. CONCLUSION: Unlike ammonium transporters in most organisms, which import NH3/NH4+ as a nitrogen source, those of Dictyostelium export ammonia/ammonium as a waste product from extensive catabolism of exogenously derived and endogenous proteins. Localization on proteolytic organelles and on the neutral contractile vacuole suggests that Dictyostelium ammonium transporters may have unique subcellular functions and play a role in the maintenance of intracellular ammonium distribution. A lack of correlation between the null strain phenotypes and ammonia excretion properties of the ammonium transporters suggests that it is not the excretion function that is important for coupling ammonia levels to the slug versus culmination choice, but rather a sensor and/or signaling function of these proteins that is important.

Disruption of the ifkA and ifkB genes results in altered cell adhesion, morphological defects and a propensity to form pre-stalk O cells during development of Dictyostelium.

IfkA and ifkB are two GCN2-like genes present in Dictyostelium. Disruption of either gene alone results in subtle developmental defects. However, disruption of ifkA and ifkB within the same strain results in severe morphological and patterning defects in the developing double null cells. The mutant cells aggregate in streams that give tightly clumped mounds. Fingers form from the mounds but remain attached to one another, especially at their bases. The fingers culminate to give fused and entangled structures lacking proper stalk but containing some spores. The morphological defects are consistent with an enhanced cell-cell and cell-substrate adhesiveness of the developing double null cells, which may result in inappropriate cell contacts and altered cell motility and sorting properties. In ifkA/ifkB nulls, cell type proportioning and patterning is altered in favor of ALC/pstO cell types. The bias toward the ALC/pstO cell types may be due, in part, to the nuclear localization of the transcription factor STATc in growing ifkA/ifkB null cells. STATc normally becomes localized to the nucleus during finger formation and only within the pre-stalk O zone. The precocious nuclear localization seen in the mutant cells may predispose the cells to a ALC/pstO cell fate. The findings indicate that IfkA and IfkB have redundant functions in Dictyostelium morphogenesis that involve maintaining proper cell-cell and cell-substrate adhesion and the equilibrium between different cell types for proper spatial patterning.

Function of ammonium transporter A in the initiation of culmination of development in Dictyostelium discoideum.

The histidine kinase DhkC controls a phosphorelay involved in regulating the slug versus culmination choice during the multicellular developmental program of Dictyostelium discoideum. When the relay is active, slug migration is favored due to the activation of a cyclic AMP (cAMP) phosphodiesterase and the resultant lowering of the intracellular and extracellular levels of cAMP. Ammonia signaling represents one input into the DhkC phosphorelay, and previous studies indicated that the ammonium transporter C inhibits the relay in response to low ammonia levels. Evidence is presented that another member of the family of ammonium transporters, AmtA, also regulates the slug/culmination choice. Under standard conditions of development, the wild-type strain requires a transitional period of 2 to 3 h to go from fingers to culminants, with some slugs forming and migrating briefly prior to culmination. In contrast, amtA null cells, like cells that lack DhkC, possessed a transitional period of only 1 to 2 h and rarely formed slugs. Disruption of amtA in an amtC null strain overcame the slugger phenotype of that strain and restored its ability to culminate. Strains lacking AmtA were insensitive to the ability of ammonia to promote and prolong slug migration. These findings lead to the proposal that AmtA functions in ammonia sensing as an activator of the DhkC phosphorelay in response to perceived high ammonia levels.

Ammonium transporter C of Dictyostelium discoideum is required for correct prestalk gene expression and for regulating the choice between slug migration and culmination.

Ammonium transporter C (AmtC) is one of three transporters in Dictyostelium that have been proposed to regulate entry and exit of ammonia in a cell type dependent manner and to mediate ammonia signaling. Previous work demonstrated that disruption of the amtC gene results in a slugger phenotype in which the cells remain as migrating slugs when they should form fruiting bodies. More detailed studies on the null strain revealed that differentiation of prestalk cell types was delayed and maintenance of prestalk cell gene expression was defective. There was little or no expression of ecmB, a marker for the initiation of culmination. Normal expression of CudA, a nuclear protein required for culmination, was absent in the anterior prestalk zone. The absence of CudA within the tip region was attributable to the lack of nuclear localization of the transcription factor STATa, despite expression of adenylyl cyclase A mRNA in the slug tips. Disruption of the histidine kinase gene dhkC in the amtC null strain restored STATa and CudA expression and the ability to culminate. The results suggest that the lack of nuclear translocation of STATa results from low cAMP due to a misregulated and overactive DhkC phosphorelay in the amtC null strain.

Temporal and spatial expression of ammonium transporter genes during growth and development of Dictyostelium discoideum.

Ammonia is an important signaling molecule involved in the regulation of development in Dictyostelium. During aggregation, ammonia gradients are established, and the ammonia concentration in the immediate environment or within a particular cell throughout development may vary. This is due to the rate of cellular ammonia production, its rate of loss by evaporation to the atmosphere or by diffusion into the substratum, and perhaps to cellular transport by ammonium transporters (AMTs). Recent efforts in genome and cDNA sequencing have identified three ammonium transporters in Dictyostelium. In addition to physically altering the levels of ammonia within cells, AMTs also may play a role in ammonia signaling. As an initial step in identifying such a function, the temporal and spatial expression of the three amt genes is examined. RT-PCR demonstrates that each of the three amt mRNAs is present and relatively constant throughout growth and development. The spatial expression of these three amt genes is examined during multiple stages of Dictyostelium development using in situ hybridization. A distinct and dynamic pattern of expression is seen for the three genes. In general, amtA is expressed heavily in pre-stalk cells in a dynamic way, while amtB and amtC are expressed in pre-spore regions consistently throughout development. AmtC also is expressed in the most anterior tip of fingers and slugs, corresponding to cells that mediate ammonia's effect on the choice between slug migration and culmination. Indeed, amtC null cells have a slugger phenotype, suggesting AmtC functions in the signaling pathway underlying the mechanics of this choice.

IfkA, a presumptive eIF2 alpha kinase of Dictyostelium, is required for proper timing of aggregation and regulation of mound size.

BACKGROUND: The transition from growth to development in Dictyostelium is initiated by amino acid starvation of growing amobae. In other eukaryotes, a key sensor of amino acid starvation and mediator of the resulting physiological responses is the GCN2 protein, an eIF2alpha kinase. GCN2 downregulates the initiation of translation of bulk mRNA and enhances translation of specific mRNAs by phosphorylating the translation initiation factor eIF2alpha. Two eIF2alpha kinases were identified in Dictyostelium and studied herein. RESULTS: Neither of the eIF2alpha kinases appeared to be involved in sensing amino acid starvation to initiate development. However, one of the kinases, IfkA, was shown to phosphorylate eIF2alpha from 1 to 7 hours after the onset of development, resulting in a shift from polysomes to free ribosomes for bulk mRNA. In the absence of the eIF2alpha phosphorylation, ifkA null cells aggregated earlier than normal and formed mounds and ultimately fruiting bodies that were larger than normal. The early aggregation phenotype in ifkA null cells reflected an apparent, earlier than normal establishment of the cAMP pulsing system. The large mound phenotype resulted from a reduced extracellular level of Countin, a component of the counting factor that regulates mound size. In wild type cells, phosphorylation of eIF2alpha by IfkA resulted in a specific stabilization and enhanced translational efficiency of countin mRNA even though reduced translation resulted for bulk mRNA. CONCLUSIONS: IfkA is an eIF2alpha kinase of Dictyostelium that normally phosphorylates eIF2alpha from 1 to 7 hours after the onset of development, or during the preaggregation phase. This results in an overall reduction in the initiation of protein synthesis during this time frame and a concomitant reduction in the number of ribosomes associated with most mRNAs. For some mRNAs, however, initiation of protein synthesis is enhanced or stabilized under the conditions of increased eIF2alpha phosphorylation. This includes countin mRNA.

The role of thiamine deficiency in alcoholic brain disease.

A deficiency in the essential nutrient thiamine resulting from chronic alcohol consumption is one factor underlying alcohol-induced brain damage. Thiamine is a helper molecule (i.e., a cofactor) required by three enzymes involved in two pathways of carbohydrate metabolism. Because intermediate products of these pathways are needed for the generation of other essential molecules in the cells (e.g., building blocks of proteins and DNA as well as brain chemicals), a reduction in thiamine can interfere with numerous cellular functions, leading to serious brain disorders, including Wernicke-Korsakoff syndrome, which is found predominantly in alcoholics. Chronic alcohol consumption can result in thiamine deficiency by causing inadequate nutritional thiamine intake, decreased absorption of thiamine from the gastrointestinal tract, and impaired thiamine utilization in the cells. People differ in their susceptibility to thiamine deficiency, however, and different brain regions also may be more or less sensitive to this condition.

Mitochondria from cultured cells derived from normal and thiamine-responsive megaloblastic anemia individuals efficiently import thiamine diphosphate.

BACKGROUND: Thiamine diphosphate (ThDP) is the active form of thiamine, and it serves as a cofactor for several enzymes, both cytosolic and mitochondrial. Isolated mitochondria have been shown to take up thiamine yet thiamine diphosphokinase is cytosolic and not present in mitochondria. Previous reports indicate that ThDP can also be taken up by rat mitochondria, but the kinetic constants associated with such uptake seemed not to be physiologically relevant. RESULTS: Here we examine ThDP uptake by mitochondria from several human cell types, including cells from patients with thiamine-responsive megaloblastic anemia (TRMA) that lack a functional thiamine transporter of the plasma membrane. Although mitochondria from normal lymphoblasts took up thiamine in the low micromolar range, surprisingly mitochondria from TRMA lymphoblasts lacked this uptake component. ThDP was taken up efficiently by mitochondria isolated from either normal or TRMA lymphoblasts. Uptake was saturable and biphasic with a high affinity component characterized by a Km of 0.4 to 0.6 microM. Mitochondria from other cell types possessed a similar high affinity uptake component with variation seen in uptake capacity as revealed by differences in Vmax values. CONCLUSIONS: The results suggest a shared thiamine transporter for mitochondria and the plasma membrane. Additionally, a high affinity component of ThDP uptake by mitochondria was identified with the apparent affinity constant less than the estimates of the cytosolic concentration of free ThDP. This finding indicates that the high affinity uptake is physiologically significant and may represent the main mechanism for supplying phosphorylated thiamine for mitochondrial enzymes.