Regulation Of Developmental Gene Expression

Diabetes, Digestive, Kidney Diseases

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Abstract

Dictyostelium, like mammalian cells, use 7-transmembrane receptor (7-TMR)/G protein-coupled pathways to mediate cellular signaling. During development, Dictyostelium initiates a pulsatile, extracellular release of cAMP that directs cell migration and activates downstream effectors via specific cAMP 7-TMRs (cARs). Adenylyl cyclase (AC) is transiently activated by the cAMP signal and then rapidly adapts. While Gbg and the cytosolic factor CRAC are implicated in AC activation, adaptation mechanisms for AC remain unknown. We identified a novel Ga (Ga9) in Dictyostelium that functions as a negative regulator of cAR signal transduction. ga9-null cells develop faster, form more aggregation centers, and chemotax faster to cAMP than wild-type cells, consistent with the loss of an inhibitory protein. Additionally, ga9-null cells initiate cAMP pulses more frequently, suggesting that these cells become resensitized (de-adapted) more rapidly to the cAMP signal. Ga9 does not directly inhibit AC, but functions to inhibit the signaling events upstream of AC. In contrast, cells expressing constitutively activated Ga9 are developmentally delayed, form significantly fewer signaling centers, and are restricted in their ability to activate AC and other downstream effectors. In mammalian cells, phosphorylation of 7-TMRs is linked to adaptation. Although cAR phosphorylation is contemporaneous with AC adaptation in Dictyostelium, the results of experiments with mutant cARs suggested that phosphorylation is not required for adaptation. We provide strong evidence to support a functional synergism between Ga9 and receptor phosphorylation to adapt AC. When phosphorylation-defective cAR1 is expressed in car1/car3/ga9-null cells, AC remains constitutively active in response to cAMP stimulation. Our data also support a role for Ga9 in a global inhibitory network and the potential for Ga-mediated sensory adaptation in other organisms. Dictyostelium is sensitive to a variety of secreted factors that regulate chemotaxis and development. We identified a new aggregation promotion factor (APF) complex secreted by starving cells that augments development. We determined that APF is distinct from other secreted factors that regulate early development, such as the secreted PDE, PSF, CMF, countin. Cells starved at densities lower than 50,000 cells/cm2 do not aggregate; this affect is abrogated by the addition of APF to the starvation buffer, prompting cells to form signaling centers and aggregate into mounds. In addition, cells starved at high density will form territories within six hours in the presence of APF compared to ten hours in its absence.. We used these bioassays to purify APF to homogeneity. APF purifies from wild-type cells as a glycosylated, 250 kDa complex, made up of four distinct proteins. Each protein was sequenced and identified by mass spectrometry. The complex includes a novel 150 kDa protein (p150), a cysteine protease, a novel oxidase-related protein (OxyA), and PDE. When purified from pde-null cells, the molecular weight of APF shifts to 150 kDa and the peak APF activity correlates only with the presence of p150. OxyA and the cysteine protease do not co-fractionate with APF activity in conditioned buffer from pde-nulls. We disrupted the gene encoding OxyA and confirmed that OxyA does not contribute APF activity. Interestingly, oxyA-null cells form extremely large aggregation territories and have defects in the production of spores. A full-length p150 gene has been isolated and experiments to disrupt the gene are in progress. Asymmetric body axis formation is central to metazoan development. Dictyostelium establishes its body axis utilizing 7-TM cAMP receptor (CAR) mediated signal transduction pathways that share features with the metazoan Wnt/GSK3 pathway. In Dictyostelium, GSK3 is required to establish posterior cell fates but is inhibitory to anterior cell differentiation. We have shown that CAR3 and CAR4 are Frizzled-kindred receptors that antagonistically regulate GSK3 to establish these cell fate patterns. cAMP/CAR-mediated GSK3 activation is absent in car3-nulls, but is persistent in cells that lack CAR4. Tyrosine kinase ZAK1 mediates the CAR3-dependent activation of GSK3; CAR3/ZAK1 transiently increases tyrosine phosphorylation and activity of GSK3 in vivo. In contrast, GSK3 is persistently tyrosine phosphorylated and activated in cells lacking CAR4. In addition, ZAK1 is transiently activated in both wild-type and car4-null cells, suggesting that CAR4 inhibits GSK3 in a ZAK1-independent, but tyrosine phosphatase dependent manner. Our data suggest that these Frizzled-kindred receptors orchestrate differential tyrosine phosphorylation and activity of GSK3 by selectively activating tyrosine kinase and tyrosine phosphatase to establish cell fate patterns at the border of the metazoa. Although, genes can be easily disrupted in Dictyostelium by homologous recombination the creation of multiple gene disruptions is severely limited by the very small number of selectable markers available. Here we use the Cre/Lox system to recycle the Blasticidin resistance selectable marker (Bsr) for reuse in the generation of double, and potentially triple or quadruple, knock-out strains. A search of the Dictyostelium genome databases for lox-like sites found no endogenous loxP sites. Thus, the Cre-recombinase activity will be directed only at targeted sites within the genome. To create a disruption cassette, Bsr was flanked by loxP sites, with stop codons in all six reading frames engineered 5' of Bsr and outside of the loxP sites. The presence of the stop codons ensures that after Cre-mediated excision of Bsr, the target gene will not encode a full-length functional protein. The disruption cassette is located within a SmaI fragment for simple blunt-end ligation into nearly any gene of interest for disruption and subsequent Cre-mediated excision of Bsr for recycling. Using this cassette we disrupted Presenilin 2 (dPS2) within its putative cytoplasmic loop. After confirming the initial disruption, these cells were transiently transfected with a plasmid encoding the Cre-recombinase. The resulting clonally-isolated colonies were screened for Cre-mediated recombination using PCR and were tested for blasticidin sensitivity. Cre-mediated chromosomal recombination resulted in the excision of Bsr, leaving behind a single loxP site and the engineered stop codons. Additionally, we found that the plasmid encoding the Cre-recombinase was not retained. The resulting dPS2-disrupted, blasticidin sensitive strain was used to generate a second disruption in the dPS1 gene by reusing the Bsr marker. Thus, the Cre/loxP system can be applied to the recycling of the Bsr marker in the creation of multiply disrupted strains. Animal cells contain intracellular lipid droplets that store triacylglycerols and cholesteryl esters which, upon hydrolysis (lipolysis), give rise to compounds important in energy metabolism, steroid hormone synthesis, membrane biosynthesis and cell signaling. In vertebrates, the droplets in most cells are coated with ADRP, whereas in adipocytes and steroidogenic cells, the droplets are coated with Perilipins. Our studies have implicated these proteins in neutral lipid storage and hydrolysis and we have shown that Perilipin and ADRP share amino acid sequence elements that direct lipid droplet targeting. We also have found that TIP47 and proteins from Dictyostelium and Drosophila also shares these sequence similarities. These proteins, tagged with GFP at their N-termini, target exclusively to lipid droplets when expressed in mammalian cells. We speculate that these related proteins universally serve as essential regulators for lipogenesis, lipolysis, or packaging and trafficking of neutral lipid storage droplets.