Regulation Of Developmental Gene Expression

盘基骨柱与哺乳动物细胞一样，通过7-跨膜受体(7-TMR)/G蛋白偶联途径介导细胞信号传导。在发育过程中，盘基骨柱启动脉冲式的细胞外释放cAMP，指导细胞迁移，并通过特定的cAMP 7-TMRs （cARs）激活下游效应物。腺苷酸环化酶（AC）被cAMP信号瞬时激活，然后迅速适应。虽然Gbg和细胞质因子CRAC与AC激活有关，但AC的适应机制尚不清楚。我们在盘基骨柱中发现了一种新的Ga (Ga9)，它可以作为cAR信号转导的负调节因子。与野生型细胞相比，ga9-null细胞发育更快，形成更多聚集中心，对cAMP的化学反应更快，这与抑制蛋白的缺失一致。此外，ga9-null细胞更频繁地启动cAMP脉冲，这表明这些细胞对cAMP信号更迅速地重新敏感（去适应）。Ga9并不直接抑制AC，而是起到抑制AC上游信号事件的作用。相反，表达组成性激活的Ga9的细胞发育迟缓，形成的信号中心明显减少，激活AC和其他下游效应物的能力受到限制。在哺乳动物细胞中，7-TMRs的磷酸化与适应有关。尽管在盘基骨柱中，cAR磷酸化与AC适应是同时发生的，但突变cAR的实验结果表明，适应并不需要磷酸化。我们提供了强有力的证据来支持Ga9和受体磷酸化之间的功能协同作用，以适应AC。当磷酸化缺陷的cAR1在cAR1 /car3/ Ga9缺失的细胞中表达时，AC在cAMP刺激下保持组成性活性。我们的数据还支持Ga9在全球抑制网络中的作用，以及在其他生物体中ga介导的感觉适应的潜力。盘基骨柱对多种调节趋化性和发育的分泌因子敏感。我们发现了一种新的聚集促进因子（APF）复合物，由饥饿细胞分泌，可以促进发育。我们确定APF不同于其他调节早期发育的分泌因子，如分泌的PDE、PSF、CMF、计数。在低于50,000个细胞/cm2的密度下饥饿的细胞不聚集；在饥饿缓冲液中加入APF可以消除这种影响，促使细胞形成信号中心并聚集成丘。此外，高密度饥饿的细胞在有APF存在的情况下会在6小时内形成领地，而在没有APF的情况下则需要10小时。我们使用这些生物测定法纯化APF至均匀性。APF从野生型细胞中纯化为糖基化的250 kDa复合物，由四种不同的蛋白质组成。通过质谱法对每个蛋白进行测序和鉴定。该复合物包括一种新的150kda蛋白（p150）、一种半胱氨酸蛋白酶、一种新的氧化酶相关蛋白（OxyA）和PDE。当从无pde的细胞中纯化时，APF的分子量转移到150 kDa， APF活性峰值仅与p150的存在相关。在pde-null的条件缓冲液中，OxyA和半胱氨酸蛋白酶不会与APF活性共分离。我们破坏了编码OxyA的基因，并证实OxyA不参与APF活性。有趣的是，无氧细胞形成非常大的聚集区域，在孢子的产生中有缺陷。一个完整的p150基因已经被分离出来，破坏该基因的实验正在进行中。不对称的体轴形成是后生动物发育的核心。盘基骨柱利用7-TM cAMP受体（CAR）介导的信号转导通路建立其体轴，这些信号转导通路与后生动物Wnt/GSK3通路具有共同的特征。在盘基骨柱中，GSK3是建立后细胞命运所必需的，但对前细胞分化有抑制作用。我们已经证明，CAR3和CAR4是卷曲同源受体，它们拮抗调节GSK3以建立这些细胞命运模式。cAMP/ car介导的GSK3激活在无car3的细胞中不存在，但在缺乏CAR4的细胞中持续存在。酪氨酸激酶ZAK1介导GSK3依赖car3的激活；在体内，CAR3/ZAK1可瞬时增加酪氨酸磷酸化和GSK3活性。相反，GSK3在缺乏CAR4的细胞中持续酪氨酸磷酸化和激活。此外，ZAK1在野生型和无CAR4的细胞中都被短暂激活，这表明CAR4以一种与ZAK1无关但依赖酪氨酸磷酸酶的方式抑制GSK3。我们的数据表明，这些frizzled亲缘受体通过选择性激活酪氨酸激酶和酪氨酸磷酸酶来协调不同的酪氨酸磷酸化和GSK3的活性，从而在后生动物的边界建立细胞命运模式。虽然在盘基骨柱中，基因可以很容易地通过同源重组被破坏，但多基因破坏的产生受到可选择标记数量非常少的严重限制。在这里，我们使用Cre/Lox系统回收Blasticidin抗性选择标记（Bsr），以便在产生双重，潜在的三重或四倍敲除菌株中重复使用。在盘基骨门基因组数据库中寻找lox样位点没有发现内源性loxP位点。因此，cre -重组酶的活性将仅针对基因组内的目标位点。为了创建一个中断盒，Bsr被放置在loxP位点的两侧，在Bsr的5'和loxP位点之外的所有六个阅读框中都有停止密码子。终止密码子的存在确保了在cre介导的Bsr切除后，靶基因不会编码全长功能蛋白。中断盒位于smi片段内，用于简单的钝端连接到几乎任何感兴趣的基因上进行中断和随后的cre介导的Bsr切除以进行循环。使用这个卡带，我们破坏了早老素2 （dPS2）在其假定的细胞质环内。在确认初始破坏后，将这些细胞短暂转染编码cre -重组酶的质粒。利用PCR技术对克隆分离的菌落进行cre介导重组筛选，并对杀胚素进行敏感性测试。cre介导的染色体重组导致Bsr的切除，留下单个loxP位点和工程终止密码子。此外，我们发现编码cre -重组酶的质粒没有被保留。由此产生的dps2中断，囊胚杀虫素敏感菌株通过重复使用Bsr标记来产生dPS1基因的第二次中断。因此，Cre/loxP系统可以应用于Bsr标记物的循环利用，以产生多重破坏菌株。动物细胞内含有储存三酰基甘油和胆固醇酯的脂滴，这些脂滴在水解（脂解）后产生在能量代谢、类固醇激素合成、膜生物合成和细胞信号传导中重要的化合物。在脊椎动物中，大多数细胞中的液滴被ADRP包裹，而在脂肪细胞和甾体生成细胞中，液滴被Perilipins包裹。我们的研究表明这些蛋白参与中性脂质储存和水解，并且我们已经表明Perilipin和ADRP共享直接脂滴靶向的氨基酸序列元件。我们还发现TIP47与盘基骨门和果蝇的蛋白质也具有这些序列相似性。这些蛋白在其n端标记有GFP，在哺乳动物细胞中表达时只针对脂滴。我们推测，这些相关蛋白普遍充当脂肪生成、脂肪分解或中性脂储存滴的包装和运输的必要调节因子。

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.