Regulation Of Developmental Gene Expression

我们的实验室正在研究的分子过程至关重要的发展一个终末分化的有机体从同质群体的全能性细胞。我们正在使用分子、遗传和生化技术以及真核系统盘基骨柱模型来定义细胞自主和非自主的信号转导途径，这些途径指定细胞命运和模式形成。在盘基骨柱中，7-跨膜（7-TM）结构域受体家族通过其配体（分泌的化学吸引剂-形态因子cAMP）的刺激，建立了基本的发育组织，即前（柄前）/后（孢子前）轴。作为一种化学引诱剂，cAMP激活cAMP受体/G蛋白信号，动员单个细胞形成多细胞聚集体；在发育后期，cAMP受体信号调节细胞命运规范。我们关注cAMP受体（CARs）家族常见和独特的信号通路，以阐明在更复杂的发育程序中可能保守的机制和电路。GSK3作为发育开关，在后生动物中建立细胞命运。在盘基骨柱中，激活的GSK3刺激孢子前/孢子通路，同时抑制柄前/柄分化。GSK3的激活和去激活分别由酪氨酸激酶ZAK1和依赖car4的PTPase介导。尽管zak1-null基因与gsk3-null基因具有许多相同的表型，但它们的前柄模式并不相同。此外，在没有ZAK1的情况下，观察到残留的酪氨酸磷酸化和GSK3的激活。我们在GSK3激活途径中发现了另一个酪氨酸激酶ZAK2；没有其他家庭成员。像ZAK1一样，ZAK2也会磷酸化并激活GSK3，但两者都通过GSK3起作用，它们在细胞分化中的作用是不同的。ZAK2和GSK3自主抑制前秆A亚群的分化。ZAK1在这些细胞中没有调节作用。相反，ZAK1和GSK3，而不是ZAK2，能拮抗另一类前柄B细胞的分化。此外，ZAK2/GSK3是非自主需要的，而ZAK1/GSK3是自主作用的，促进孢子前/孢子细胞的命运。最后，我们发现酪氨酸磷酸化/激活GSK3是发育过程中细胞极性和趋化性所必需的。我们提出，GSK3的组合调控可以在盘形骨和其他系统中不同地引导细胞极性、定向细胞迁移和细胞命运规范。细胞以化学引诱剂梯度定向移动，在整个细胞体中差异小于2%。这些浅的细胞外梯度被放大为非常陡峭的细胞内梯度，信号成分特异性地定位于运动细胞的前缘或后方。尽管受体分布均匀，并按比例激活，这种情况仍会发生。复杂的反馈回路位于受体信号的下游，整合了激活和抑制途径，被认为可以调节这种陡峭的细胞内梯度。我们已经在盘基骨柱中发现了一种新的信号功能，涉及一种Galpha亚基（Ga9），可以拮抗趋化反应。最引人注目的是，缺乏Ga9的细胞是超极化和高趋化的，而表达组成激活的Ga9的细胞则表现出相互的表型。从机制上讲，Ga9在受体刺激后非常迅速地发挥作用，负调控多种下游途径，最终建立肌动蛋白和肌球蛋白的不对称动员。我们认为功能相似的ga介导的抑制信号可能调节大多数真核细胞的化学引诱反应。分泌因子，如激素和细胞因子，在许多发育过程中是必不可少的。我们现在已经确定了一种新的分泌因子APF（聚集促进因子），它可以增强盘基骨菌的早期发育。利用一种生物测定法，在非常低的细胞密度下拯救细胞的发育缺陷，我们纯化了APF到均匀性。APF活性不同于其他已知因素。它是一个糖基化的250 kDa复合物，该复合物的每个蛋白质都通过质谱法鉴定。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。从pde-nulls纯化的APF是完全活性的，但分离的分子质量为150 kDa。在pde-null中，OxyA和半胱氨酸蛋白酶不与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。我们分离了全长p150基因，发现p150的过表达会导致APF活性的过表达。p150作为一个完整的膜前体蛋白单次合成，在膜上或膜内裂解释放活性APF。在另一个单独的项目中，我们研究脂肪细胞中的脂肪分解，它控制脂肪酸的释放，为身体的各种组织提供能量。这一反应是由蛋白激酶A （PKA）激活、激素敏感脂肪酶（HSL）（一种胞质酶）和包裹在脂肪细胞脂滴表面的脂磷脂（Perilipin）介导的。脂肪细胞脂溶激活的关键步骤是激素敏感脂肪酶（HSL）从细胞质转移到脂质储存滴的表面。我们已经证明，在HSL中，PKA在659或660丝氨酸上的磷酸化是影响易位反应所必需的。当这些丝氨酸残基同时突变为丙氨酸时，不会发生易位。此外，催化丝氨酸423的突变消除了HSL易位，表明失活酶在PKA激活后不会迁移到脂滴。为了评估Perilipin是否直接参与pka介导的脂肪分解，我们在中国仓鼠卵巢成纤维细胞中表达了Perilipin基因的两种主要剪接变体Perilipin A和B的天然和突变形式的编码结构。脂脂蛋白定位于脂滴表面，取代了通常包裹在脂滴表面的脂肪分化相关蛋白。当PKA处于静止状态时，Perilipin A对三酰甘油水解的抑制率为87%，但PKA的激活和Perilipin A的磷酸化会产生7倍的脂解激活。Perilipin n端PKA位点的突变消除了PKA介导的脂溶反应。相比之下，perilipin B仅对脂肪分解发挥最小的保护作用，并且对PKA激活无反应。由于中国仓鼠卵巢细胞不含pka激活的脂肪酶，我们得出结论，仅表达Perilipin A就足以使pka介导的脂肪分解在这些细胞中发生。此外，数据表明，Perilipin A独特的c端部分负责防止脂肪分解，而n端PKA位点的磷酸化减弱了这种保护作用。与野生型小鼠的脂肪细胞相比，来自无perilipin动物的脂肪细胞具有更高的脂肪分解基础率，但不能对脂肪分解刺激做出最大反应。这种缺陷位于-肾上腺素能受体-腺苷酸环化酶复合物的下游。我们发现，HSL在无濒海鞘平的小鼠中与低水平的脂滴表面基本相关，但无濒海鞘平小鼠胚胎成纤维细胞衍生的脂肪细胞中不存在从细胞质到脂滴的刺激易位。我们还通过引入gfp标记的HSL（含或不含Perilipin A）在无脂肪的中国仓鼠卵巢细胞系中重建了HSL易位反应。在蛋白激酶A的激活下，HSL- gfp仅在表达完全磷酸化的Perilipin A的细胞中易位到脂滴，证实了Perilipin是引发HSL易位反应的必要条件。

Our laboratory is investigating molecular processes critical for developing a terminally differentiated organism from a homogeneous population of totipotent cells. We are using molecular, genetic, and biochemical techniques and the model eukaryotic system Dictyostelium to define cell autonomous and non-autonomous signal transduction pathways that specify cell fate and pattern formation. In Dictyostelium, stimulation of a family of 7-transmembrane (7-TM) domain receptors by its ligand, the secreted chemoattractant-morphogen cAMP, establishes the fundamental developmental organization, an anterior (prestalk)/posterior (prespore) axis. As a chemoattractant, cAMP activates cAMP receptor/G protein signaling to mobilize individual cells to form multicellular aggregates; later in development, cAMP receptor signaling regulates cell fate specification. We focus on signaling pathways, both common and unique to the family of cAMP receptors (CARs), to elucidate mechanisms and circuits that may be conserved in more complex developmental programs. GSK3 acts as developmental switch to establish cell fates in the metazoa. In Dictyostelium, activated GSK3 stimulates prespore/spore pathways, while suppressing prestalk/stalk differentiation. Activation/de-activation of GSK3 is mediated by tyrosine kinase ZAK1 and a CAR4-dependent PTPase, respectively. Although zak1-nulls share many of the phenotypes of gsk3-nulls, their prestalk patterns are not identical. Further, residual tyrosine phosphorylation and activation of GSK3 is observed in the absence of ZAK1. We identified ZAK2, the other tyrosine kinase in the GSK3 activation pathway; no additional family members exist. Like ZAK1, ZAK2 will phosphorylate and activate GSK3, but while both act via GSK3, their roles in cellular differentiation are distinct. ZAK2 and GSK3 repress the differentiation the prestalk A sub-population autonomously. ZAK1 has no regulatory role in these cells. Conversely, ZAK1 and GSK3, but not ZAK2, antagonize the differentiation of a different prestalk class, prestalk B cells. Further, ZAK2/GSK3 is required non-autonomously, while ZAK1/GSK3 acts autonomously, to promote prespore/spore cell fates. Finally, we show that tyrosine phosphorylation/activation of GSK3 is required for cell polarity and chemotaxis during development. We propose that combinatorial regulation of GSK3 can differentially guide cell polarity, directional cell migration, and cell fate specification in Dictyostelium and potentially other systems. Cells move directionally in chemoattractant gradients that differ by less than 2% across the cell body. These shallow extracellular gradients are amplified into very steep intracellular gradients, with signaling components localized specifically to the leading edge or rear of moving cells. This occurs despite uniform receptor distribution and proportionate activation. Complex feedback loops that are downstream of receptor signaling and that integrate activating and inhibiting pathways are suggested to regulate this steep intracellular gradient. We have identified a novel signaling function in Dictyostelium involving a Galpha subunit (Ga9) that antagonizes chemotactic response. Most dramatically, cells lacking Ga9 are hyperpolarized and hyperchemotactic, whereas cells expressing constitutively activated Ga9 exhibit a reciprocal phenotype. Mechanistically, Ga9 functions very rapidly following receptor stimulation to negatively regulate multiple downstream pathways that ultimately establish the asymmetric mobilizations of actin and myosin. We suggest that functionally similar Ga-mediated inhibitory signaling may modulate chemoattractant responses in most eukayotic cells. Secreted factors, such as hormones and cytokines, are essential for many developmental processes. We have now identified a novel, secreted factor APF (aggregation promotion factor) that augments early Dictyostelium development. Using a bioassay that rescues the developmental defect of cells placed at very low cell density, we purified APF to homogeneity. APF activity is distinct from other known factors. It is a glycosylated, 250 kDa complex and each protein of the complex was identified by mass spectrometry. The complex includes a novel 150 kDa protein (p150), a cysteine protease, a novel oxidase-related protein (OxyA), and PDE. APF purified from pde-nulls is fully active, but fractionates with a molecular mass of 150 kDa. OxyA and the cysteine protease do not co-fractionate with APF activity in pde-nulls. We disrupted the gene encoding OxyA and confirmed that OxyA does not contribute to APF activity. We isolated the full-length p150 gene and showed that overexpression of p150 leads to an overexpression of APF activity. p150 is synthesized as a single pass, integral membrane precursor protein and cleavage at or within the membrane releases active APF. In a separate project we study lipolysis in adipocytes that governs the release of fatty acids for the supply of energy to various tissues of the body. This reaction is mediated by protein kinase A (PKA) activation, hormone-sensitive lipase (HSL), a cytosolic enzyme, and Perilipin, which coats the lipid droplet surface in adipocytes. A key step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. We have demonstrated that PKA phosphorylation at either serine 659 or 660 within HSL, is required to effect the translocation reaction. Translocation does not occur when these serines residues are mutated simultaneously to alanines. Also, mutation of the catalytic serine 423 eliminates HSL translocation, showing that the inactive enzyme does not migrate to the lipid droplet upon PKA activation. To assess whether the Perilipins participate directly in PKA-mediated lipolysis, we expressed constructs coding for native and mutated forms of the two major splice variants of the Perilipin gene, Perilipins A and B, in Chinese hamster ovary fibroblasts. Perilipins localize to lipid droplet surfaces and displace the adipose differentiation-related protein that normally coats the droplets in these cells. Perilipin A inhibits triacylglycerol hydrolysis by 87% when PKA is quiescent, but activation of PKA and phosphorylation of Perilipin A engenders a 7-fold lipolytic activation. Mutation of PKA sites within the N-terminal region of Perilipin abrogates the PKA-mediated lipolytic response. In contrast, perilipin B exerts only minimal protection against lipolysis and is unresponsive to PKA activation. Since Chinese hamster ovary cells contain no PKA-activated lipase, we conclude that the expression of Perilipin A alone is sufficient to confer PKA-mediated lipolysis in these cells. Moreover, the data indicate that the unique C-terminal portion of Perilipin A is responsible for its protection against lipolysis and that phosphorylation at the N-terminal PKA sites attenuates this protective effect. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the beta-adrenergic receptor-adenylyl cyclase complex. We showed that HSL is basally associated with lipid droplet surfaces at a low level in perilipin-nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without Perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable Perilipin A, confirming that Perilipin is required to elicit the HSL translocation reaction.