SIGNALING PATHWAYS THAT REGULATE DEVELOPMENT

我们(和其他)对cAMP细胞表面受体的研究首次将7-TMR与形态发生信号联系起来。在多细胞聚合体中，cAMP受体(CAR)信号调节激活/去激活蛋白激酶GSK3的通路，进而建立发育模式和细胞命运规范。在某些方面，网柄藻胞外cAMP是Wnt的功能类似物，Wnt是一种分泌配体，在后生动物发育过程中调节多条但不连续的途径。WNTs组织平面细胞极性，协调细胞迁移，但也作为GSK3的有效抑制剂发挥作用，GSK3是决定细胞命运的发育开关。这一途径中许多基因的突变会导致小鼠和人类的胚胎死亡和肿瘤形成。 我们已经证明了ZAK1是GSK3的一个激活的酪氨酸激酶，现在已经发现了一个相关的酪氨酸激酶ZAK2，它也调节Dictyostelials发育过程中的GSK3功能；没有额外的家族成员存在。我们已经证明，在不同的分化细胞群体中，ZAK2和ZAK1对GSK3的酪氨酸磷酸化/激活是调节GSK3的不同需要。ZAK2也可以以非自主的方式调节细胞类型的分化。最后，我们发现Dictyostelius对cAMP的有效极化依赖于Zak介导的GSK3的酪氨酸磷酸化。我们的结果扩展了GSK3信号在发育过程中的复杂性，并表明GSK3的组合调控可以不同地指导Dictyostelius和潜在的其他系统中的细胞极性、定向细胞迁移和细胞命运的指定。 哺乳动物细胞中β-连环蛋白的稳定是Wnt刺激的启动子(如Topflash)在哺乳动物细胞中活性所必需的。我们已经证明Galpao和Galphaq通过Wnt介导β-连环蛋白的稳定。RNAi耗竭或PTX处理使Galpao失活仅部分抑制β-连环素的稳定，但PTX完全抑制Wnt/Topflash的激活。由于胞内β-连环蛋白的水平似乎不能简单地预测基因表达的反式激活功能，我们研究了经WNT和/或PTX处理的哺乳动物细胞中的β-连环蛋白复合体。WNT可促进单体β-连环蛋白在胞浆中的积聚，并与细胞核内的Lef/Tcf转录因子相互作用。经PTX处理后，b-连环蛋白从膜上与E-钙粘蛋白结合释放到胞浆，在胞浆中与β-连环蛋白二聚化，并有可能隔离。尽管PTX处理细胞的WNT刺激也引起胞浆和细胞核中β-连环蛋白的增加，但大多数β-连环蛋白与α-连环蛋白形成复合体，不能诱导WNT/Topflash的诱导。这些数据与其他研究一致，这些研究推断，α-连环蛋白作为β-连环蛋白信号抑制物发挥作用，阻止β-连环蛋白/TCF复合体与DNA结合。 我们现在正在将我们对网柄苔藓发育细胞信号的研究扩展到伽马分泌酶/早老素(PS)途径。我们的研究旨在进一步剖析这些机制，并确定早老素信号转导的新靶点。我们分析Dictyostelius中这些通路的能力有助于理解在人类中保守的基本信号事件。γ-分泌酶/PS复合体由早老素(PS)、尼古丁(NCT)、Aph1和Pen2组成，负责1型单程跨膜蛋白的膜内蛋白分解，对多种细胞和发育功能至关重要。在某些情况下，蛋白分解诱导转录因子的释放，而在另一些情况下，PS信号转导的机制尚不清楚。伽马分泌酶的主要底物包括β-淀粉样前体蛋白和Notch，伽玛分泌酶复合体的突变显然与阿尔茨海默病、Notch信号缺陷和胚胎死亡有关。由于Dictyostelius没有这些传统底物的同源物，它提出了一个独特的系统来揭示新的PS功能。对PS、Aph1、Pen2和NCT基因的一系列单突变和双突变的分析表明，PS复合体在Dictyostants发育过程中对细胞命运的指定起着两个重要但不同的作用。已经确定了可能的新型底物。 网柄网柄菌在研究细胞和发育功能的许多方面都是异常强大的。尽管靶向基因干扰的高效率使研究人员能够表征许多特定的基因，但在单个细胞内创造多个突变来研究基因之间的上位关系或不同途径之间的冗余一直是困难的。我们开发了一个强大的系统来产生多个基因(敲除)突变，其中细胞对额外的定向或随机突变的转化保持敏感，并用于突变或标记蛋白的功能表达研究。

Our (and other) studies of the cell surface receptors for cAMP were the first to link 7-TMRs with morphogen signaling. In the multicellular aggregate, cAMP receptor (CAR) signaling regulates pathways that activate/de-activate protein kinase GSK3, which, in turn, establishes developmental patterning and cell fate specification. In certain respects, extracellular cAMP in Dictyostelium serves as a functional analog of Wnt, a secreted ligand that regulates multiple, but discrete pathways, during metazoan development. The Wnts organize planar cell polarity and coordinate cell migration, but also function as effective inhibitors of GSK3, a developmental switch for cell fate determination. Mutations of many genes in this pathway result in embryonic lethality and tumorigenesis in mice and humans. We had shown that ZAK1 is an activating tyrosine kinase of GSK3 and have now identified a related tyrosine kinase, ZAK2, that also regulates GSK3 function during Dictyostelium development; no additional family members exist. We have shown that tyrosine phosphorylation/activation of GSK3 by ZAK2 and ZAK1 are differentially required to regulate GSK3 within distinct differentiated cell populations. ZAK2 can also act in a non-autonomous manner to regulate cell-type differentiation. Finally, we show that efficient polarization of Dictyostelium toward cAMP depends on ZAK-mediated tyrosine phosphorylation of GSK3. Our results extend the complexity of GSK3 signaling during development and suggest that combinatorial regulation of GSK3 can differentially guide cell polarity, directional cell migration, and cell fate specification in Dictyostelium and potentially other systems. beta-catenin stabilization in mammalian cells is required for Wnt stimulated promoter (e.g. Topflash) activity in mammalian cells. We have shown that Galphao and Galphaq mediate beta-catenin stabilization by Wnt. Inactivation of Galphao by RNAi depletion or by PTX treatment only partially inhibits beta-catenin stabilization, but PTX completely suppresses Wnt/Topflash activation. Since levels of cytosolic beta-catenin do not appear to simply predict transactivating function for gene expression, we investigated beta-catenin protein complexes in mammalian cells treated with Wnt and/or with PTX. Wnt promoted the accumulation of monomeric beta-catenin in the cytosol and interaction with Lef/Tcf transcription factors in the nucleus. Treatment with PTX induced the release of b-catenin from association with E-cadherin at the membrane to the cytosol, where it can dimerize with and potentially sequester beta-catenin. Although Wnt stimulation of PTX-treated cells also induced an increase in cytosolic and nuclear beta-catenin, most beta-catenin was in complex with alpha-catenin and was unable to effect induction of Wnt/Topflash. These data are consistent with other studies that infer that alpha-catenin functions as a beta-catenin signaling inhibitor, blocking beta-catenin/TCF complexes from binding to DNA. We are now extending our studies on developmental cellular signaling in Dictyostelium to the gamma-secretase/Presenilin (PS) pathway. Our studies are intended to further dissect these mechanisms and to identify novel targets of Presenilin signaling. Our ability to analyze these pathways in Dictyostelium can contribute to understanding essential signaling events that are conserved in humans. The gamma-secretase/PS complex, comprised of Presenilin (PS), Nicastrin (Nct), Aph1 and Pen2, is responsible for intramembrane proteolysis of type 1 single-pass transmembrane proteins and is essential for a variety of cellular and developmental functions. In some instances proteolysis induces the release of a transcription factor, while in others, mechanisms of PS signal transduction remain unknown. Principle substrates of gamma-secretase include beta-Amyloid Precursor Protein and Notch, and mutations in the gamma-secretase complex have been clearly linked to Alzheimer's disease, defects in Notch signaling, and embryonic lethality. Since Dictyostelium does not have orthologs of these traditional substrates, it presents a unique system to reveal novel PS functions. Analyses of a series of single and double mutations of the PS, Aph1, Pen2, and Nct genes show that the PS complex plays two important, yet distinct, roles in control of cell fate specification during Dictyostelium development. Putative novel substrates have been identified. Dictyostelium is exceptionally powerful for studying numerous aspects of cellular and developmental function. While the high efficiency of targeted gene disruption has enabled researchers to characterize many specific genes, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or redundancies between various pathways. We developed a robust system for production of multiple gene (knock-out) mutations, where cells remain sensitive to transformation for additional targeted or random mutagenesis and for functional expression studies of mutated or tagged proteins.