SIGNALING PATHWAYS IN CONTROL OF GROWTH AND DEVELOPMENT

控制生长和发育的信号通路

• 批准号: 8741590
• 负责人: ALAN R KIMMEL
• 金额: $ 125.52万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8741590
• 关键词: Actins; Adenylate Cyclase; Amino Acids; Bacteria; Binding; Binding Sites; Biochemical; Bioinformatics; CHARGE syndrome; Cell Differentiation process; Cell Line; Cell physiology; Cells; Chemotactic Factors; Chemotaxis; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Cleaved cell; Complex; Coupled; Cyclic AMP; Cyclic AMP Receptors; Cyclic GMP; Cytoskeleton; DNA; DNA Binding; DNA Sequence; DNA-Binding Proteins; DNA-Directed RNA Polymerase; DNA-Protein Interaction; Data; Defect; Development; Dictyostelium; Dictyostelium discoideum; Digestion; Disease; Ensure; Epitopes; Eukaryota; Family; Feedback; Fluorescence Resonance Energy Transfer; Folate; Frequencies; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Gene Expression; Gene Expression Profile; Gene Mutation; Gene Targeting; Generations; Genes; Genome; Growth; Growth Factor; Growth and Development function; Guanosine Triphosphate; Haploidy; Human; Image; Individual; Knock-in Mouse; Knock-out; Laboratories; Leukocytes; Ligand Binding; Ligands; MAP Kinase Gene; MAPK1 gene; Maps; Mediating; Micrococcal Nuclease; Modeling; Molecular; Molecular Genetics; Mono-S; Movement; Natural Immunity; Neurotransmitters; Nucleosomes; Nutrient; Organism; Orthologous Gene; Pathway interactions; Pattern; Phagocytosis; Phenotype; Phosphorylation; Phosphotransferases; Physiological Processes; Population; Positioning Attribute; Process; Production; Protein Family; Proto-Oncogene Proteins c-akt; Protocols documentation; Pseudopodia; Recycling; Regulation; Relative (related person); Research; Resolution; Resources; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Source; Specificity; Stimulus; System; Techniques; Total Internal Reflection Fluorescent; Transmembrane Domain; attenuation; base; blasticidin S; cell growth; chemokine; chemokine receptor; endonuclease; extracellular; fMet-Leu-Phe receptor; feeding; fungus; gene function; genetic manipulation; genome-wide; helicase; homologous recombination; human disease; insight; macrophage; morphogens; mutant; neutrophil; novel; polymerization; ras Guanine Nucleotide Exchange Factors; receptor; receptor function; recombinase; response; transcription factor; transcriptome sequencing

Dictyostelium discoideum is an exceptionally powerful eukaryotic model to study many aspects of growth, development, and fundamental cellular processes. Its small-sized, haploid genome allows highly efficient targeted homologous recombination for gene disruption and knock-in epitope tagging. We had developed a robust system for the generation of multiple gene mutations in Dictyostelium by recycling the Blasticidin S selectable marker after transient expression of the Cre recombinase. We have now further optimized the system for higher efficiency and, additionally, coupled it to both knock-out and knock-in gene targeting, allowing the characterization of multiple and cooperative gene functions in a single cell line. Micrococcal nuclease (MNase) is an endonuclease that cleaves native DNA at high frequency, but is blocked in chromatin by sites of intimate DNA-protein interaction, including nucleosomal regions. Protection from MNase cleavage has often been used to map transcription factor binding sites and nucleosomal positions on a single gene basis; however, by combining MNase digestion with high-throughput, paired-end DNA sequencing, we map protein-DNA interaction regions across the entire genome. Biochemical and bioinformatic protocols have been optimized for global mono-nucleosome positioning at 160 bp spacing coverage, but are applicable to mapping more broadly or for site-specific binding of transcription factors at 50 bp resolution. Control of chromatin structure is critical for multicellular development and regulation of cell differentiation. The CHD (Chromodomain-Helicase-DNA binding) protein family is one of the major ATP-dependent, chromatin remodeling factors that regulate nucleosome positioning and access of transcription factors and RNA polymerase to the eukaryotic genome. There are 3 mammalian CHD subfamilies and their impaired functions are associated with several human diseases. We have identified three CHD orthologs (ChdA, ChdB, and ChdC) in Dictyostelium discoideum. These CHDs are expressed throughout development, but with unique patterns. Null mutants lacking each CHD have distinct, non-redundant phenotypes that reflect their expression patterns and suggest functional specificity. Accordingly, utilizing genome-wide (RNA-seq) transcriptome profiling for each null strain, we showed that the different CHDs regulate independent gene sets during both growth and development. ChdC is an apparent ortholog of the mammalian Class III CHD group that is associated with the human CHARGE syndrome, and GO analyses of aberrant gene expression in chdC-nulls suggest defects in both cell-autonomous and non-autonomous signaling, which have been confirmed through analyses of chdC-nulls developed in pure populations or with low levels of WT cells. We have provided novel insight into the broad function of CHDs in the regulation development and disease, through chromatin-mediated changes in directed gene expression. Migratory cells, like mammalian leukocytes and Dictyostelium, utilize G protein coupled receptor(GPCR) signaling to regulate MAPK/ERK, PI3K, TORC2/AKT, adenylyl cyclase, and actin polymerization, which collectively direct chemotaxis. Upon ligand binding, mammalian GPCRs are phosphorylated at cytoplasmic residues, uncoupling G protein pathways, but activating others. Still, connections between GPCR phosphorylation and chemotaxis are unclear. In developing Dictyostelium, secreted cAMP serves as a chemoattractant, with extracellular cAMP propagated as oscillating waves to ensure directional migratory signals. cAMP oscillations derive from transient excitatory responses of adenylyl cyclase, which then rapidly adapts. We have studied chemotactic signaling in Dictyostelium that express non-phosphorylatable cAMP receptors and show through chemotaxis modeling, single-cell FRET imaging, pure and chimeric population wavelet quantification, biochemical analyses, and TIRF microscopy, that receptor phosphorylation is required to regulate adenylyl cyclase adaptation, long-range oscillatory cAMP wave production, and cytoskeletal actin response. Phosphorylation defects, thus, promote hyperactive actin polymerization at the cell periphery, misdirected pseudopodia, and the loss of directional chemotaxis. Our data indicate that chemoattractant receptor phosphorylation is required to co-regulate essential pathways for migratory cell polarization and chemotaxis. Our results significantly extend the understanding of GPCR phosphorylation function, providing strong evidence that this evolutionarily conserved mechanism is required in a signal attenuation pathway that is necessary to maintain persistent directional movement of Dictyostelium, neutrophils, and other migratory cells. Global stimulation of Dictyostelium with different chemoattractants elicits multiple transient signaling responses, including synthesis of cAMP and cGMP, actin polymerization, activation of kinases ERK2, TORC2, and PI3K, and Ras-GTP accumulation; mechanisms that down-regulate these responses are poorly understood. We examined transient activation of TORC2 in response to chemically distinct chemoattractants, cAMP and folate, and suggest that TORC2 is regulated by adaptive, de-sensitizing responses to stimulatory ligands that are independent of downstream, feedback or feed-forward circuits. Cells with acquired insensitivity to either folate or cAMP remain fully responsive to TORC2 activation if stimulated with the other ligand. Thus, TORC2 responses to cAMP or folate are not cross-inhibitory. Using a series of signaling mutants, we have shown that folate and cAMP activate TORC2 through an identical GEF/Ras pathway, but separate receptors and G protein couplings. Since the common GEF/Ras pathway also remains fully responsive to one chemoattractant after de-sensitization to the other, GEF/Ras must act downstream and independently of adaptation to persistent ligand stimulation. When initial chemoattractant concentrations are immediately diluted, cells rapidly regain full responsiveness. We suggest that ligand adaptation functions in upstream inhibitory pathways that involve chemoattractant-specific receptor/G protein complexes and regulate multiple response pathways.

盘基网柄菌是一种异常强大的真核模型，可用于研究生长、发育和基本细胞过程的许多方面。其小型单倍体基因组可实现高效的靶向同源重组，以实现基因破坏和敲入表位标记。我们开发了一个强大的系统，通过在 Cre 重组酶瞬时表达后回收杀稻瘟菌素 S 选择标记，在盘基网柄菌中产生多个基因突变。我们现在进一步优化了该系统以提高效率，此外，将其与敲除和敲入基因靶向结合起来，从而可以表征单个细胞系中的多个协同基因功能。 微球菌核酸酶 (MNase) 是一种核酸内切酶，可高频切割天然 DNA，但在染色质中被 DNA-蛋白质密切相互作用位点（包括核小体区域）阻断。防止 MNase 裂解的保护通常用于在单基因基础上绘制转录因子结合位点和核小体位置图；然而，通过将 MNase 消化与高通量双端 DNA 测序相结合，我们绘制了整个基因组中蛋白质-DNA 相互作用区域的图谱。生化和生物信息学方案已针对 160 bp 间距覆盖范围的全局单核小体定位进行了优化，但适用于更广泛的作图或 50 bp 分辨率的转录因子的位点特异性结合。 染色质结构的控制对于多细胞发育和细胞分化的调节至关重要。 CHD（染色质结构域-解旋酶-DNA 结合）蛋白家族是主要的 ATP 依赖性染色质重塑因子之一，可调节核小体定位以及转录因子和 RNA 聚合酶进入真核基因组。哺乳动物 CHD 有 3 个亚科，它们的功能受损与多种人类疾病有关。我们在盘基网柄菌中鉴定了三种 CHD 直系同源物（ChdA、ChdB 和 ChdC）。这些先天性心脏病在整个发育过程中都有表达，但具有独特的模式。缺乏每种 CHD 的无效突变体具有独特的、非冗余的表型，反映了它们的表达模式并表明了功能特异性。因此，利用每个无效菌株的全基因组 (RNA-seq) 转录组分析，我们发现不同的 CHD 在生长和发育过程中调节独立的基因集。 ChdC 是与人类 CHARGE 综合征相关的哺乳动物 III 类 CHD 组的明显直系同源物，对 chdC 缺失中异常基因表达的 GO 分析表明细胞自主和非自主信号传导存在缺陷，这一点已通过对纯群体或低水平 WT 细胞中开发的 chdC-null 进行分析。通过染色质介导的定向基因表达变化，我们对先心病在调控发展和疾病中的广泛功能提供了新的见解。 迁移细胞，如哺乳动物白细胞和盘基网柄菌，利用 G 蛋白偶联受体 (GPCR) 信号传导来调节 MAPK/ERK、PI3K、TORC2/AKT、腺苷酸环化酶和肌动蛋白聚合，这些共同指导趋化性。配体结合后，哺乳动物 GPCR 在细胞质残基处被磷酸化，解偶联 G 蛋白途径，但激活其他途径。尽管如此，GPCR 磷酸化和趋化性之间的联系尚不清楚。在盘基网柄菌的发育过程中，分泌的 cAMP 作为化学引诱剂，细胞外 cAMP 以振荡波的形式传播，以确保定向迁移信号。 cAMP 振荡源自腺苷酸环化酶的瞬时兴奋反应，然后迅速适应。我们研究了盘基网柄菌中表达非磷酸化 cAMP 受体的趋化信号传导，并通过趋化性建模、单细胞 FRET 成像、纯嵌合群体小波定量、生化分析和 TIRF 显微镜显示，受体磷酸化是调节腺苷酸环化酶适应所必需的、长程振荡 cAMP 波产生和细胞骨架肌动蛋白反应。因此，磷酸化缺陷会促进细胞外周肌动蛋白聚合过度活跃、伪足定向错误以及定向趋化性丧失。我们的数据表明，趋化剂受体磷酸化是共同调节迁移细胞极化和趋化性的重要途径所必需的。我们的结果显着扩展了对 GPCR 磷酸化功能的理解，提供了强有力的证据，证明这种进化上保守的机制是信号衰减途径所必需的，而信号衰减途径是维持盘基网柄菌、中性粒细胞和其他迁移细胞持续定向运动所必需的。 用不同的趋化剂对盘基网柄菌进行整体刺激，会引发多种瞬时信号反应，包括 cAMP 和 cGMP 的合成、肌动蛋白聚合、激酶 ERK2、TORC2 和 PI3K 的激活以及 Ras-GTP 的积累；下调这些反应的机制尚不清楚。我们检查了 TORC2 对化学上不同的化学引诱剂、cAMP 和叶酸的瞬时激活，并表明 TORC2 受到对刺激配体的适应性、脱敏反应的调节，这些刺激配体独立于下游、反馈或前馈回路。如果用其他配体刺激，对叶酸或 cAMP 获得性不敏感的细胞仍然对 TORC2 激活完全敏感。因此，TORC2 对 cAMP 或叶酸的反应不具有交叉抑制性。使用一系列信号突变体，我们发现叶酸和 cAMP 通过相同的 GEF/Ras 途径激活 TORC2，但受体和 G 蛋白偶联是分开的。由于共同的 GEF/Ras 通路在对另一种化学引诱剂脱敏后仍对另一种化学引诱剂保持完全响应，因此 GEF/Ras 必须在下游发挥作用，并且独立于对持续配体刺激的适应。当初始化学引诱剂浓度立即稀释时，细胞迅速恢复完全反应能力。我们认为配体适应在上游抑制途径中发挥作用，涉及趋化剂特异性受体/G 蛋白复合物并调节多种反应途径。