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.

DICEDYOSTELIUM DISCOIDEUM是一种非常有力的真核模型，可研究生长，发育和基本细胞过程的许多方面。它的小型单倍体基因组允许具有高效的靶向同源重组，以用于基因破坏和敲门表位标记。我们通过回收Blasticidin的可选标记物在CRE重组酶的短暂表达后回收可选的标记物，从而开发了一个可靠的系统，以用于在Dictyostelium中产生多个基因突变。现在，我们已经进一步优化了该系统以提高效率，并将其耦合到敲除基因靶向，从而可以表征单个细胞系中多重和合作基因功能。 微球菌核酸酶（MNase）是一种核酸酶，以高频裂解天然DNA，但在包括核小体区域在内的亲密DNA-蛋白质相互作用的位点在染色质中阻塞。对MNase裂解的保护通常已用于以单个基因为基础来绘制转录因子结合位点和核小体位置。但是，通过将MNase消化与高通量，配对的DNA测序相结合，我们绘制整个基因组中的蛋白-DNA相互作用区域。生化和生物信息学方案已针对在160 bp间距覆盖范围的全局单核体定位进行了优化，但适用于更广泛的绘制或以50 bp分辨率的转录因子的特定地点结合。 染色质结构的控制对于细胞分化的多细胞发育和调节至关重要。 CHD（染色体 - 羟基酶-DNA结合）蛋白家族是主要依赖ATP的染色质重塑因子之一，可调节核小体定位以及转录因子的访问以及RNA聚合酶对真核基因组的访问。有3种哺乳动物的CHD亚家族，其功能受损与几种人类疾病有关。我们已经确定了迪斯特尔迪斯特尔的三个CHD直系同源物（CHDA，CHDB和CHDC）。这些CHD在整个开发过程中都表达出来，但具有独特的模式。缺乏每个CHD的空突变体具有不同的，非冗余的表型，反映其表达模式并提出功能特异性。因此，利用整个基因组（RNA-seq）的转录组分析，我们表明不同的CHD在生长和发育过程中调节独立的基因集。 CHDC是与人类电荷综合征相关的哺乳动物III级基因组的明显直系同源物，并且GO对CHDC-Nulls中异常基因表达的分析表明，通过在CHDC-Nulls在Pure Lows Cells Cill Cill Cills coll low later cells cill cell sallys conde sallys中证实了细胞自主和非自主信号的缺陷。我们通过染色质介导的定向基因表达的变化对CHD在调节发育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柱状骨），利用G蛋白耦合受体（GPCR）信号传导来调节MAPK/ERK，PI3K，TORC2/AKT，Adenylyl Cyclase和肌动蛋白的聚合，从而调节集体指导化合物。配体结合后，哺乳动物GPCR在细胞质残基上磷酸化，解开G蛋白途径，但激活其他途径。尽管如此，GPCR磷酸化与趋化性之间的连接尚不清楚。在开发dictyostelium的过程中，分泌的营地用作趋化剂，细胞外营地传播为振荡波，以确保方向性的迁移信号。 cAMP振荡来自腺苷环酶的短暂兴奋反应，然后迅速适应。我们已经研究了表达不可磷酸化的cAMP受体的dictyostelium中的趋化信号传导，并通过趋化模型，单细胞FRET成像，纯和嵌合群体小波小波的定量，生化分析和TIRF显微镜进行了调节磷酸化的养殖酶磷酸化和磷酸化疗法的磷酸化疗法，并且是长期磷酸化酶的磷酸化，这是长期循环，促磷酸化的磷酸化，并显示出长期磷酸化的磷酸化，从而调节了长期循环，从而长期循环，并将其显示为细胞骨骼肌动蛋白反应。因此，磷酸化缺陷会促进细胞周围，误导性假霉菌和方向性趋化性丧失的过度活跃肌动蛋白聚合。我们的数据表明，趋化剂受体磷酸化需要共同调节迁移细胞极化和趋化性的基本途径。我们的结果显着扩展了对GPCR磷酸化功能的理解，提供了有力的证据表明，在信号衰减途径中需要这种进化保守的机制，这是维持dictyostelium，中性粒细胞和其他迁移细胞的持续方向运动所必需的。 用不同的化学吸引剂刺激dictyostelium的全球刺激会引起多个瞬态信号反应，包括CAMP和CGMP的合成，肌动蛋白聚合，激活激活ERK2，TORC2和PI3K，以及RAS-GTP的积累；下调这些反应的机制知之甚少。我们检查了TORC2的瞬时激活，以响应化学上不同的化学吸引剂，cAMP和叶酸，并表明TORC2受自适应，去敏感性的反应对刺激配体的反应，这些反应独立于下游，反馈或前馈电路。如果用其他配体刺激，对叶酸或cAMP的无敏细胞对TORC2的激活保持完全反应。因此，TORC2对营地或叶酸的反应不是交叉抑制。使用一系列信号突变体，我们表明叶酸和cAMP通过相同的GEF/RAS途径激活TORC2，但分开的受体和G蛋白耦合。由于去敏化后，常见的GEF/RAS途径也对一种化学吸收剂充分响应，因此GEF/RAS必须在下游起作用，并且必须独立于适应持续的配体刺激。当初始趋化剂浓度立即稀释时，细胞会迅速恢复完全反应性。我们建议配体适应在上游抑制途径中起作用，涉及趋化因子特异性受体/G蛋白复合物并调节多个反应途径。