Characterization of PDGFR dimer-specific dynamics in the craniofacial mesenchyme

颅面间质中 PDGFR 二聚体特异性动力学的表征

基本信息

批准号：
10361222
负责人：
Katherine Ann Fantauzzo
金额：
$ 12.53万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10361222
关键词：
Ablation Adult Affinity Affinity Chromatography Alleles Authorization documentation Award Binding Bioinformatics Biological Assay Biology Branchial arch structure C-terminal Cell Lineage Cell Proliferation Cell physiology Cells Cellular biology Cephalic Cleft Lip Cleft Palate Collaborations Colorado Communication Complement Complex Congenital Abnormality Defect Dental Schools Development Disease Ectoderm Embryo Equipment Etiology Event Fluorescence Fluorescence Microscopy Frozen Sections Funding Gene Expression Genes Genetic Epistasis Genetic Transcription Goals Hemorrhage Human Immunoprecipitation In Vitro Incidence Independent Scientist Award Intracellular Signaling Proteins Joints Knock-in LEOPARD Syndrome Laboratory Research Leadership Life Ligands Ligation Live Birth Malignant Neoplasms Mass Spectrum Analysis Maxilla Medical Medicine Mesenchyme Microscopy Morphogenesis Mus Mutant Strains Mice N-terminal Nasal septum structure Neural Crest Cell Noonan Syndrome Ontology Output Pattern Phenotype Plasmids Platelet-Derived Growth Factor Platelet-Derived Growth Factor Receptor Play Postdoctoral Fellow Process Property Protein Dephosphorylation Proteins Proteome Receptor Protein-Tyrosine Kinases Research Role Signal Transduction Signaling Molecule Techniques Technology Terminator Codon Testing Time Training Training Programs Transcript Travel United States Universities Vascular Diseases Venus Wages Width career development cohesion craniofacial craniofacial development craniofacial structure design differential expression dimer experimental study gain of function mutation homologous recombination in vivo innovation insight meetings migration mutant mutant mouse model nanobodies novel novel therapeutics osteoblast differentiation palatal shelves platelet-derived growth factor BB programs receptor recruit response spatiotemporal training opportunity transcriptome transcriptome sequencing working group

项目摘要

Project Summary Craniofacial development is a complex morphogenetic process, disruptions in which result in highly prevalent human birth defects. Signaling through the platelet-derived growth factor receptors (PDGFRs) plays a critical role in this process in humans and mice. PDGFRalpha and PDGFRBeta have recently been shown to genetically and physically interact in the craniofacial mesenchyme to form functional heterodimers with unique signal molecule binding properties and the ability to generate more robust intracellular signaling and mitogenic responses in vitro than those generated by homodimeric receptor complexes. The aim of this proposal is to examine the in vivo dynamics of PDGFR dimer-specific formation, as well as the resulting effects on gene expression and cell activity in the craniofacial mesenchyme. First, PDGFR-bimolecular fluorescence complementation (BiFC) fragment alleles will be generated containing the N- or C-terminal regions of the Venus fluorescent protein. Venus expression will be analyzed in craniofacial structures by fluorescence microscopy to examine the spatiotemporal dynamics of PDGFR homodimer versus heterodimer formation. Second, the effect of SHP-2 binding to PDGFRalpha on downstream signaling will be determined through genetic epistasis experiments and, in parallel, BiFC and affinity purification will be employed to selectively purify PDGFRalpha/Beta heterodimers and identify PDGFR dimer-specific interacting proteins by mass spectrometry. Finally, RNA-sequencing will be performed to define the transcriptional program induced downstream of PDGFR dimer-specific activation in the maxillary processes. Additional experiments, technologies and training opportunities are introduced in this Independent Scientist Award application that significantly extend and enhance the original research plan and allow for career development. These include cell biology and superresolution microscopy training to characterize the subcellular localization and internalization dynamics of the various PDGFR dimers; training in the application of bioinformatics approaches for RNA-sequencing analysis; and participation in leadership and scientific communication training programs. This training plan will take place within the School of Dental Medicine at the University of Colorado Anschutz Medical Campus, which has a strong, well-established research program in craniofacial biology and provides ample opportunities for training and collaboration. University support includes guaranteed salary support beyond the award period; provision of funds for post-doctoral fellow recruitment, shared equipment usage, course tuition and travel to scientific meetings; permission for the candidate to spend essentially full-time conducting research; and participation in joint lab meetings and working groups designed to facilitate the development of the candidate's research program. Combined, these activities will contribute towards the goal of establishing a recognized research laboratory committed to understanding the signaling hierarchies that govern mammalian craniofacial development and how disruption of these signaling events leads to human craniofacial birth defects.

项目摘要颅面发育是一个复杂的形态发生过程，干扰导致高度普遍的人类先天缺陷。通过血小板衍生的生长因子受体（PDGFR）发出信号传导在这一过程中的关键作用在人类和小鼠中。 PDGFRALPHA和PDGFRBETA最近已显示为遗传和物理在颅面间充质中相互作用，形成具有独特的功能异二聚体信号分子结合特性以及产生更健壮的细胞内信号传导和有丝分裂的能力体外反应比同二聚体受体复合物产生的反应。该提议的目的是检查PDGFR二聚体特异性形成的体内动力学以及对基因的影响颅面间充质中的表达和细胞活性。首先，PDGFR分子荧光将生成互补（BIFC）片段等位基因，其中包含包含N-或C末端区域金星荧光蛋白。金星表达将通过荧光在颅面结构中进行分析显微镜检查PDGFR同二聚体与异二聚体形成的时空动力学。其次，将通过遗传确定SHP-2与PDGFRALPHA对下游信号传导的影响上位性实验，并同时将使用BIFC和亲和力纯化来选择性纯化 PDGFRALPHA/BETA异二聚体并通过质谱识别PDGFR二聚体特异性相互作用的蛋白质。最后，将执行RNA测序以定义转录程序在下游的下游上颌过程中的PDGFR二聚体特异性激活。其他实验，技术和培训这项独立科学家奖的申请中引入了机会，该奖项大大扩展和增强原始研究计划并允许职业发展。这些包括细胞生物学和超分辨率显微镜训练以表征细胞亚本地化和内在化动力学各种PDGFR二聚体；在应用生物信息学方法中进行RNA测序分析的培训；并参与领导力和科学沟通培训计划。该培训计划将发生在科罗拉多州安索兹大学医学校园的牙科医学院内颅面生物学方面的强大，公认的研究计划，并为培训提供了足够的机会和协作。大学的支持包括颁奖期限之后的保证工资支持；提供博士后招聘，共享设备使用，课程学费和前往科学的资金会议；候选人的许可，本质上花费全职进行研究；并参与联合实验室会议和旨在促进候选人研究发展的工作组程序。这些活动结合在一起，将有助于建立公认的研究的目标实验室致力于了解哺乳动物颅面的信号层次结构发展以及这些信号事件的破坏如何导致人类颅面的先天缺陷。