Molecular mechanisms that regulate target cell sensitivity to Hedgehog morphogens

调节靶细胞对刺猬形态发生素敏感性的分子机制

• 批准号: 10373751
• 负责人: Jennifer Kong
• 金额: $ 8.11万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10373751
• 关键词: Advisory Committees; Area; Biochemistry; Biology; COVID-19; CRISPR screen; Cell Proliferation; Cell surface; Cells; Cilia; Complex; Congenital Abnormality; Congenital Heart Defects; Core Facility; Cues; Defect; Development; Developmental Biology; Embryo; Embryonic Development; Embryonic Heart; Erinaceidae; Event; Funding; Future; Genetic; Heart; Knockout Mice; Knowledge; Left; Ligands; Light; Malignant Neoplasms; Mass Spectrum Analysis; Membrane; Mentors; Mentorship; Microscopy; Molecular; Mus; Organ; Pathway interactions; Pattern; Play; Proteins; Proteomics; Regulation; Research; Research Personnel; Role; Signal Transduction; Situs Inversus; Tissues; Training; Universities; Work; cardiogenesis; career; extracellular; genome-wide; member; morphogens; mouse model; novel; programs; protein transport; response; skills; smoothened signaling pathway; targeted treatment; tissue regeneration; ubiquitin ligase

PROJECT SUMMARY/ABSTRACT Hedgehog (Hh) signaling is essential for the development of many tissues and organs. Hh ligands are morphogens and thus direct cell fate decisions in a manner dependent on signaling strength. Precise regulation of this signaling strength is critical for embryonic development, as even modest disruptions in the signaling amplitude can result in severe birth defects. Many studies have shown that signaling strength is influenced by both morphogen concentration and duration of morphogen exposure. However, an equally important layer of regulation remains unrecognized and understudied: How does a target cell regulate its sensitivity to a morphogen? The central focus of my postdoctoral research has been to study the mechanisms that modulate a cell’s sensitivity to extracellular morphogens. Initially, I used genome-wide CRISPR screens to discover three novel Hh signaling attenuators: Mosmo, Megf8, and Mgrn1. Through support from the K99, I showed that these three proteins form a membrane-tethered ubiquitin ligase complex (the MMM complex) that suppresses a cell’s sensitivity to Hh morphogens by clearing the Hh effector Smoothened (SMO) from the cell surface and primary cilium. Using knockout mouse models, I also showed that the MMM complex is essential for proper left-right patterning and embryonic heart development. Collectively, my work has begun to (1) unravel a new mechanism through which target cells modify their responses to extracellular cues by altering the landscape of proteins at the cell surface and (2) shed light on the mechanisms that underlie the complex genetics of heterotaxy and congenital heart defects. COVID-19 related research restrictions and university closures severely delayed my career plans and personal development. While my research progress reflects the successful completion of Aims 2 and 3 in my original proposal, COVID-19 research restrictions severely delayed progress on Aim1, which sought to determine the mechanism through which the MMM complex regulates protein trafficking events. A funding extension would allow me to develop critical new skills in mass spectrometry and advanced microscopy to understand how the MMM complex regulates the trafficking of proteins to the cell surface and primary cilium. I will accomplish this with training from my mentor Dr. Rajat Rohatgi (biochemistry), my co-mentor Dr. Tim Stearns (cilia biology), and Dr. Ryan Leib (the proteomics director of the Stanford mass spectrometry core facility and member of my scientific advisory committee). A funding extension would also allow me to further train under Dr. Cecilia Lo, to become more proficient at analyzing embryonic mouse hearts for developmental defects. In summary, the training and mentorship I will receive during the extended K99 period will equip me with the knowledge necessary to transition into the study of the molecular mechanisms that contribute to heterotaxy and congenital heart defects. Ultimately, this training will be critical in my transition into an independent investigator and help me establish a vibrant research program in the fields of Cell Signaling and Developmental Biology.

项目概要/摘要 Hedgehog (Hh) 信号传导对于许多组织和器官的发育至关重要。 Hh 配体是 形态发生素，从而以依赖于信号强度的方式指导细胞命运决定。精确的 这种信号强度的调节对于胚胎发育至关重要，因为即使是轻微的破坏 信号幅度可能导致严重的出生缺陷。许多研究表明信号强度 受形态发生素浓度和形态发生素暴露时间的影响。然而，一个同样 重要的调控层仍未得到认识和研究：靶细胞如何调节其 对形态发生素的敏感性？我博士后研究的重点是研究其机制 调节细胞对细胞外形态发生素的敏感性。最初，我使用全基因组 CRISPR 筛选 发现三种新型 Hh 信号衰减器：Mosmo、Megf8 和 Mgrn1。通过K99的支持，我 表明这三种蛋白质形成膜束缚的泛素连接酶复合物（MMM 复合物）， 通过清除细胞中的 Hh 效应子平滑 (SMO) 来抑制细胞对 Hh 形态发生素的敏感性 表面和初级纤毛。使用基因敲除小鼠模型，我还表明 MMM 复合物是必不可少的 用于正确的左右模式和胚胎心脏发育。总的来说，我的工作已经开始 (1) 揭示了一种新机制，通过该机制，靶细胞可以通过改变细胞外信号来改变其对细胞外信号的反应 细胞表面蛋白质的景观以及（2）揭示了该复合物背后的机制 异位性和先天性心脏缺陷的遗​​传学。 COVID-19 相关研究限制和大学 关闭严重延迟了我的职业计划和个人发展。虽然我的研究进展反映了 成功完成我最初提案中的目标 2 和 3，COVID-19 研究受到严格限制 推迟了 Aim1 的进展，该项目试图确定 MMM 复合物的机制 调节蛋白质贩运事件。资金延期将使我能够大规模开发关键的新技能 光谱测定法和先进的显微镜来了解 MMM 复合物如何调节 蛋白质到达细胞表面和初级纤毛。我将通过我的导师 Rajat 博士的培训来实现这一目标 Rohatgi（生物化学）、我的合作导师 Tim Stearns 博士（纤毛生物学）和 Ryan Leib 博士（蛋白质组学） 斯坦福大学质谱核心设施主任和我的科学顾问委员会成员）。一个 延期资助也让我能够在卢博士的指导下进一步接受培训，以更加熟练地掌握 分析小鼠胚胎心脏的发育缺陷。总而言之，我将接受的培训和指导 在延长的 K99 期间收到的课程将为我提供过渡到学习所需的知识 导致异位性和先天性心脏缺陷的分子机制。最终，这 培训对于我向独立调查员的过渡至关重要，并帮助我建立一个充满活力的 细胞信号传导和发育生物学领域的研究计划。