Molecular mechanisms that regulate target cell sensitivity to Hedgehog morphogens

项目摘要/摘要 刺猬(HH)信号对许多组织和器官的发育是必不可少的。HH配体是 因此，决定细胞命运的方式取决于信号强度。精确 对这种信号强度的调节对胚胎发育至关重要，因为即使是轻微的干扰 信号幅度可能导致严重的出生缺陷。许多研究表明，信号强度是 受形态因子浓度和暴露时间的影响。然而，一个同样的 重要的一层调控仍未被认识和研究不足：靶细胞如何调控其 对形态生成素敏感吗？我的博士后研究的中心焦点一直是研究 调节细胞对细胞外形态因子的敏感性。最初，我使用全基因组CRISPR筛查来 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了一种膜系泛素连接酶复合体(MMM复合体)，该复合体 通过清除细胞中的HH效应物平滑(SMO)来抑制细胞对HH形态因子的敏感性 表面纤毛和初级纤毛。使用基因敲除的小鼠模型，我也证明了MMM复合体是必不可少的 用于正确的左右排列和胚胎心脏发育。总体而言，我的工作已经开始(1) 解开靶细胞通过改变其对细胞外提示的反应的新机制 细胞表面蛋白质的图景和(2)阐明了构成复合体的机制 异位畸形和先天性心脏缺陷的遗传学。与新冠肺炎相关的研究限制和大学 关闭严重推迟了我的职业计划和个人发展。虽然我的研究进展反映了 顺利完成我原提案中的目标2和目标3，新冠肺炎研究受到严格限制 Aim1的进展延迟，该研究试图确定MMM复合体的机制 管理蛋白质交易事件。延长资助期限将使我能够大量发展关键的新技能 光谱和先进显微镜，以了解MMM复合体如何调节 蛋白质进入细胞表面和初生纤毛。我将通过我的导师Rajat博士的培训来实现这一点 Rohatgi(生物化学)，我的共同导师Tim Stearns博士(纤毛生物学)和Ryan Leib博士(蛋白质组学 斯坦福大学质谱学核心设施主任和我的科学咨询委员会成员)。一个 延长资助期限还将使我能够在罗康瑞博士的指导下进一步培训，使我更精通 分析胚胎小鼠心脏的发育缺陷。总而言之，我将进行的培训和指导 在延长的K99期间收到的将使我具备过渡到学习所需的知识 导致异位畸形和先天性心脏缺陷的分子机制。归根结底，这 培训将是我转变为独立调查员的关键，并帮助我建立一个充满活力的 细胞信号和发育生物学领域的研究计划。

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.