Molecular Mechanisms Underlying Cytoneme Formation by Sonic Hedgehog-Producing Cells

Sonic Hedgehog 产生细胞形成细胞因子的分子机制

• 批准号: 10678288
• 负责人: Christina Adele Daly
• 金额: $ 4.37万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL GRADUATE SCHOOL OF BIOMEDICAL SCIENCES, LLC
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10678288
• 关键词: Actin-Binding Protein; Actins; Address; Appearance; Automobile Driving; Binding; Biochemical; Biochemistry; Biological; Brothers; Cell Adhesion; Cell Communication; Cell Differentiation process; Cell surface; Cell-Adhesion Molecule Receptors; Cells; Communication; Cultured Cells; Cytoskeletal Modeling; Cytoskeleton; Defect; Development; Developmental Process; Disease; Electron Microscopy; Embryo; Embryonic Development; Fibroblasts; Filopodia; Fixatives; Funding; Genetic; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Image; Imaging Techniques; In Vitro; Integral Membrane Protein; Knowledge; Laboratories; Ligands; Limb Development; Link; Malignant - descriptor; Malignant Neoplasms; Mediating; Methods; Molecular; Mus; Mutant Strains Mice; Neural Tube Development; Neural tube; Oncogenes; Pathology; Pathway interactions; Pattern; Phosphorylation; Phosphotransferases; Physiological; Play; Polymers; Process; Proteins; Protocols documentation; Regulation; Role; SHH gene; Signal Pathway; Signal Transduction; Signaling Molecule; Signaling Protein; Sonic Hedgehog Pathway; Structure; Techniques; Testing; Thinness; Tissues; Translating; adult stem cell; autocrine; bioimaging; confocal imaging; defined contribution; developmental disease; genetic manipulation; imaging approach; in vivo; kinase inhibitor; malformation; morphogens; polymerization; preservation; protein protein interaction; receptor; response; rho GTP-Binding Proteins; sample fixation; smoothened signaling pathway; sonic hedgehog receptor; stem cell niche; tumor

ABSTRACT Proper physiological development relies on cell-to-cell communication via secreted morphogens. These potent signaling molecules signal at both short and long range in temporal- and tissue-specific manners to determine cell fate and pattern tissues. Sonic Hedgehog (SHH), a well-studied vertebrate morphogen, plays an integral role in a variety of developmental processes including neural tube patterning and limb development. Defects in the pathway result in developmental malformations, while aberrant activation has been associated with developmental disease and malignant transformation. SHH-producing cells extend thin, actin-based projections to transport SHH to receiving cells. These specialized filopodia are termed “cytonemes” due to their thread-like appearance. Cytonemes are proposed to play a key role in tightly regulating morphogen concentration gradients during development. However, little is known about the molecular mechanisms driving cytoneme formation. This is mostly due to the difficulty to study the delicate structures using conventional fixation methods. To permit mechanistic studies in vitro, our lab developed a modified electron microscopy fixative (MEM- fix) protocol that preserves cytoneme integrity for confocal analysis. Using MEM-fix, along with a combination of advanced imaging and biochemical techniques, our lab has found that expression of SHH can promote cytoneme initiation in cultured murine fibroblasts. Furthermore, we identified a requirement for the SHH coreceptors and adhesion proteins, Cell adhesion-associated, Down- regulated by Oncogenes (CDON) and Brother of CDON (BOC), along with Dispatched (DISP) deployment receptor in SHH-mediated cytoneme formation and stability. These findings along with identification of interactions between SHH, DISP, and BOC as well as DISP and CDON point towards cytoneme-initiating signaling occurring downstream of SHH binding to these transmembrane proteins. However, the specific interactions and intracellular signaling pathways that initiate cytoneme outgrowth have yet to be identified. My proposed project is focused on determining the SHH-activated signals occurring in response to its association with BOC and DISP that drive cytoneme initiation in SHH- producing cells. The first goal of my project is to elucidate the specific cell surface interactions between DISP, BOC, CDON, and SHH that signal to intracellular cytoskeletal regulators. The second aspect of my project is to elucidate the regulatory molecules that directly promote actin nucleation and polymerization in response to SHH. To address these outstanding questions, I am combining targeted genetic and biochemical techniques with advanced imaging approaches to methodically interrogate the signal cascade resulting in SHH-mediated induction of cytoneme formation. I will confirm findings in vivo by using our recently developed protocol facilitating imaging of cytonemes in mouse embryos.

摘要 正常的生理发育依赖于通过分泌的形态发生素进行的细胞间通讯。这些 有效的信号分子以时间和组织特异性方式在短距离和长距离上发出信号 以确定细胞命运和组织模式。Sonic Hedgehog（SHH），一种被广泛研究的脊椎动物 形态发生素，在包括神经管在内的多种发育过程中起着不可或缺的作用 模式和肢体发育。该途径的缺陷导致发育畸形，而 异常激活与发育性疾病和恶性转化有关。 产生SHH的细胞延伸薄的、基于肌动蛋白的突起以将SHH运输到接收细胞。这些 特化的丝状伪足由于其线状外观而被称为“细胞丝”。细胞丝是 提出了在严格调节形态发生剂浓度梯度的过程中发挥关键作用， 发展然而，驱动细胞丝形成的分子机制知之甚少。 这主要是由于使用传统固定方法难以研究精细结构。 为了进行体外机制研究，我们的实验室开发了一种改良的电子显微镜固定液（MEM- Fix）方案，该方案保持了用于共聚焦分析的细胞丝完整性。使用MEM-fix，沿着 结合先进的成像和生化技术，我们的实验室发现， SHH可促进培养的小鼠成纤维细胞的细胞丝起始。此外，我们还发现了一个 需要SHH辅助受体和粘附蛋白，细胞粘附相关，Down- 受癌基因（CDON）和CDON兄弟（BOC）调节，沿着调度（DISP） 部署受体在SHH介导的细胞丝形成和稳定性。这些发现沿着 SHH、DISP和BOC之间以及DISP和CDON之间相互作用的鉴定表明， 细胞丝起始信号传导发生在SHH结合这些跨膜蛋白的下游。 然而，启动细胞丝生长的特异性相互作用和细胞内信号传导途径 还有待确认我提出的项目集中在确定SHH激活的信号 在SHH中与BOC和DISP相关，而BOC和DISP驱动细胞丝起始， 产生细胞。我的项目的第一个目标是阐明特定的细胞表面之间的相互作用， DISP、BOC、CDON和SHH，其向细胞内细胞骨架调节剂发出信号。的第二方面 我的项目是阐明直接促进肌动蛋白成核的调节分子， 聚合反应响应SHH。为了解决这些悬而未决的问题，我将有针对性地 遗传和生化技术与先进的成像方法，有条不紊地询问 导致SHH介导诱导细胞丝形成的信号级联。我将确认 体内使用我们最近开发的协议，促进成像的小鼠胚胎中的cytonemes。