Cell-type specific assembly of apical extracellular matrices

顶端细胞外基质的细胞类型特异性组装

• 批准号: 10749768
• 负责人: Helen Frances Schmidt
• 金额: $ 6.95万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10749768
• 关键词: Address; Apical; Binding; Biological; Biological Assay; Biology; Biomedical Research; Caenorhabditis elegans; Candidate Disease Gene; Cell Nucleus; Cells; Cellular biology; Chronic Obstructive Pulmonary Disease; Classification; Collagen; Communities; Complex; Confocal Microscopy; Data; Destinations; Development; EGF gene; Educational process of instructing; Environment; Epithelium; Extracellular Matrix; Fibronectins; Fluorescent in Situ Hybridization; Future; Gene Structure; Genes; Genetic; Genetic Transcription; Genomics; Glycocalyx; Goals; Institution; Intestines; Laboratories; Labyrinth; Lobular Neoplasia; Lung; Malignant Neoplasms; Membrane Proteins; Microscopy; Modeling; Monitor; Mucins; Nature; Organ; Organism; Pattern; Pennsylvania; Peptide Hydrolases; Process; Protein Family; Proteins; Proteolysis; RNA Interference; Research; Resolution; Resources; Role; Running; Shapes; Signal Pathway; Site; Sorting; Specific qualifier value; Structure; Surface; System; Techniques; Tertiary Protein Structure; Testing; Training; Tube; Tubular formation; Ulcerative Colitis; Universities; Vulva; Work; Zona Pellucida; candidate identification; career; cell type; design; differential expression; experimental study; extracellular; fluorescence imaging; gene product; gene regulatory network; genomic platform; human disease; inner ear diseases; insight; knock-down; mutant; notch protein; promoter; recruit; sample fixation; single cell technology; single nucleus RNA-sequencing; transcription factor; transcriptome; transcriptomics; undergraduate student

Project Summary/Abstract The development and function of tubular organs, including the lungs, intestines, and inner ear, depends in part on an apical extracellular matrix (aECM) that lines the organ interior. aECMs contain many components that are shared across organisms such as mucins, collagens, and zona pellucida domain (ZP) proteins. Matrix proteins are often arranged into distinct layers or spatial domains within an organ; however, these structures are often destroyed by fixation, limiting the ability to study how they are formed. This proposal uses the well characterized and highly tractable model of the developing C. elegans vulva to understand how aECM is specified and assembled. Due to its transparent body and the ease of fluorescently tagging proteins, C. elegans is an ideal system to study aECM in a live organism. Many of the aECM domains in the vulva are cell type specific, allowing us to directly connect the assembly of matrix proteins to the biology of the cells they coat. This study focuses on two ZP matrix proteins, LET-653 and NOAH-1, that localize to the matrix of specific vulva cells. Like ZP proteins in some other systems, such as the mammalian inner ear, the localization of LET-653 and NOAH-1 is not well explained by their transcription patterns. ZP proteins require protease cleavage to properly localize and must also bind surface factors at their destination site. The identity of these ZP protein partners is unknown. Aim 1 will dissect the relative contributions of each cell type to the expression, processing and export, and matrix localization of ZP proteins. It will also identify the transcription factors required for cell type specific matrix fate. Aim 2 will identify candidate ZP protein partners with differential expression between cell types by single nucleus RNA sequencing (snRNA-Seq) and test their requirement for ZP matrix assembly. Ultimately, these experiments will uncover a gene regulatory network that specifies apical matrix identity and identify ZP protein partners that could be applicable to many systems. This project is designed to provide training in epithelial and matrix biology, as well as relevant techniques in single cell transcriptomics, genetics, and microscopy. It is sponsored by Dr. Meera Sundaram, an expert in matrix biology and C. elegans vulva development, and co-sponsored by Dr. John Murray, an expert in gene regulatory networks and single cell technologies in C. elegans. The research will be conducted at the University of Pennsylvania, an institution with a collaborative and stimulating intellectual environment as well as first-rate facilities and resources to conduct biomedical research. The proposed training plan incorporates many professional development opportunities available through the university, sponsors, and C. elegans community. At the conclusion of the proposed research and professional activities, the applicant will be prepared for a career incorporating teaching undergraduates and running her own laboratory.

项目总结/摘要 管状器官的发育和功能，包括肺、肠和内耳， 部分位于排列在器官内部的顶端细胞外基质（aECM）上。aECM包含许多组件 这些蛋白质在生物体中共有，例如粘蛋白、胶原蛋白和透明质酸结构域（ZP）蛋白。矩阵 蛋白质通常在器官内排列成不同的层或空间域;然而，这些结构 通常被固定所破坏，限制了研究它们是如何形成的能力。 该建议使用了发展中的C. elegans 外阴了解aECM是如何指定和组装。由于其透明的身体和方便， 荧光标记蛋白，C. elegans是研究活生物体中aECM的理想系统。许多 外阴中的aECM结构域是细胞类型特异性的，使我们能够直接连接基质的组装， 蛋白质与它们所覆盖的细胞的生物学联系在一起。本研究的重点是两个ZP基质蛋白，LET-653和 NOAH-1，其定位于特定外阴细胞的基质。像其他系统中的ZP蛋白一样， 哺乳动物内耳，LET-653和NOAH-1的定位不能很好地解释其转录 模式. ZP蛋白需要蛋白酶切割以正确定位，并且还必须在其位置结合表面因子。 目的地。这些ZP蛋白伴侣的身份是未知的。目标1将解剖亲属 每种细胞类型对ZP蛋白的表达、加工和输出以及基质定位的贡献。 它还将确定细胞类型特异性基质命运所需的转录因子。目标2将确定 单核RNA在细胞类型之间差异表达的候选ZP蛋白伴侣 测序（snRNA-Seq）并测试它们对ZP基质组装的要求。最终，这些实验将 揭示基因调控网络，指定顶端基质身份，并确定ZP蛋白伴侣， 它可以应用于许多系统。 该项目旨在提供上皮和基质生物学方面的培训， 单细胞转录组学、遗传学和显微镜技术。它由Meera Sundaram博士赞助， 基质生物学专家和C. Elegans外阴发育，并由John Murray博士共同赞助， 基因调控网络和单细胞技术在C.优美的这项研究将在 宾夕法尼亚大学，一个具有协作和激励智力环境的机构，以及 作为一流的设施和资源进行生物医学研究。拟议的培训计划包括 通过大学，赞助商和C提供许多专业发展机会。elegans 社区在拟议的研究和专业活动结束时，申请人将 她为自己的职业生涯做好了准备，包括教本科生和经营自己的实验室。