Migratory Morphology: A Function of Fibrous Extracellular Matrix Geometry Sensing

迁移形态：纤维细胞外基质几何传感的功能

• 批准号: 8824068
• 负责人: Justin L. Brown
• 金额: $ 20.26万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-26 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8824068
• 关键词: Adhesions; Architecture; Binding; Binding Proteins; Biocompatible Materials; Biological Assay; Biology; Biomimetic Materials; Bulla; Caliber; Cell Adhesion; Cell Culture Techniques; Cell physiology; Cell-Cell Adhesion; Cells; Cellular biology; Co-Immunoprecipitations; Development; Discipline; Engineering; Extracellular Matrix; Fiber; Fluorescence Resonance Energy Transfer; Focal Adhesion Kinase 1; Focal Adhesions; Foundations; Future; Geometry; Grant; Guanosine Triphosphate Phosphohydrolases; Guidelines; In Vitro; Knowledge; Lateral; Location; Mediating; Membrane; Mesenchymal; Mesenchymal Stem Cells; Modeling; Monomeric GTP-Binding Proteins; Morphology; Neoplasm Metastasis; Organ; PTK2 gene; Pathologic Processes; Pattern; Phosphorylation; Phosphotransferases; Photons; Physical environment; Physiological Processes; Proteins; Regenerative Medicine; Role; Science; Spatial Distribution; Stem cells; Time; Tissue Engineering; Tissues; Vesicle; Western Blotting; Work; Wound Healing; caveolin 1; cell motility; citrate carrier; design; in vivo; in vivo Model; insight; migration; nanofiber; next generation; novel; public health relevance; response; time use; tissue regeneration; tumor

DESCRIPTION: The mechanism by which a cell interacts with its physical environment underlies fundamental cell functions of cell adhesion and migration. Understanding these relationships has broad ranging implications from informing the design of next generation engineered tissues to aiding in the understanding of tumor metastases. Cell migration has long been studied in the context of a petri dish; however, recent work has shown that cells migrate in vivo through a unique mechanism, lobopodial migration, not observed in standard in vitro cell culture. This application seeks to develop a model of in vivo migration, and examine two fundamental unanswered questions related to migratory morphologies. This grant seeks to merge the disciplines of regenerative medicine, materials science and cell biology to determine a mechanism by which MSCs sense and respond to the curvature of nanofibers present in the extracellular matrix and leading to altered migratory morphology. Specifically, this application seeks to identify if MSCs are capable of actively wrapping around a nanofiber and if they can then sense the diameter of the nanofiber through focal adhesion or vesicle-stabilizing mechanisms leading to altered migratory morphology. Preliminary evidence in support of this grant has indicated that there is a correlation between fiber diameter and focal adhesion size/maturity. The fiber diameters corresponding to the largest adhesions also demonstrated increased RhoA activity and cytoskeletal stiffness. Additionally, nanofiber diameter demonstrated a correlation with the vesicle-stabilizing protein Arfaptin 2; when fiber diameter approached the typical size of an Arfaptin 2 decorated vesicle the level of Rac-1 activation increased in an Arfaptin 2 specific manner (Arfaptin 2 is known to associate with active Rac-1). Specific Aim 1 will produce nanofiber substrates that demonstrate a range of diameters from 1.0�m - 100nm; while maintaining consistency with all other geometric parameters of a nanofiber substrate and will examine the activation and localization of the GTPases cdc42, Rac-1 and RhoA to determine the migratory morphology on each fiber diameter. Additionally, Specific Aim 1 will use time-correlated single photon counting to determine whether the membrane near the nanofiber is under tension or compression. Specific Aim 2 examines the role of the focal adhesion proteins FAK and Src on sensing the diameter of nanofibers leading to altered migratory morphology. Specific Aim 3 examines the role of the vesicle-stabilizing proteins, Arfaptin 2 and Caveolin 1&2, on sensing the diameter of nanofibers leading to altered migratory morphology. Successful completion of this application will provide design guidelines for future biomaterial architectures for applications from wound healing to tissue regeneration and advance biology through identification of an intracellular sensing mechanism of the extracellular matrix and expanded current knowledge of why unique migratory morphologies are observed in vivo.

产品说明：细胞与其物理环境相互作用的机制是细胞粘附和迁移的基本细胞功能的基础。理解这些关系具有广泛的意义，从为下一代工程组织的设计提供信息到帮助理解肿瘤转移。细胞迁移长期以来一直在培养皿中进行研究;然而，最近的工作表明，细胞在体内通过一种独特的机制迁移，即叶足迁移，而在标准的体外细胞培养中没有观察到。本申请旨在开发体内迁移模型，并研究与迁移形态学相关的两个基本未回答的问题。这项资助旨在合并再生医学，材料科学和细胞生物学的学科，以确定MSC感知和响应细胞外基质中存在的纳米纤维曲率并导致迁移形态改变的机制。具体地，本申请试图鉴定MSC是否能够主动地包裹在血管周围，以及它们是否可以通过导致改变的迁移形态的粘着斑或囊泡稳定机制来感知血管的直径。支持该资助的初步证据表明，纤维直径与焦点粘合尺寸/成熟度之间存在相关性。纤维直径对应于最大的粘连也表现出增加的RhoA活性和细胞骨架刚度。此外，纤维直径证明与囊泡稳定蛋白Arfaptin 2相关;当纤维直径接近Arfaptin 2修饰囊泡的典型尺寸时，Rac-1活化水平以Arfaptin 2特异性方式增加（已知Arfaptin 2与活性Rac-1相关）。Specific Aim 1将生产直径范围为1.0 μ m -100 nm的纤维基质;同时保持与纤维基质的所有其他几何参数的一致性，并将检查GTP酶cdc 42，Rac-1和RhoA的激活和定位，以确定每个纤维直径上的迁移形态。此外，具体目标1将使用时间相关单光子计数来确定隔膜附近的膜是处于拉伸还是压缩状态。具体目标2研究了粘着斑蛋白FAK和Src在感知纳米纤维直径导致迁移形态改变方面的作用。具体目标3研究了囊泡稳定蛋白Arfaptin 2和Caveolin 1和2在感知纳米纤维直径导致迁移形态改变方面的作用。成功完成这一申请将提供设计指南，为未来的生物材料架构的应用，从伤口愈合组织再生和先进的生物学通过识别细胞外基质的细胞内传感机制，并扩大目前的知识，为什么独特的迁移形态在体内观察。