Mechanoregulation of Basal Keratinocyte Migration in Wounded Tissue

项目摘要 上皮的动态平衡是通过作用于整个组织的细胞的机械力的平衡来维持的- 比例。损伤破坏了这种机械平衡，目前还不清楚如何改变体内平衡的机械信号 影响伤口修复所需的细胞行为。不能有效修复会导致纤维性瘢痕，慢性 无法愈合的伤口，并有助于病理。上皮损伤修复依赖于基底膜的迁移 角质形成细胞转移到受损部位。虽然我们知道基底角质形成细胞对机械力很敏感， 我们缺乏对上皮损伤如何改变活体组织力学以及这些创伤如何诱导的了解 生物物理变化随后协调伤口修复所需的基本角质形成细胞的行为。本研究 旨在通过使用斑马鱼幼体来解决这些问题，斑马鱼幼体可以进行实时的、活体内的成像 它们的光学透明性。初步的活体成像实验表明，上皮损伤导致快速的基底动脉 角质形成细胞迁移到伤口部位，这是有效修复所需的。角质形成细胞的基础迁移是 依赖于机械信号，如细胞肿胀引起的膜张力，并与 伤口边缘上皮组织结构的一过性和局限性破坏。基底层角质形成细胞迁移 可以通过阻断Arp2/3复合体的激活或通过Talin1基因敲除来抑制，提示可能存在联系 机械信号与体内F-肌动蛋白或粘着斑复合体重塑之间的关系。更进一步，暂时的 减弱细胞与细胞外基质的粘附会改变角质形成细胞的基础迁移，导致创面较差。 愈合，并导致上皮结构的慢性破坏。这种表型模仿了相关的病理 金德勒综合征是一种皮肤病，患者在受伤后表现出伤口愈合缺陷。这些 初步观察表明，斑马鱼幼体的基底层角质形成细胞对机械信号有反应 在损伤后的上皮组织中，通过启动有效的伤口愈合所需的迁移反应来实现。他们 此外，基底层角质形成细胞有缺陷的迁移可能与创面愈合病理有关。这个 拟议的研究将调查机械传感器Piezo1和Talin1如何调节张力传感 分别通过F-肌动蛋白和局部黏附重塑诱导创伤后角质形成细胞的基本行为。这些 随后，研究结果将被转化为研究金德勒综合征的斑马鱼模型，以确定 基础角质形成细胞行为异常在伤口愈合病理生理学中的作用。为了确保 这个项目的成功，已经制定了一个量身定做的培训计划，利用了出色的研究成果 威斯康星大学麦迪逊分校的环境。使用斑马鱼模型的专门培训 用于伤口愈合研究的生物体和用于量化上皮组织的先进体内成像技术 机械师将帮助完成所规定的目标。这项培训将有助于成功过渡到 研究独立性。

Project Summary Epithelial homeostasis is maintained by the balance of mechanical forces acting upon cells across the tissue- scale. Injury disrupts this mechanical balance and it is unclear how changing homeostatic mechanical signals impacts cell behavior needed for wound repair. Failure to efficiently repair can lead to fibrotic scarring, chronic non-healing wounds, and contribute to pathology. Epithelial wound repair relies on the migration of basal keratinocytes to the site of damage. While it is known that basal keratinocytes are sensitive to mechanical forces, we lack an understanding of how epithelial injury alters tissue mechanics in vivo and how these wound-induced biophysical changes subsequently coordinate basal keratinocyte behavior needed for wound repair. This study aims to address these issues by using larval zebrafish, which are amenable to real-time, intravital imaging due to their optical transparency. Preliminary live-imaging experiments show that epithelial injury causes rapid basal keratinocyte migration to the wound site, which is needed for efficient repair. Basal keratinocyte migration is dependent on mechanical signals, such as membrane tension due to cell swelling, and is associated with a transient and localized disruption of epithelial tissue architecture at the wound edge. Basal keratinocyte migration can be inhibited by blocking Arp2/3 complex activation or through Talin1 knockdown, suggesting a potential link between mechanical signaling and F-actin or focal adhesion complex remodeling in vivo. Further, transiently weakening cell adhesion to the extracellular matrix alters basal keratinocyte migration, causing poor wound healing, and resulting in chronic disruption of epithelial architecture. This phenotype mimics pathology associated with Kindler Syndrome, a skin disease in which patients show wound healing defects in response to injury. These preliminary observations demonstrate that basal keratinocytes of larval zebrafish respond to mechanical signals in epithelial tissue after injury by initiating a migratory response that is required for efficient wound healing. They also suggest that defective basal keratinocyte migration may contribute to wound healing pathology. The proposed study will investigate how tension sensing by the mechanotransducers Piezo1 and Talin1 regulate wound-induced basal keratinocyte behavior by F-actin and focal adhesion remodeling, respectively. These findings will subsequently be translated to investigate a zebrafish model of Kindler Syndrome to determine the contribution of dysregulated basal keratinocyte behavior to wound healing pathophysiology. To ensure the success of this project, a tailored training plan has been developed that takes advantage of the excellent research environment at the University of Wisconsin – Madison. Dedicated training in the use of the zebrafish model organism for wound healing studies and advanced in vivo imaging techniques for quantifying epithelial tissue mechanics will aid in the completion of the stated aims. This training will facilitate a successful transition to research independence.