Role of junctional complexes in the maintenance and renewal of the epidermis

皮肤的最外层称为表皮，它为所有哺乳动物提供保护，使其免受环境的侵害。在每2-4周发生一次的表皮自我更新期间，角质形成细胞必须经历一系列精心策划的事件，包括细胞增殖、分化、迁移和程序性细胞死亡。长期目标：为了适应表皮的动态更新，在接触角质形成细胞之间发现的粘附连接（紧密连接、粘附连接和桥粒）和通讯连接（间隙）被破坏并以高度调节的方式重建。粘合剂和间隙连接共享一些共同的构建块，促进它们的相互作用和串扰，我们提出这对皮肤维护和屏障功能至关重要。我们的长期目标是充分了解粘附和通信连接如何协调工作，以协调表皮的更新。 短期目标：啮齿类动物表皮含有至少六种连接蛋白（Cx 26、Cx 30、Cx30.3、Cx 31、Cx31.1和Cx43;本文称为“角质形成细胞连接蛋白”），其在时间和空间上表达以在接触的角质形成细胞之间形成间隙连接的复杂阵列。连接蛋白以其规范形式允许代谢物的调节和直接的细胞-细胞通道，但也可能具有非规范作用，其中细胞表面的功能性半通道参与旁分泌信号传导或与粘附连接的成分串扰以影响表皮完整性和屏障功能。我们的短期目标是询问和确定每个角质形成细胞连接蛋白是如何部署在角质形成细胞调节表皮的稳态。假设和方法：我们的假设是角质形成细胞连接蛋白具有独特的细胞生命周期，并使用经典和非经典功能来精细地调节表皮稳态和屏障功能。我们的研究将集中于具有分化能力的大鼠表皮角质形成细胞，其表现出以下稳健特征：a）适合于未标记和标记的野生型和功能有限的角质形成细胞连接蛋白的异位表达，B）理想地研究连接蛋白相互作用组和与粘附连接蛋白的串扰，c）对高分辨率活细胞成像和评估连接蛋白生命周期的方法有弹性，d）适合于评估连接蛋白半通道和细胞间通道功能，和e）适合于确定连接蛋白对角质形成细胞表型和器官型表皮形成的影响。 重要性：皮肤对环境损害的抵抗力主要是通过维持生命的屏障功能建立的，该屏障功能由在具有通信能力的角质形成细胞之间形成的粘附连接提供。我们正准备研究粘合剂和通信网络如何协同工作以适应表皮自我更新的机制。

The outer most layer of the skin is called the epidermis which provides all mammals with protection against environmental insults. During the self-renewal of the epidermis that occurs every 2-4 weeks, keratinocytes must undergo a well-orchestrated series of events that involve cell proliferation, differentiation, migration, and programmed cell death. Long-term Objective: To accommodate the dynamic turnover of the epidermis, adhesive junctions (tight, adherens and desmosomes) and communication junctions (gap) found between contacting keratinocytes are destroyed and rebuilt in a highly regulated fashion. Adhesive and gap junctions share some common building blocks that facilitates their interplay and crosstalk that we proposed is critical for skin maintenance and barrier function. Our long-term goal is to fully understand how both adhesive and communication junctions work in harmony to orchestrate the renewal of the epidermis. Short-term Objective: Rodent epidermis contains at least six connexins (Cx26, Cx30, Cx30.3, Cx31, Cx31.1 and Cx43; termed hereon as "keratinocyte connexins") that are temporally and spatially expressed to form a complex array of gap junctions between contacting keratinocytes. Connexins in their canonical form allow the regulated and direct cell-cell passage of metabolites but may also have non-canonical roles where functional hemichannels at the cell surface engage in paracrine signalling or crosstalk with constituents of adhesive junctions to impact epidermis integrity and barrier function. Our short-term goal is to interrogate and determine how each keratinocyte connexin is deployed in keratinocytes to regulate the homeostasis of the epidermis. Hypothesis and Approach: It is our hypothesis that keratinocyte connexins have unique cellular life cycles and use both canonical and non-canonical functions to exquisitely regulate epidermal homeostasis and barrier function. Our studies will focus on differentiation-competent rat epidermal keratinocytes that exhibit robust features of being, a) amenable to the ectopic expression of untagged and tagged wildtype and functionally-limited keratinocyte connexins, b) ideal to investigate the connexin interactome and crosstalk with adhesive junction proteins, c) resilient to high-resolution live cell imaging and approaches to assess the life cycle of connexins, d) suitable for assessing both connexin hemichannel and intercellular channel function, and e) appropriate for determining the impact of connexins on the phenotype of keratinocytes and organotypic epidermis formation. Significance: The resistance of skin to environmental insults is largely established by life-sustaining barrier function provided by adhesive junctions that form between communication-competent keratinocytes. We are uniquely poised to investigate the mechanisms underpinning how adhesive and communication networks work together to accommodate the self-renewal of the epidermis.