Mechanotransduction-inflammation coupling: Piezo1-dependent cellular morphology changes in disease

项目总结/摘要 该R35计划旨在描述Piezo 1机械敏感离子的先前未被认识的作用 使用关节软骨细胞（AC）作为炎症条件下调节细胞活性的通道， 模型系统机械转导是一个高度动态和普遍存在的过程，几乎所有哺乳动物， 细胞在机械刺激下表现出快速和强有力的离子流入。在疾病状态下，细胞呈现异常 通过改变的机械敏感性和生化转导揭示的机械转导。Piezo 1是一个 新型机械激活（MA）Ca 2+渗透通道在心血管、肌肉、 骨骼和神经组织AC中Piezo 1的增强功能表达与软骨相关 紊乱有趣的是，AC呈现出从球形AC到具有点状突起的细胞的形态分化。 骨关节炎的过程。肌动蛋白丝（F-肌动蛋白）网络一直专注于理解形态， AC的逻辑分化和机械稳定性，但我们的初步数据表明Piezo 1的潜在作用 在IL 1 α介导的炎症条件下，F-actin重塑过程中的作用。 在这里，我们提出了Piezo 1介导的形态学变化和促凋亡的范式转变概念。 炎症条件下的囊肿形成。机械传导和炎症都影响或重新影响细胞的功能。 导致了一系列生理过程和异常。机械中存在知识缺口- 非转导-炎症偶联，形态学依赖性细胞机械敏感性，Piezo 1介导 形态学变化，Piezo 1在圆形AC膜皮质上的定位，以及Piezo 1-recruit- 成纤维细胞样形状的AC的过程中的部分。该拟议方案将确定：（一）如果激活压电- 终止局部膜弯曲并触发形成细胞突起和重组细胞骨架网络， (ii)特异性炎性细胞因子触发形态学变化并增强Piezo 1通道， cesses，这反过来又改变了Piezo 1的门控特性，以及（iii）如果Piezo 1介导的炎症反应， sponse增加机械疲劳和死亡。全细胞膜片钳方法可以记录 通道活动，但AC提出了密封膜的技术挑战，主要是由于它们的圆 形态和小尺寸（直径约10 μm）。因此，我们定制了一种新的显微镜，名为 “机械显微镜”，通过结合原子力显微镜（AFM），高速比率Ca 2 + im- 老化显微镜和共聚焦显微镜，以同时记录细胞形态和生化 在炎症条件下响应机械提示的信号。此外，我们将利用超级... 分辨率STED显微镜和Piezo 1-TdTomato报告小鼠询问膜弯曲的动力学， 在治疗特定的炎性细胞因子后，AC中的Piezo 1定位。 我们的计划将在概念上揭示Piezo 1依赖性炎症反应的基本过程， 海绵体导致细胞形态分化。此外，我们的研究结果将提供新的见解， 机械传导-炎症耦合机制，并导致新的治疗策略，以防止 在软骨疾病的早期阶段的形态学变化。

PROJECT SUMMARY / ABSTRACT This R35 program aims to delineate a previously unappreciated role of Piezo1 mechanosensitive ion channels in regulating cellular activity under inflammatory conditions using articular chondrocytes (ACs) as a model system. Mechanotransduction is a highly dynamic and ubiquitous process, and virtually all mammalian cells exhibit rapid and robust ion influx upon mechanical stimulus. In disease states, cells present abnormal mechanotransduction revealed by altered mechanosensitivity and biochemical transduction. Piezo1 is a novel mechanically-activated (MA) Ca2+-permeating channel abundantly expressed in cardiovascular, musculo- skeletal, and neural tissues. Augmented functional expression of Piezo1 in ACs is associated with cartilage disorders. Interestingly, ACs present a morphological differentiation from spherical ACs to cells with point-like processes in osteoarthritis. The actin filament (F-actin) networks have been focused on understanding morpho- logical differentiation and mechanical stability of AC, yet our preliminary data suggest a potential role of Piezo1 in F-actin remodeling in process formation under IL1α-mediated inflammatory conditions. Here, we propose a paradigm-shifting concept of Piezo1-mediated morphological changes and pro- cess formation in inflammatory conditions. Both mechanotransduction and inflammation influence or are re- sponsible for a wide array of physiological processes and abnormalities. A knowledge gap exists in the mecha- notransduction-inflammation coupling, morphology-dependent cellular mechanosensitivity, Piezo1-mediated morphological changes, Piezo1 localization on the membrane cortex of round-shape ACs, and Piezo1-recruit- ment in processes of fibroblast-like shaped ACs. This proposed program will identify: (i) if activated Piezo1 al- ters local membrane curvature and triggers to form cellular protrusions and reorganize cytoskeleton meshwork, (ii) specific inflammatory cytokines triggering morphological changes and augmenting Piezo1 channels in pro- cesses, which in turn altering the gating properties of Piezo1, and (iii) if Piezo1-mediated inflammatory re- sponse increases mechanical fatigue and death. The whole-cell patch-clamp method allows to record ion channel activities, yet ACs present technical challenges on sealing the membrane primarily due to their round morphology and small size (~10 μm in diameter). Thus, we have custom-built a novel microscopy, named ‘Mechano-microscopy’, by combining an atomic force microscope (AFM), a high-speed ratiometric Ca2+ im- aging microscope, and a confocal microscope to simultaneously record cellular morphology and biochemical signals in response to mechanical cues under inflammatory conditions. In addition, we will utilize the super- resolution STED microscope and Piezo1-TdTomato reporter mice to interrogate dynamics of membrane curva- ture and Piezo1 localization in ACs after treating specific inflammatory cytokines. Our program will conceptually unveil fundamental processes of Piezo1-dependent inflammatory re- sponses leading to cellular morphological differentiation. In addition, our results will provide new insights into the mechanotransduction-inflammation coupling mechanisms and lead to new therapeutic strategies to prevent morphology changes at the early stages of cartilage disorders.