Bartle Emily I, Urner Tara M, Raju Siddharth S, Mattheyses Alexa L
Department of Cell Biology, Emory University, Atlanta, Georgia.
Department of Cell Biology, Emory University, Atlanta, Georgia.
Biophys J. 2017 Dec 5;113(11):2519-2529. doi: 10.1016/j.bpj.2017.09.028.
Desmosomes are macromolecular cell-cell junctions that provide adhesive strength in epithelial tissue. Desmosome function is inseparably linked to structure, and it is hypothesized that the arrangement, or order, of desmosomal cadherins in the intercellular space is critical for adhesive strength. However, due to desmosome size, molecular complexity, and dynamics, the role that order plays in adhesion is challenging to study. Herein, we present an excitation resolved fluorescence polarization microscopy approach to measure the spatiotemporal dynamics of order and disorder of the desmosomal cadherin desmoglein 3 (Dsg3) in living cells. Simulations were used to establish order factor as a robust metric for quantifying the spatiotemporal dynamics of order and disorder. Order factor measurements in keratinocytes showed the Dsg3 extracellular domain is ordered at the individual desmosome, single cell, and cell population levels compared to a series of disordered controls. Desmosomal adhesion is Ca dependent, and reduction of extracellular Ca leads to a loss of adhesion measured by dispase fragmentation assay (λ = 15.1 min). Live cell imaging revealed Dsg3 order decreased more rapidly (λ = 5.5 min), indicating that cadherin order is not required for adhesion. Our results suggest that rapid disordering of cadherins can communicate a change in extracellular Ca concentration to the cell, leading to a downstream loss of adhesion. Fluorescence polarization is an effective bridge between protein structure and complex dynamics and the approach presented here is broadly applicable to studying order in macromolecular structures.
桥粒是大分子细胞间连接结构，可在上皮组织中提供黏附强度。桥粒功能与结构紧密相连，据推测，细胞间空间中桥粒钙黏蛋白的排列或顺序对黏附强度至关重要。然而，由于桥粒的大小、分子复杂性和动态性，顺序在黏附中所起的作用难以研究。在此，我们提出一种激发分辨荧光偏振显微镜方法，用于测量活细胞中桥粒钙黏蛋白桥粒芯糖蛋白3（Dsg3）有序和无序状态的时空动态变化。通过模拟建立了顺序因子，作为量化有序和无序时空动态变化的可靠指标。角质形成细胞中的顺序因子测量结果显示，与一系列无序对照相比，Dsg3细胞外结构域在单个桥粒、单细胞和细胞群体水平上均呈有序状态。桥粒黏附依赖于钙离子，通过分散酶裂解试验测量（λ = 15.1分钟），细胞外钙离子减少会导致黏附丧失。活细胞成像显示Dsg3的有序性下降得更快（λ = 5.5分钟），这表明钙黏蛋白的有序性并非黏附所必需。我们的结果表明，钙黏蛋白的快速无序化可将细胞外钙离子浓度的变化传递给细胞，从而导致下游黏附丧失。荧光偏振是蛋白质结构与复杂动态变化之间的有效桥梁，本文提出的方法广泛适用于研究大分子结构中的有序性。
