Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907, USA.
Int J Mol Sci. 2021 Dec 28;23(1):317. doi: 10.3390/ijms23010317.
In plants, secretion of cell wall components and membrane proteins plays a fundamental role in growth and development as well as survival in diverse environments. Exocytosis, as the last step of the secretory trafficking pathway, is a highly ordered and precisely controlled process involving tethering, docking, and fusion of vesicles at the plasma membrane (PM) for cargo delivery. Although the exocytic process and machinery are well characterized in yeast and animal models, the molecular players and specific molecular events that underpin late stages of exocytosis in plant cells remain largely unknown. Here, by using the delivery of functional, fluorescent-tagged cellulose synthase (CESA) complexes (CSCs) to the PM as a model system for secretion, as well as single-particle tracking in living cells, we describe a quantitative approach for measuring the frequency of vesicle tethering events. Genetic and pharmacological inhibition of cytoskeletal function, reveal that the initial vesicle tethering step of exocytosis is dependent on actin and myosin XI. In contrast, treatments with the microtubule inhibitor, oryzalin, did not significantly affect vesicle tethering or fusion during CSC exocytosis but caused a minor increase in transient or aborted tethering events. With data from this new quantitative approach and improved spatiotemporal resolution of single particle events during secretion, we generate a revised model for the role of the cortical cytoskeleton in CSC trafficking.
在植物中，细胞壁成分和膜蛋白的分泌对于生长、发育以及在各种环境中的生存都起着至关重要的作用。胞吐作用是分泌途径的最后一步，是一个高度有序且受到精确控制的过程，包括囊泡在质膜（PM）上的锚定、对接和融合，以进行货物传递。尽管在酵母和动物模型中，胞吐作用过程和机制已经得到了很好的描述，但在植物细胞中，支持胞吐作用晚期的分子参与者和特定分子事件在很大程度上仍然未知。在这里，我们使用功能性荧光标记的纤维素合酶（CESA）复合物（CSCs）向 PM 的递送来作为分泌的模型系统，以及活细胞中的单颗粒跟踪，描述了一种用于测量囊泡锚定事件频率的定量方法。细胞骨架功能的遗传和药理学抑制表明，胞吐作用的初始囊泡锚定步骤依赖于肌动蛋白和肌球蛋白 XI。相比之下，用微管抑制剂吖啶橙处理不会显著影响 CSC 胞吐作用过程中的囊泡锚定或融合，但会导致短暂或中止的锚定事件略有增加。利用这种新的定量方法的数据以及分泌过程中单颗粒事件的时空分辨率的提高，我们生成了一个关于皮质细胞骨架在 CSC 运输中的作用的修订模型。
