Both Clathrin-Mediated and Membrane Microdomain-Associated Endocytosis Contribute to the Cellular Adaptation to Hyperosmotic Stress in Arabidopsis.

Zheng Wu,Yi Man,Yue Zhang,Xiaojuan Li,Chengyu Fan,Ruili Li,Yanping Jing

doi:10.3390/ijms222212534

Abstract

As sessile organisms, plants must directly deal with an often complex and adverse environment in which hyperosmotic stress is one of the most serious abiotic factors, challenging cellular physiology and integrity. The plasma membrane (PM) is the hydrophobic barrier between the inside and outside environments of cells and is considered a central compartment in cellular adaptation to diverse stress conditions through dynamic PM remodeling. Endocytosis is a powerful method for rapid remodeling of the PM. In animal cells, different endocytic pathways are activated in response to osmotic stress, while only a few reports are related to the endocytosis response pathway and involve a mechanism in plant cells upon hyperosmotic stress. In this study, using different endocytosis inhibitors, the microdomain-specific dye di-4-ANEPPDHQ, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), and confocal microscopy, we discovered that internalized Clathrin Light Chain-Green Fluorescent Protein (CLC-GFP) increased under hyperosmotic conditions, accompanied by decreased fluorescence intensity of CLC-GFP at the PM. CLC-GFP tended to have higher diffusion coefficients and a fraction of CLC-GFP molecules underwent slower diffusion upon hyperosmotic stress. Meanwhile, an increased motion range of CLC-GFP was found under hyperosmotic treatment compared with the control. In addition, the order of the PM decreased, but the order of the endosome increased when cells were in hyperosmotic conditions. Hence, our results demonstrated that clathrin-mediated endocytosis and membrane microdomain-associated endocytosis both participate in the adaptation to hyperosmotic stress. These findings will help to further understand the role and the regulatory mechanism involved in plant endocytosis in helping plants adapt to osmotic stress.

Highlights

Cells must deal with stochastic and challenging changes in their environment
Using an artificially constructed Clathrin Light Chain-Green Fluorescent Protein (CLC-GFP) fusion protein, which was previously used and proved to be functional [26,27], and the endocytic tracer dye FM4-64, we found that internalized clathrin light chain (CLC)-GFP colocalized with FM4-64 signals and increased under hyperosmotic stress (Figure 1a)
Since hyperosmotic stress directly causes cell dehydration and plasma-wall separation, the restoration of cell turgor pressure and plasma membrane remodeling are essential for maintaining plant cell life, and endocytosis is a key factor for this process [4]

Summary

Introduction

Cells must deal with stochastic and challenging changes in their environment. A prominent example is alterations in osmolarity. Deviations from homeostatic conditions are often unfavorable to cellular life and evoke stress responses, enabling cell adaptation and survival under adverse environments [1,2,3]. The plasma membrane (PM) is considered a central compartment in cellular adaptation to diverse stress conditions since it is the barrier between the inside and outside environments of cells [4]. Dynamic PM remodeling is a crucial process in cellular adaptation to various stress conditions. Endocytosis provides a major route of entry for membrane proteins, lipids, and extracellular molecules into the cell. Clathrin-mediated endocytosis (CME) is the major endocytic pathway in plants. In addition to CME, plant cells have additional endocytic pathways in the clathrin-independent route. Flot1-assisted membrane microdomainassociated endocytosis discovered in Arabidopsis thaliana has been found to be involved in the regulation of signal transduction via endocytosis [10,11,12,13]

Methods

Results

Conclusion