Single Event Resolution of Plant Plasma Membrane Protein Endocytosis by TIRF Microscopy.

Grégory Vert,Alexander Johnson

doi:10.3389/fpls.2017.00612

Abstract

Endocytosis is a key process in the internalization of extracellular materials and plasma membrane proteins, such as receptors and transporters, thereby controlling many aspects of cell signaling and cellular homeostasis. Endocytosis in plants has an essential role not only for basic cellular functions but also for growth and development, nutrient delivery, toxin avoidance, and pathogen defense. The precise mechanisms of endocytosis in plants remain quite elusive. The lack of direct visualization and examination of single events of endocytosis has greatly hampered our ability to precisely monitor the cell surface lifetime and the recruitment profile of proteins driving endocytosis or endocytosed cargos in plants. Here, we discuss the necessity to systematically implement total internal reflection fluorescence microcopy (TIRF) in the Plant Cell Biology community and present reliable protocols for high spatial and temporal imaging of endocytosis in plants using clathrin-mediated endocytosis as a test case, since it represents the major route for internalization of cell-surface proteins in plants. We developed a robust method to directly visualize cell surface proteins using TIRF microscopy combined to a high throughput, automated and unbiased analysis pipeline to determine the temporal recruitment profile of proteins to single sites of endocytosis, using the departure of clathrin as a physiological reference for scission. Using this ‘departure assay’, we assessed the recruitment of two different AP-2 subunits, alpha and mu, to the sites of endocytosis and found that AP2A1 was recruited in concert with clathrin, while AP2M was not. This validated approach therefore offers a powerful solution to better characterize the plant endocytic machinery and the dynamics of one’s favorite cargo protein.

Highlights

Endocytosis is the process of transporting cell surface or extracellular materials, including proteins, lipids and nutrients, into the cell via invaginations of small vesicles that pinch off from the plasma membrane
Due to the nature of the pinholes, a lot of photons are discarded and the z-resolution is still limited to ∼500 nm. This makes spinning disk microscopy unsuitable for tracking weak signals and focusing with great precision on cell surface endocytosis accessory proteins (EAPs) aggregating at sites of clathrin-mediated endocytosis (CME) (Mettlen and Danuser, 2014)
Taking together that total internal reflection fluorescence microcopy (TIRF) is possible in plants and that plant images are suitable for automated particle detection programs, we developed a reliable method for directly studying single events of CME in plants with high temporal and spatial resolution

Summary

INTRODUCTION

Endocytosis is the process of transporting cell surface or extracellular materials, including proteins, lipids and nutrients, into the cell via invaginations of small vesicles that pinch off from the plasma membrane. CME is best understood in mammalian and yeast systems (McMahon and Boucrot, 2011; Lu et al, 2016) where over 60 conserved key EAPs have been characterized and for which details of the physiological role and precise temporal dynamics are defined (McMahon and Boucrot, 2011; Merrifield and Kaksonen, 2014; Lu et al, 2016). Due to the nature of the pinholes, a lot of photons are discarded and the z-resolution is still limited to ∼500 nm This makes spinning disk microscopy unsuitable for tracking weak signals and focusing with great precision on cell surface EAPs aggregating at sites of CME (Mettlen and Danuser, 2014). This provides the plant endocytosis community with powerful imaging pipelines to greatly increase our understanding of plant CME, bridging the gap in knowledge between other model systems

MATERIALS AND METHODS

RESULTS AND DISCUSSION

CONCLUSION