Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

Published: May 12, 2023

doi:

¹Department of Biology,Rutgers University, ²Pharmacology and Toxicology Department,University of Arkansas for Medical Sciences

Summary

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

Abstract

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Introduction

Membrane traffic is a conserved cellular process that distributes membrane components (also known as cargoes), including proteins, lipids, and other biological products, between different organelles within a eukaryotic cell or across the plasma membrane to and from the extracellular space¹. This process is facilitated by a collection of membranes and organelles named the endomembrane system, which consists of the nuclear membrane, the endoplasmic reticulum, the Golgi apparatus, the vacuole/lysosomes, the plasma membrane, and multiple endosomes¹. The endomembrane system enables the modification, packaging, and transport of membrane components using dynamic vesicles that shuttle between these organelles. Membrane trafficking events are crucial to cell development, growth, and environmental adaptation and, thus, are under stringent and complex regulation². Currently, multiple approaches in molecular biology, chemical biology, microscopy, and mass spectrometry have been developed and applied to the field of membrane trafficking and have greatly advanced the understanding of the spatiotemporal regulation of the endomembrane system³^,⁴. Molecular biology is used for classical genetic manipulations of the putative players involved in membrane trafficking, such as altering the gene expression of the protein of interest or labeling the protein of interest with certain tags. Tools in chemical biology include the usage of molecules that specifically interfere with the traffic of certain routes⁴^,⁵. Mass spectrometry is powerful for identifying the components in an organelle that has been mechanically isolated by biochemical approaches³^,⁴. However, membrane traffic is a dynamic, diverse, and complex biological process¹. To visualize the membrane trafficking process in live cells under various conditions, light microscopy is an essential tool. Continuous progress has been made in advanced microscope techniques to overcome the challenges in measuring the efficiency, kinetics, and diversity of the events⁴. Here, this study focuses on the widely adopted methodologies in chemical/pharmacological biology, molecular biology, and microscopy to study membrane trafficking events in a naturally simplified and experimentally accessible system, the stomatal developmental process.

Stomata are micropores on plant aerial surfaces that open and close to facilitate gas exchange between the inner cells and the environment⁶^,⁷^,⁸. Hence, stomata are essential for photosynthesis and transpiration, two events that are crucial for plant survival and growth. Stomatal development is dynamically adjusted by environmental cues to optimize the plant's adaptation to the surroundings⁹. Dating back to studies in 2002, the identification of the receptor protein Too Many Mouths (TMM) opened the door to a new era of investigating the molecular mechanisms of stomatal development in the model plant Arabidopsis thaliana¹⁰. After just a few decades, a classical signaling pathway has been identified. From upstream to downstream, this pathway includes a group of secretory peptide ligands in the epidermal patterning factors (EFP) family, several cell-surface leucine-rich-repeat (LRR) receptor kinases in the EREECTA (ER) family, the LRR receptor protein TMM, a MAPK cascade, and several bHLH transcription factors including SPEECHLESS (SPCH), MUTE, FAMA, and SCREAM (SCRM)¹¹^,¹²^,¹³^,¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸^,¹⁹^,²⁰^,²¹^,²²^,²³^,²⁴^,²⁵^,²⁶. Previous work indicates that one of the receptor kinases, ER-LIKE 1 (ERL1), demonstrates active subcellular behaviors upon EPF perception²⁰. ERL2 also dynamically traffics between the plasma membrane and some intracellular organelles²⁷. Blocking the membrane trafficking steps causes abnormal stomatal patterning, resulting in stomatal clusters on the leaf surface²⁸. These results suggest that membrane traffic plays an essential role in stomatal development. This study describes a protocol to spatiotemporally investigate the ERL1 dynamics using protein-protein subcellular co-localization analysis combined with pharmacological treatment using some membrane trafficking inhibitors.

Protocol

1. Preparation of the solutions Prepare seed sterilization solution by mixing 15 mL of bleach with 35 mL of distilled water and 50 µL of Triton X-100. Prepare brefeldin A (BFA) solution by dissolving the BFA powder in ethanol to a final concentration of 10 mM (stock). Prepare wortmannin (Wm) solution by dissolving the Wm powder in DMSO to a final concentration of 10 mM (stock). 2. Sowing the seeds Aliquot 10-50 see…

Representative Results

A previous study indicated that ERL1 is an active receptor kinase that undergoes dynamic membrane trafficking events20. ERL1 is a transmembrane LRR-receptor kinase on the plasma membrane. Newly synthesized ERL1 in the endoplasmic reticulum is processed in the Golgi bodies and further transported to the plasma membrane. The ERL1 molecules on the plasma membrane can perceive EPF ligands using their extracellular LRR domain18. Upon activation by the inhibitory EPFs, including …

Discussion

The endomembrane system separates the cytoplasm of a eukaryotic cell into different compartments, which enables the specialized biological function of these organelles. To deliver cargo proteins and macromolecules to their final destination at the right time, numerous vesicles are guided to shuttle between these organelles. Highly regulated membrane trafficking events play fundamental roles in the viability, development, and growth of cells. The mechanism regulating this crucial and complicated process is still poorly un…

Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the National Science Foundation (IOS-2217757) (X.Q.) and the University of Arkansas for Medical Sciences (UAMS) Bronson Foundation Award (H.Z.).

Materials

10 mL syringes	VWR	BD309695	Vacuum samples
Brefeldin A (BFA)	Sigma	B7651	membrane trafficking drug
Confocal Microscope	Leica	Lecia SP8 TCS with LAS-X software package	Imaging
Dissecting Forceps	VWR	82027-402	Genetic cross
Fiji	NIH	https://imagej.net/Fiji	Image processing
Leica LAS AF software	Leica	http://www.leica-microsystems.com	Image processing
transgenic seeds of ERL1-YFP			Qi, X. et al. The manifold actions of signaling peptides on subcellular dynamics of a receptor specify stomatal cell fate. Elife. 9, doi:10.7554/eLife.58097, (2020).
transgenic seeds of RFP-Ara7			Ebine, K. et al. A membrane trafficking pathway regulated by the plant-specific RAB GTPase ARA6. Nat Cell Biol. 13 (7), 853-859, doi:10.1038/ncb2270, (2011).
Wortmannin (Wm)	Sigma	W1628	membrane trafficking drug

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He, Q., Zhang, H., Qi, X. Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells. J. Vis. Exp. (195), e65257, doi:10.3791/65257 (2023).