Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

Summary

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

Abstract

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Introduction

Peripherally-associated plasma-membrane proteins are the key components of cell signaling pathways. One of their fundamental roles is their transient plasma membrane association and dissociation, which is important for the signal transduction between plasma membrane and cytoplasm. Peripherally-associated plasma membrane proteins can be attached on plasma membrane by lipid anchors (N-myristoylation, S-acylation, or prenylation) or by lipid binding domains (interacting with phosphatidylinositol phosphates, phosphatidic acid, etc.).

Plasma-membrane binding properties of these proteins can be examined in vivo, e.g., w....

Protocol

1. Preparation of Biological Material

1. Prepare biological material expressing the fluorescently tagged protein of interest, as well as a cytoplasmic marker. Follow the procedures mentioned in the Introduction.

2. Confocal Laser Scanning Microscopy

1. Stain the material prepared in section 1 with FM 4-64 dye^7⁠.
   1. Apply a staining protocol that is appropriate for the studied material. For tobacco BY-2 cells, stain 200 μL of cell suspension with 0.2 μL of 10 mM FM 4-64 solution in dimethylsulfoxide.
      Note: The typical staining concentration of FM 4-6....

Results

DREPP¹⁰ is a plant-specific peripherally-associated plasma-membrane protein that is associated with the plasma membrane via an N-myristoylation and an electrostatic interaction with phosphatidylinositol phosphates^11,12. DREPP was described as a component of calcium-signaling machinery in the plant cell and also interacts with the cytoskeleton^13,14. .......

Discussion

The method described here generates a more accurate estimation of plasma membrane partitioning for peripherally associated proteins compared to other approaches based on measuring fluorescence intensities⁵. The major improvement of this method is that it takes into account the light diffraction and superposition of the plasma-membrane and the cytoplasmic signals. Although these method results are in correlation with results of a simple method based on the comparison of fluorescence intensity at th.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FM 4-64	ThermoFisher Scientific	T13320	Plasma membrane dye
Dimethyl sulfoxide	Sigma-Aldrich	D4540 Sigma	Dye solvent
Ordinary equipment (microscopic slides, pipettes, tips, tubes)			Equipment for cell labelling and microscopy
Confocal laser scanning microscope
Ordinary computer