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., when a fluorescently-tagged protein is modified by a site-directed mutagenesis of key amino acids, or when it is treated with various inhibitors affecting lipid signaling. The distributions of peripheral plasma membrane proteins are mostly being evaluated qualitatively, especially in cases, when protein re-distribution is obvious. The presented method is optimal for situations when protein re-distribution is only partial and quantitative evaluation is necessary. A frequently used approach of when plasma membrane association is estimated from confocal laser scanning microscopy images as a ratio of fluorescence intensities at the plasma-membrane and in the cytoplasm^1,2, is simple, but not accurate. Fluorescence intensities at the plasma membrane reflect a superposition of the plasma-membrane and cytoplasm signal due to the light diffraction characteristic for the particular fluorescence microscopy technique and optical elements used³. Consequently, the cytoplasmic signal is included also in the membrane region. For this reason, FM 4-64 staining pattern cannot be used as a mask for a membrane signal selection⁴. Furthermore, simple measurements of membrane signal at the position defined by the FM 4-64 staining maximum always systematically overestimate the real plasma-membrane signal of peripherally-associated plasma-membrane protein due to the superposition of the membrane and cytoplasmic compound. The maximum of observed signals for fluorescently-tagged peripherally-associated proteins also does not co-localize with the maximum of the plasma membrane marker (i.e., FM 4-64 styryl dye), but is shifted towards the cytoplasm. Another limitation is based on the fact that the FM 4-64 emission peak is wider in comparison with the emission peaks for green fluorescent proteins such as GFP due to the wavelength-dependency of light diffraction³.

In the method described here, the tagged protein signal is fitted by two empirical functions describing a hypothetical distribution of the plasma membrane and cytoplasm signal, respectively. This signal decomposition is applied to linear fluorescence profiles that are applied to the cell surface perpendicularly to the plasma membrane in source images, which are regular, two-channel confocal sections of fluorescently-tagged protein expressing cells labeled with FM 4-64 dye.

The first function used for fitting describes a diffraction of a cytoplasm signal on the cell edge. It is obtained from previously acquired fluorescence profiles that were measured in cells expressing a cytoplasm protein marker tagged by the same chromophore as the plasma membrane peripherally-associated protein of interest. The second function describing a diffraction of a plasma-membrane signal is derived from the fluorescence of FM 4-64. This signal is firstly approximated by a Gaussian function that is being used for an approximate modeling of light diffraction of a point source. Secondly, this model, valid for red FM 4-64 emission, is mathematically transformed to the form that is relevant for an emission wavelength of the chromophore used for the tagging of peripherally-associated proteins of interest at the plasma membrane. Both functions are normalized by the maximal intensity and by the mean from 10% of the highest values for FM 4-64 signal and cytoplasmic protein signal, respectively. By this signal decomposition (non-linear least square fitting method), the ratio of the plasma membrane and the cytoplasm fraction of the examined protein can be estimated easily and accurately. The real physical dimension of computed partitioning coefficient is in the range of micrometer, because cytoplasmic volume concentration is compared with surface concentration on the plasma membrane. It defines the distance from the plasma membrane to the cytoplasm, within which the same amount of proteins is localized as in the adjacent area of the plasma membrane. This value is equivalent to the partitioning coefficient K₂ introduced previously⁵. The method is very quick, requiring only single confocal sections acquired using routine confocal laser scanning microscope, and it is not computationally demanding. The analysis core has been implemented in a portable R package and an additional ImageJ macro was written to provide graphical user interface to run the analysis from the intuitive dialogs. Software and more detailed description of the method (published previously⁶) can be found at http://kfrserver.natur.cuni.cz/lide/vosolsob/Peripheral/.

The method is suitable for isolated cells, protoplasts, and tissues, where the plasma membrane of individual cells is clearly distinguishable, expressing a fluorescently-tagged construct of examined peripherally-associated protein. A chromophore compatible with FM 4-64 staining must be used. FM 4-64 emits red fluorescence; therefore, examined protein can be tagged by a fluorescence protein with blue, green, or yellow emission (e.g., GFP, CFP, YFP). Stable transformation of biological material is recommended because it enables less artificial and more reproducible observations of protein distribution. It is necessary that the examined protein has a relatively homogeneous cytoplasmic distribution. The localization of a protein in the endoplasmic reticulum or another intracellular membrane compartment can produce artificial results.

Additionally, the same biological material expressing a cytoplasmic marker must be used for the comparison. Cells can be transformed by a free chomophore (the same as used for peripheral protein tagging, e.g., free GFP) or by tagged protein of interest with abolished membrane binding capacity. Membrane binding capacity can be abolished, for example, by trimming of the membrane-binding domain or by site-directed mutagenesis of key amino acid residua (e.g., sites for N-myristoylation, S-acylation, or prenylation, etc.).

For confocal scanning microscopy, cells must be labeled by a membrane marker like FM 4-64 dye. If FM 4-64 staining is not suitable for the studied material (due to interfering autofluorescence, poor dye penetration, etc.), the plasma membrane can be labeled, for example, by integral plasma membrane protein tagged to an appropriate chomophore (mCherry, RFP, etc.). It is essential that the marker has negligible localization in the intracellular membrane compartments (endomembranes).

If working with fixed samples and antibodies, fixable analogue FM 4-64FX or plasma membrane labeling by antibody against an appropriate target can be used. In this case, it is essential to evaluate results very carefully because fixation procedures can lead to selective loss of proteins from both the cytoplasm and the plasma membrane.

Protocol

1. Preparation of Biological Material 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 Stain the material prepared in section 1 with FM 4-64 dye7⁠. Apply a staining protocol that is appropriate for the studied material. For tobacco BY-2 cells, stain 200 …

Representative 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 phosphates11,12. DREPP was described as a component of calcium-signaling machinery in the plant cell and also interacts with the cytoskeleton13,14. …

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

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