Name: determination of plasma membrane partitioning for peripherally-associated proteins
Uploaded: 2018-06-15T14:45:00
Description: Watch this Scientific Journal Video about Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins at JoVE.com

This project was supported by NPU I, LO1417 (Ministry of Education, Youth and Sports of the Czech Republic).

1. Preparation of Biological Material
<ol>
	<li>Prepare biological material expressing the fluorescently tagged protein of interest, as well as a cytoplasmic marker. Follow the procedures mentioned in the Introduction.</li>
</ol>
2. Confocal Laser Scanning Microscopy
<ol>
	<li>Stain the material prepared in section 1 with FM 4-64 dye7&#8288;.
	<ol>
		<li>Apply a staining protocol that is appropriate for the studied material. For tobacco BY-2 cells, stain 200 ...

<ol>
	<li>Barbosa, I. C. R., Shikata, H., Zourelidou, M., Heilmann, M., Heilmann, I., Schwechheimer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipid+composition+and+a+polybasic+motif+determine+D6+PROTEIN+KINASE+polar+association+with+the+plasma+membrane+and+tropic+responses.">Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.</a> Development. 143 (24), 4687-4700 (2016).</li><li>Kato, M., Aoyama, T., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ca2+-binding+protein+PCaP2+located+on+the+plasma+membrane+is+involved+in+root+hair+development+as+a+possible+signal+transducer.">The Ca2+-binding protein PCaP2 located on the plasma membrane is involved in root hair development as a possible signal transducer.</a> Plant J. 74 (4), 690-700 (2013).</li><li>Kubitscheck, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fluorescence Microscopy: From Principles to Biological Applications. , (2013).</li><li>Janecki, A. J., Janecki, M., Akhter, S., Donowitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+plasma+membrane+expression+of+a+fusion+protein+of+Na/H+exchanger+NHE3+and+green+fluorescence+protein+(GFP)+in+living+PS120+fibroblasts.">Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts.</a> J Histochem Cytochem. 48 (11), 1479-1492 (2000).</li><li>Peitzsch, R. M., McLaughlin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+acylated+peptides+and+fatty+acids+to+phospholipid+vesicles:+pertinence+to+myristoylated+proteins.">Binding of acylated peptides and fatty acids to phospholipid vesicles: pertinence to myristoylated proteins.</a> Biochemistry. 32 (39), 10436-10443 (1993).</li><li>Vosolsob&#283;, S., Petr&#225;&#353;ek, J., Schwarzerov&#225;, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+plasticity+of+plasma+membrane+interaction+in+DREPP+family+proteins.">Evolutionary plasticity of plasma membrane interaction in DREPP family proteins.</a> Biochim Biophys Acta - Biomembr. 1859 (5), 686-697 (2017).</li><li>Bolte, S., Talbot, C., Boutte, Y., Catrice, O., Read, N. D., Satiat-Jeunemaitre, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FM-dyes+as+experimental+probes+for+dissecting+vesicle+trafficking+in+living+plant+cells.">FM-dyes as experimental probes for dissecting vesicle trafficking in living plant cells.</a> J Microsc. 214 (2), 159-173 (2004).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>R Development Core Team. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> R: A language and environment for statistical computing. , (2008).</li><li>Logan, D. C., Domergue, O., Teyssendier de la Serve, B., Rossignol, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+family+of+plasma+membrane+polypeptides+differentially+regulated+during+plant+development.">A new family of plasma membrane polypeptides differentially regulated during plant development.</a> Biochem Mol Biol Int. 43 (5), 1051-1062 (1997).</li><li>Ide, Y., Nagasaki, N., Tomioka, R., Suito, M., Kamiya, T., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+properties+of+a+novel,+hydrophilic+cation-binding+protein+associated+with+the+plasma+membrane.">Molecular properties of a novel, hydrophilic cation-binding protein associated with the plasma membrane.</a> J Exp Bot. 58 (5), 1173-1183 (2007).</li><li>Nagasaki-Takeuchi, N., Miyano, M., Maeshima, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+plasma+membrane-associated+protein+of+Arabidopsis+thaliana+AtPCaP1+binds+copper+ions+and+changes+its+higher+order+structure.">A plasma membrane-associated protein of Arabidopsis thaliana AtPCaP1 binds copper ions and changes its higher order structure.</a> J Biochem. 144 (4), 487-497 (2008).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MDP25,+A+Novel+Calcium+Regulatory+Protein,+Mediates+Hypocotyl+Cell+Elongation+by+Destabilizing+Cortical+Microtubules+in+Arabidopsis.">MDP25, A Novel Calcium Regulatory Protein, Mediates Hypocotyl Cell Elongation by Destabilizing Cortical Microtubules in Arabidopsis.</a> Plant Cell. 23 (12), 4411-4427 (2011).</li><li>Qin, T., Liu, X., Li, J., Sun, J., Song, L., Mao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+microtubule-destabilizing+protein+25+functions+in+pollen+tube+growth+by+severing+actin+filaments.">Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube growth by severing actin filaments.</a> Plant Cell. 26 (1), 325-339 (2014).</li><li>Nagata, T., Nemoto, Y., Hasezawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+BY-2+Cell+Line+as+the+&amp;#34;HeLa&amp;#34;+Cell+in+the+Cell+Biology+of+Higher+Plants.">Tobacco BY-2 Cell Line as the &amp;#34;HeLa&amp;#34; Cell in the Cell Biology of Higher Plants.</a> Int Rev Cytol. 132, 1-30 (1992).</li><li>Hellens, R. P., Anne Edwards, E., Leyland, N. R., Bean, S., Mullineaux, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pGreen:+A+versatile+and+flexible+binary+Ti+vector+for+Agrobacterium-mediated+plant+transformation.">pGreen: A versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation.</a> Plant Mol Biol. 42 (6), 819-832 (2000).</li><li>Vermeer, J. E. M., Thole, J. M., Goedhart, J., Nielsen, E., Munnik, T., Gadella, T. W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+phosphatidylinositol+4-phosphate+dynamics+in+living+plant+cells.">Imaging phosphatidylinositol 4-phosphate dynamics in living plant cells.</a> Plant J. 57 (2), 356-372 (2009).</li><li>La&#328;kov&#225;, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Dynamics+of+Plant+Plasma+Membrane+Proteins+with+Fluorescence+Recovery+and+Raster+Image+Correlation+Spectroscopy.">Determination of Dynamics of Plant Plasma Membrane Proteins with Fluorescence Recovery and Raster Image Correlation Spectroscopy.</a> Microsc Microanal. 22 (2), 290-299 (2016).</li></ol>

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...

An erratum was issued for: <a href="https://www.jove.com/video/57837/determination-plasma-membrane-partitioning-for-peripherally">Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins</a>. The Affiliations section was updated.
One of the affiliations was updated from:
Department of Experimental Plant Biology, University of Science, Charles University
to:
Department of Experimental Plant Biology, Faculty of Science, Charles University

An erratum was issued for: <a href="https://www.jove.com/video/57837/determination-plasma-membrane-partitioning-for-peripherally">Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins</a>. The Affiliations section was updated.

Erratum: Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

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 cytoplasm1,2, 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.

<table border="0" cellpadding="0" cellspacing="0" style="width:944px;" width="944">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">FM 4-64</td>
			<td style="width:135px;">ThermoFisher Scientific</td>
			<td style="width:384px;">T13320</td>
			<td style="width:184px;">Plasma membrane dye</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Dimethyl sulfoxide</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:384px;">D4540 Sigma</td>
			<td style="width:184px;">Dye solvent</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Ordinary equipment (microscopic slides, pipettes, tips, tubes)</td>
			<td style="width:135px;"></td>
			<td></td>
			<td style="width:184px;">Equipment for cell labelling and microscopy</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Confocal laser scanning microscope</td>
			<td style="width:135px;"></td>
			<td style="width:384px;"></td>
			<td style="width:184px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Ordinary computer</td>
			<td style="width:135px;"></td>
			<td style="width:384px;"></td>
			<td style="width:184px;"></td>
		</tr>
	</tbody>
</table>

determination of plasma membrane partitioning for peripherally-associated proteins

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.

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. ...

Watch this Scientific Journal Video about Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins at JoVE.com

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

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.

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

This method can help answer key questions in cell biology, including unraveling cell signaling pathways. So the main advantage of this technique is that it actually doesn't require any sophisticated, microscopical approaches. It's also very fast and therefore usable for realtime observation of protein distribution in cells.The evaluation and quantification of membrane partitioning of peripherally associated protein is complicated due to light diffraction that causes a mixing of plasma membrane and cytoplasmic fluorescence under the microscope. Begin this procedure with preparation of biological material as described in the text protocol. Stain the prepared material with FM 4-64 dye by applying a staining protocol that is appropriate for the studied material.For tobacco BY-2 cells, stain 200 microliters of cell suspension with 0.2 microliters of 10 millimolar FM 4-64 solution in dimethyl sulfoxide. To capture one equatorial confocal section per one cell, set a sequential two-channel scanning for the chromophore used as the protein tag and for FM 4-64. Also set a higher optical resolution.An example setup is a 63 times oil immersion objective with a numerical aperture of 1.3 and an image size of 1, 024 by 1, 024 pixels. Install Fiji ImageJ Distribution and the required macro peripheral. In Fiji, select plugins, then macros, then install in the main menu and specify the path to the downloaded file with the ImageJ macro peripheral.Install the R Project software and required packages. Run the R Graphical User Interface, select packages, then install packages in the main menu, and specify the nearest repository as well as package ggplot2 for installation. Download the R Package peripheral.Install them by selecting packages then install packages from local files. Process the confocal images of the cytoplasmic marker. To do so, import the images to Fiji using plugins then bioformats, then bioformats importer from the Fiji menu with default settings.Run the import options macro for an analysis setup. Activate the macro by selecting the same item after clicking the menu tool peripheral protein menu. Define the appropriate value in the field gaussian blur radius pixel.This influences picture smoothing and noise reduction. A low value is sufficient and does not cause any image information loss. Now set a value in the field profile line width pixel.A thicker line causes higher smoothing of the profile curves. A default value of 10 pixels is recommended. Set an image title, parsing by the definition of sample delimiters and exported items.Click OK.Check the title parsing in the next dialog window and click OK or back to reset. Next, perform a linear selection across the plasma membrane region in the processed images and measure the fluorescence profiles. To do so, select the Fiji tool's straight line from the Fiji toolbar.Click the image and drag a line across the plasma membrane region. The line must start in the extracellular space and should be perpendicular to the plasma membrane. Choose regions with a thicker and more homogenous layer of cortical cytoplasm.The optimal length of the linear selection is five to 10 microns. Run the take profile macro by clicking the take profile tool in the Fiji toolbar. The automatic measurement of fluorescence profiles in both channels will be performed, and the data will be displayed in the results window.According to individual signal variability, take representative numbers of the profiles for each cell. Run the plot profile data macro for graphical visualization of the measured profiles. Set the parameter in the invoked macro dialog window.Keep the check box use filtering for input data unselected. Specify if the plot should be created from recent data in the result window from a single CSV file or from all CSV files in a specified directory by clicking the appropriate radio button. Specify which grouping factors will be used for the plotting by selecting the appropriate check boxes.Select the factor I if all profiles should be displayed in unique plots. Keep the check boxes unselected if all data should be plotted in a single plot. Click OK.Specify the path and file name for saving the results or for eventual data import in the next dialog window.Confirm analysis by clicking OK in the dialog windows, showing the performed R code. Run the profile filtering setup macro if some plotted profiles have excessive signals in the extracellular or the intracellular space. Follow by setting the parameters in the invoked macro dialog window.For each channel, set the appropriate intensity threshold in the file, remove measurements with an excessive extracellular or intracellular signal. Specify the region where the intensity threshold will be applied in the field at X coordinates lower or greater than and then click OK.Run the plot profile data macro again with the check box use filtering for data input selected. Now add the data from the previously saved file.Ensure that all aberrant profiles were successfully removed, otherwise, improve the filtering parameters. Run the create model macro to create a model of the plasma membrane and the cytoplasm fluorescence distribution based on the calibration data. After setting the parameters in the invoked macro dialog window as detailed in the text protocol, click OK.Specify the input file path and name in the next dialog windows and confirm the analysis by clicking OK in the dialog window showing the performed R code.Next, process the images of the cells expressing the protein of interest. Import the images and set up the analysis as before. The smoothing and line width should be identical.Proceed to measure the profiles. Check the accuracy of the profiles by plotting, and set the filtering if desired as described earlier. Run the calculate distribution macro for the calculation of a protein partitioning between the plasma membrane and in the cytoplasm.Specify input data and model sources and eventually set a data filtering. Keep the check box activated for remove results with residual variability greater than if a result filtering is desired. Specify the threshold of the maximum allowed residual variability that can be unexplained by the signal decomposition of the individual profile measurement.Select sample grouping factors, which may be used for creating a box plot. Click OK, specify the input or output paths, and confirm the analysis by clicking OK in the dialog window showing the performed R code. Shown here is an image of an FM 4-64 stained cell expressing a plant protein, which is associated with the plasma membrane via N-myristoylation and electrostatic interaction to phosphoinositides.These interactions were experimentally disturbed by wortmannin. The importance of N-myristoylation was tested by mutation in glycine on the second amino acid position which serves as N-myristoylation site. The effect of a removing of both plasma membrane binding abilities was tested by truncation of the N-terminal domain of the protein.The resulting box plot shows a decrease of a plasma membrane affinity after mutation of N-myristoylation site and after drug treatment. Their mutual effect is comparable to the effect of removing the N-terminal domain. After watching this video, you should have a good understanding of how to carry out the main steps of the analysis in a proper order and how to set their parameters.Following this analysis, statistical method like ANOVA can be performed for statistical evaluation of obtained data. Though this method was developed for plant suspension cells, it can also be applied to other cells or tissues with smooth and clearly distinguishable plasma membranes.

Research

JoVE Journal

Biology

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy (FCS)

Determination of Lipid Raft Partitioning of Fluorescently-tagged Probes in Living Cells by Fluorescence Correlation Spectroscopy FCS

In the past fifteen years the notion that cell membranes are not homogenous and rely on microdomains to exert their functions has become widely accepted. ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...