Saeid Ghavami was supported by a Health Science Centre Operating Grant, CHRIM operating grant and Research Manitoba New Investigator Operating Grant. Javad Alizadeh was supported by Research Manitoba studentship. Shahla Shojaei was supported by a Health Science Foundation Operating grant and the MITACS Accelerate postdoctoral fellowship. Adel Rezaei Moghadam was supported by an NSERC operating grant which was held by Joseph W. Gordon. Amir A. Zeki was supported by the NIH/NHLBI K08 award (1K08HL114882-01A1). Marek J. Los kindly acknowledges the support from LE STUDIUM Institute for Advanced Studies (region Centre-Val de Loire, France) through its Smart Loire Valley General Program and co-funded by the Marie Sklodowska-Curie Actions, grant #665790. Simone da Silva Rosa was supported by UMGF studentship.

1. Determination of RhoA Localization Using Membrane/Cytosol Fractionation
<ol>
	<li>Cell culture and simvastatin treatment
	<ol>
		<li>Seed 50,000 of U251 cells in a 100 mm dish and culture them in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) (high glucose, 10% fetal bovine serum [FBS]).</li>
		<li>When 30% confluent, treat the cells by removing the medium and adding simvastatin-containing medium to it (10 &#181;M of simvastatin dissolved in dimethyl sulfoxide [DMSO]), and incubate for 36 h at 37...

<ol>
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A., Golub, T. R., Lander, E. S., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+analysis+of+metastasis+reveals+an+essential+role+for+RhoC.">Genomic analysis of metastasis reveals an essential role for RhoC.</a> Nature. 406 (6795), 532-535 (2000).</li><li>Ghavami, S., 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=Statin-triggered+cell+death+in+primary+human+lung+mesenchymal+cells+involves+p53-PUMA+and+release+of+Smac+and+Omi+but+not+cytochrome+c.">Statin-triggered cell death in primary human lung mesenchymal cells involves p53-PUMA and release of Smac and Omi but not cytochrome c.</a> Biochimica et Biophysica Acta. 1803 (4), 452-467 (2010).</li><li>Cordle, A., Koenigsknecht-Talboo, J., Wilkinson, B., Limpert, A., Landreth, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+statin-mediated+inhibition+of+small+G-protein+function.">Mechanisms of statin-mediated inhibition of small G-protein function.</a> Journal of Biological Chemistry. 280 (40), 34202-34209 (2005).</li><li>Waiczies, S., Bendix, I., Zipp, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geranylgeranylation+but+not+GTP-loading+of+Rho+GTPases+determines+T+cell+function.">Geranylgeranylation but not GTP-loading of Rho GTPases determines T cell function.</a> Science Signaling. 1 (12), pt3 (2008).</li><li>Waiczies, S., 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=Geranylgeranylation+but+not+GTP+loading+determines+rho+migratory+function+in+T+cells.">Geranylgeranylation but not GTP loading determines rho migratory function in T cells.</a> Journal of Immunology. 179 (9), 6024-6032 (2007).</li><li>Satori, C. 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Here we describe an accurate method to measure small GTPase prenylation and GTP binding shown as small GTPase subcellular localization (membrane versus cytosol) and Rho GTP loading. Small GTPases are expressed in eukaryotic cells and play essential roles in cellular proliferation, motility, and structure. Both prenylation and GTP binding are involved in the regulation of GTPase activity; therefore, assays to evaluate the prenylation and GTP binding of these proteins are important tools for cell biologists<sup cl...

Rho GTPases are a group of small proteins (21 - 25 kDa), which are well conserved throughout evolution, and form a unique subfamily in the Ras superfamily of small GTPases. In each subfamily within this superfamily, there is a shared G domain core that is involved in the GTPase activity and nucleotide exchange1. The difference between the Rho family and the other Ras subfamilies is the presence of a &#34;Rho insert domain&#34; within the 5th &#946; strand and the 4th &#945; helix in the small GTPase domain2.
Based on the recent classification, Rho GTPases are considered a family of signaling proteins that fit into the Ras GTPase superfamily3. Mammalian Rho GTPases have 22 members based on their specific function and general characterization4 in which RhoA, Rac1, and Cdc42 are among the most-studied members in this group. Rho GTPases are linked to intracellular signaling pathways via a tightly regulated mechanism which is dependent on molecular switches via protein posttranslational modifications5.
GTP loading and hydrolysis are essential mechanisms in the activation/deactivation cycle of small Rho GTPases and are regulated via GTPase-activating proteins (GAPs). GAPs are responsible for the GTP hydrolysis and work in concert with guanine nucleotide exchange factors (GEFs) which are responsible for the GTP-loading reaction. Rho GDP dissociation inhibitors (GDIs) provide further regulation of small Rho GTPases via binding to the GDP-bound Rho GTPases. This inhibits GDP dissociation and facilitates sequestering of small Rho GTPases away from the active intracellular membrane sites. There is also further regulation of Rho GTPase proteins involving the prenylation of GDIs which regulates both nucleotide hydrolysis and exchange and controls GDP/GTP cycling1,6,7,8.
Both GTP-loading and Rho GTPase prenylation are involved in the movement of Rho GTPase between cytosol and cell membranes by changing the lipophilic properties of these proteins1,9. The abovementioned regulators interact with phospholipids of the cell membrane and other modulating proteins of the GDP/GTP exchange activity10. Moreover, GDIs, dissociation inhibitors, block both the GTP hydrolysis and the GDP/GTP exchange. GDIs inhibit the dissociation of the inactive Rho proteins from GDP and, therefore, their interaction with downstream effectors. GDIs also regulate the cycling of GTPases between the cytosol and membrane in the cell. The activity of Rho GTPases depends to a great extent on their movement to the cell membrane; thus, GDIs are regarded as critical regulators that can sequester GTPases in the cytoplasm through hiding their hydrophobic region/domains11,12.
For Rho GTPase to have an optimum signaling and function in all stages of its activation cycle, the dynamic cycle of GTP-loading/GTP hydrolysis is crucial. Any kind of alterations in this process may result in subsequent changes in cell functions regulated by Rho GTPase, such as cell polarity, proliferation, morphogenesis, cytokinesis, migration, adhesion, and survival13,14.
The current protocol provides readers with a detailed method to monitor small RhoA GTPase activation via the investigation of their prenylation and GDP/GTP loading. This method can also be used to detect the prenylation and GTP binding of a wide range of small GTPases. The GTPase-linked immunosorbent assay can be used to measure the level of activation of other kinds of GTPases, such as Rac1, Rac2, Rac3,H-, K-, or N-Ras, Arf, and Rho15. The pharmacological agent simvastatin is used as an example, as it was recently reported to be involved in the regulation of small Rho GTPase prenylation and activity8,9,14,16.

<table border="0" cellpadding="0" cellspacing="0" style="width:1055px;" width="1054">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">DMEM high Glucose</td>
			<td style="width:228px;">VWR (Canada)</td>
			<td style="width:123px;">VWRL0101-0500</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:228px;">VWR (Canada)</td>
			<td style="width:123px;">CA45001-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td style="width:228px;">VWR (Canada)</td>
			<td style="width:123px;">97062-806</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA (Ethylenediamine tetraacetic acid)</td>
			<td style="width:228px;">VWR (Canada)</td>
			<td style="width:123px;">CA71007-118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EGTA (Ethylene glycol bis(2-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid)</td>
			<td style="width:228px;">VWR (Canada)</td>
			<td style="width:123px;">CAAAJ60767-AE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DTT (DL-Dithiothreitol)</td>
			<td style="width:228px;">VWR (Canada)</td>
			<td style="width:123px;">CA97061-340</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ammonium Persulfate</td>
			<td style="width:228px;">VWR (Canada)</td>
			<td style="width:123px;">CABDH9214-500G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-Hydroxymethylaminomethane</td>
			<td style="width:228px;">VWR (Canada)</td>
			<td style="width:123px;">CA71009-186</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30% Acrylamide/Bis Solution</td>
			<td style="width:228px;">Biorad (Canada)</td>
			<td style="width:123px;">1610158</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TEMED</td>
			<td style="width:228px;">Biorad (Canada)</td>
			<td style="width:123px;">1610801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor cocktail</td>
			<td style="width:228px;">Sigma/Aldrich (Canada)</td>
			<td style="width:123px;">P8340-5ML</td>
			<td>1:75 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rho-GTPase Antibody Sampler Kit</td>
			<td style="width:228px;">Cell Signaling (Canada)</td>
			<td style="width:123px;">9968</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pan-Cadherin antibody</td>
			<td style="width:228px;">Cell Signaling (Canada)</td>
			<td style="width:123px;">4068</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH antibody</td>
			<td style="width:228px;">Santa Cruz Biotechnology (USA)</td>
			<td style="width:123px;">sc-69778</td>
			<td>1:3000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RhoA G-LISA Activation Assay (Luminescence format)</td>
			<td style="width:228px;">Cytoskeleton Inc. (USA)</td>
			<td style="width:123px;">BK121</td>
			<td>Cytoskeleton I. G-LISA Activation Assays Technical Guide. 2016.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">RhoA Antibody</td>
			<td style="width:228px;">Cell Signaling</td>
			<td style="width:123px;">2117</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">ECL</td>
			<td style="width:228px;">Amersham-Pharmacia Biotech</td>
			<td style="width:123px;">RPN2209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">Anti-Rabbit IgG (whole molecule) Peroxidase antibody</td>
			<td style="width:228px;">Sigma</td>
			<td style="width:123px;">A6154-1ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">SpectraMax iD5 Multi-Mode Microplate Reader</td>
			<td style="width:228px;">Molecular Devices&#160;</td>
			<td style="width:123px;">1612071A</td>
			<td style="width:172px;">Spectrophotometer</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:62px;width:532px;">Nonidet P-40</td>
			<td style="width:228px;">Sigma</td>
			<td style="width:123px;">11332473001</td>
			<td style="width:172px;">non-denaturing detergent, octylphenoxypolyethoxyethanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">DMSO</td>
			<td style="width:228px;">Sigma</td>
			<td style="width:123px;">D8418-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">PBS</td>
			<td style="width:228px;">Sigma</td>
			<td style="width:123px;">P5493-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:532px;">Phophatase Inhibitor cocktail</td>
			<td style="width:228px;">Sigma</td>
			<td style="width:123px;">P5726-5ML</td>
			<td>1:75 Dilution</td>
		</tr>
	</tbody>
</table>

detection of small gtpase prenylation and gtp binding using membrane fractionation and  gtpase-linked immunosorbent assay

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of diverse cellular functions, including cytoskeletal dynamics, cell motility, cell polarity, axonal guidance, vesicular trafficking, and cell cycle control. Changes in Rho GTPase signaling play an essential regulatory role in many pathological conditions, such as&#160;cancer, central nervous system diseases, and immune system-dependent diseases. The posttranslational modification of Rho GTPases (i.e., prenylation by mevalonate pathway intermediates) and GTP binding are key factors which affect the activation of this protein. In this paper, two essential and simple methods are provided to detect a broad range of Rho GTPase prenylation and GTP binding activities. Details of the technical procedures that have been used are explained step by step in this manuscript.

Membrane Fractionation:
Ultracentrifugation was used for the fractionation of membrane and cytosol components. As shown in Figure 1,&#160;the supernatant contains the cytosolic fraction and the pellet contains the membrane fraction. The abundance of RhoA in cytosolic andmembrane fractions obtained from U251 cells was examined after the treatment with simvastatin using immunoblotting...

Watch this Scientific Journal Video about Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay at JoVE.com

Detection of Small GTPase Prenylation and GTP Binding Using Membrane Fractionation and  GTPase-linked Immunosorbent Assay

Here we describe a protocol to investigate the prenylation and guanosine-5'-triphosphate (GTP)-loading of Rho GTPase. This protocol consists of two detailed methods, namely membrane fractionation and a GTPase-linked immunosorbent assay. The protocol can be used for measuring the prenylation and GTP loading of different other small GTPases.

The Rho GTPase family belongs to the Ras superfamily and includes approximately 20 members in humans. Rho GTPases are important in the regulation of ...

The small Rho GTPase is a protein of a superfamily with diverse cellular function. For example, these proteins are involved in the regulation of cell fate, cellular response to a stress, cellular motility, and cell cycle. The activity of a small Rho GTPase protein are regulated by post-translational modification.These modifications are involved in tight regulation of their activity. As an example, protein prenylation and GTP bond are the most important post-translation modification of these proteins. My lab has recently modified and developed simple method to investigate the post-translational modification of these superfamily proteins.For this purpose, we have used 251 glioblastoma cell lines, and targeting them with simvastatin, and measured post-translational modification of these proteins. The method involves sub-cellular fractionation and GTP bound protein to this protein superfamily. For this purpose, we have used localization of RhoA, and GTP bound to RhoA to measure the post-translational modification of this protein.Remove cells from the 37 degree incubator. Look at the cells under the microscope to confirm confluency. The cells should be at 70 to 80 percent confluent.Wash the cells once with cold PBS. Add five milliliters of EDTA, and place the cells back into the 37 degree incubator for five minutes. After five minutes of incubation, collect EDTA with cells into a 15 mil tube.Place the tube in an ice box, and proceed to the centrifuge. Set up the centrifuge to 1500g at four degrees, and spin the cells for five minutes. Following the spin, remove the supernate, and add one milliliter of cold PBS.Triturate the cells well. Transfer the cell mixture to a new labeled 1.5 milliliter tube. Following transfer, proceed to the centrifuge.Set the centrifuge to 1500g at four degrees, and spin the cells for five minutes. Check the pellet size. Place the samples on ice.Discard the supernate completely, without disturbing the pellet. Add Buffer One. Mix well by pipetting up and down.Proceed to sonication. Set the sonicator for five cycles, five seconds each, and repeat three times. Sonication should proceed on ice, but is not shown in the video, for illustrative purposes.Proceed to the ultracentrifuge. Use an ultracentrifuge to separate the cell homogenates into cytoplasmic and membrane fractions. Set the centrifuge to 100, 000g for 35 minutes, at four degrees.Check the pellet size. Separate the supernate. Collect the supernate completely, being careful to not disturb the pellet.The supernate is the cytosolic fraction. Place the supernate in a newly labeled tube. Add Buffer Two to the pellet.This is the membrane fraction. Mix well by pipetting up and down. Proceed to protein determination and Western blot sample preparation.Load the samples on to an SDS-PAGE using a 15%gel. Confirm protein transfer by visualizing the molecular weight marker, or using a Ponceau stain. Add primary antibodies for immunoblotting analysis.We use Rac1/2/3, Cdc42, and RhoA. Bring the culture plates out of the incubator. Look at the cells under the microscope to confirm confluency.The cells should be 70 to 80%confluent. Place the culture plate on ice. Aspirate the media and wash the cells with ice cold PBS pH'd to 7.2.Aspirate the PBS. Tilt the plates on ice for an additional minute to remove all remnants of PBS. Residual PBS adversely affects this assay.Lyse the cells in an appropriate volume of ice cold lysis buffer containing protease and phosphatase inhibitors. Harvest cell lysate with the cell scraper. Incline the culture plate for this technique.Transfer the lysate into a labeled, ice-cold cryo tube, and keep on ice. Mix thoroughly using a vortex. Keep 10 microliters of the lysate for a protein assay.Snap freeze the remaining cell lysate in liquid nitrogen. Transfer the snap frozen cryotubes to a minus 80 freezer. Store these samples.Note, samples should not be stored longer than 14 days. Prepare cell lysates from the other culture plates by repeating steps one to eight, one by one. Work quickly, and never leave the samples on ice for longer than 10 minutes.Never handle all culture plates simultaneously. Measure the protein concentration. Calculate the volume of lysis buffer to normalize the concentration of protein between the samples.Note:one milligram per milliliter is usually the best, however, 0.3 to two milligrams per milliliter can be detectable. Prepare a blank control by adding 60 microliters of lysis buffer, and 60 microliters of binding buffer to a microtube. Prepare a positive control by adding 12 microliters of Rho control protein, 48 microliters of lysis buffer, and 60 microliters of binding buffer.Take the Rho affinity plate out of its bag and place on ice. Dissolve the powder in the wells with 100 microliters of ice cold distilled water. Keep the plate on ice.Thaw the snap frozen cell lysates in a water bath set to 25 degrees Celsius. Add the calculated volume of ice cold lysis buffer to each sample to normalize the protein concentration. Transfer 60 microliters of normalized ice cold samples to microtubes, and add 60 microliters of binding buffer.Mix thoroughly, and keep on ice. Completely remove the water from the microplate by vigorous flicking, followed by five to seven hard taps on a lab mat. Add 50 microliters of the normalized samples, a blank control, and a positive control to the wells in duplicate.Place the plate on an orbital shaker for 30 minutes at four degrees Celsius. Note:the speed of the shaker should be set to 300 rpm. Clear the samples from the plate by flicking, and wash twice with 200 microliters of room temperature washing buffer.Vigorously remove the washing buffer from the wells after each wash by flicking, followed by tapping, and keep the plate on the bench at room temperature. Add 200 microliters of room temperature antigen-presenting buffer into each well, and incubate at room temperature for two minutes. Flick out the solution from the wells, and wash three times with 200 microliters of room temperature washing buffer.Add 50 microliters of freshly prepared one to 250 anti-RhoA primary antibody to each well. Place the plate on an orbital shaker for 45 minutes at 300 rpm, set to 25 degrees Celsius. Flick out the solution from the wells, and wash three times with 200 microliters of washing buffer at room temperature.Add 50 microliters of freshly prepared one to 250 anti-RhoA secondary antibody to each well. Place the plate on top of an orbital shaker for 45 minutes, at 300 rpm, set to 25 degrees Celsius. Prepare the HRP detection reagent by mixing equal volumes of Reagent A and Reagent B.Flick out the solution from each well, and wash three times with 200 microliters of room temperature washing buffer.Add 50 microliters of freshly prepared HRP detection reagent to each well. Read the luminescence signal within three to five minutes to obtain a maximal signal. Analyze the results using an appropriate software package.Our results show that simvastatin decreased the localization of RhoA in the membrane fraction. What increased is accumulation in the cytosolic fraction. Interestingly, simvastatin increased GTP bound to the RhoA.Why?We expect simvastatin decrease this activity. It is very important they measure both sub-cellular localization of this protein, and measure GTP binding to this protein to have a final evaluation of the activity of the small Rho GTPases.

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Cell Biology

Biochemistry

Unit 1

Signaling Networks of Kinase Receptors

SCIENTISTS IN ACTION

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Quantification of Bacterial Histidine Kinase Autophosphorylation Using a Nitrocellulose Binding Assay

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.

We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.

We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their ...

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.

Brain Membrane Fractionation: An Ex Vivo Approach to Assess Subsynaptic Protein Localization

Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments.

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate ...

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of the kinase activity of LRRK2 has been implicated in the pathogenesis of PD, it is essential to establish a method to evaluate the physiological levels of the kinase activity of LRRK2. Recent studies revealed that LRRK2 phosphorylates members of the Rab GTPase family, including Rab10, under physiological conditions. Although the phosphorylation of endogenous Rab10 by LRRK2 in cultured cells could be detected by mass spectrometry, it has been difficult to detect it by immunoblotting due to the poor sensitivity of currently available phosphorylation-specific antibodies for Rab10. Here, we describe a simple method of detecting the endogenous levels of Rab10 phosphorylation by LRRK2 based on immunoblotting utilizing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) combined with a phosphate-binding tag (P-tag), which is N-(5-(2-aminoethylcarbamoyl)pyridin-2-ylmetyl)-N,N&#39;,N&#39;-tris(pyridin-2-yl-methyl)-1,3-diaminopropan-2-ol. The present protocol not only provides an example of the methodology utilizing the P-tag but also enables the assessment of how mutations as well as inhibitor treatment/administration or any other factors alter the downstream signaling of LRRK2 in cells and tissues.

The present study describes a simple method of detecting endogenous levels of Rab10 phosphorylation by leucine-rich repeat kinase 2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been shown to be linked with familial Parkinson&#39;s disease (FPD). Since abnormal activation of ...

Fractionation for Resolution of Soluble and Insoluble Huntingtin Species

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a key pathological feature of HD is the aberrant accumulation of mutant HTT (mHTT) protein into high molecular weight complexes and intracellular inclusion bodies composed of fragments and other proteins. Conventional methods to measure and understand the contributions of various forms of mHTT-containing aggregates include fluorescence microscopy, western blot analysis, and filter trap assays. 
However, most of these methods are conformation specific, and therefore may not resolve the full state of mHTT protein flux due to the complex nature of aggregate solubility and resolution.
For the identification of aggregated mHTT and various modified forms and complexes, separation and solubilization of the cellular aggregates and fragments is mandatory. Here we describe a method to isolate and visualize soluble mHTT, monomers, oligomers, fragments, and an insoluble high molecular weight (HMW) accumulated mHTT species. HMW mHTT tracks with disease progression, corresponds with mouse behavior readouts, and has been beneficially modulated by certain therapeutic interventions1. This approach can be used with mouse brain, peripheral tissues, and cell culture but may be adapted to other model systems or disease contexts.

A method is described for fractionation of insoluble and soluble mutant huntingtin species from mouse brain and cell culture. The method described is useful for characterization and quantification of huntingtin protein flux and aids in analyzing protein homeostasis in disease pathogenesis and in the presence of perturbations

The accumulation of misfolded proteins is central to pathology in Huntington's disease (HD) and many other neurodegenerative disorders. Specifically, a ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...