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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9.7K 次观看.  University of Manitoba. 小 Rho GTPase 是具有多种细胞功能的超级家庭蛋白质。例如，这些蛋白质参与调节细胞命运、细胞对压力的反应、细胞活力和细胞周期。小罗GTPase蛋白的活性由翻译后修饰调节。这些修改涉及严格监管其活动。例如，蛋白质预位和GTP键是这些蛋白质最重要的翻译后修饰。我的实验室最近修改并开发了简单的方法来研究这些超家庭蛋白质的翻译后修饰。为此，我们使用...