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W tym Artykule

  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

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.

Streszczenie

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 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.

Wprowadzenie

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 "Rho insert domain" within the 5th β strand and the 4th α 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.

Protokół

1. Determination of RhoA Localization Using Membrane/Cytosol Fractionation

  1. Cell culture and simvastatin treatment
    1. Seed 50,000 of U251 cells in a 100 mm dish and culture them in Dulbecco's modified Eagle's medium (DMEM) (high glucose, 10% fetal bovine serum [FBS]).
    2. When 30% confluent, treat the cells by removing the medium and adding simvastatin-containing medium to it (10 µM of simvastatin dissolved in dimethyl sulfoxide [DMSO]), and incubate for 36 h at 37 °C8. Use DMSO alone as a vehicle control.
      NOTE: 10 million cells are needed for the cytosol and membrane fractionation of the cells.
  2. Collection of cells
    1. Remove the cells from the 37 °C incubator. Look at the cells under a microscope to confirm the confluency.
      NOTE: The cells should be 70% - 80% confluent.
    2. Aspirate the medium, wash the cells 1x with cold phosphate-buffered saline (PBS). Add 5 mL of ethylenediaminetetraacetic acid (EDTA) buffer (KCl: 400 mg/L, NaCl: 6800 mg/L, NaHCO3: 2200 mg/L, NaH2PO4.H2O: 140 mg/L, D-glucose: 1,000 mg, EDTA disodium: 373 mg/L) per plate and place the cells back into the 37 °C incubator for 5 min.
    3. After 5 min of incubation, collect the EDTA with the cells in a 15 mL tube containing the same amount of medium as EDTA.
      NOTE: Having medium in the tube neutralizes the EDTA and prevents any further digestion of the cell membranes.
    4. Place the tube in an ice box and proceed to the centrifuge.
    5. Set up the centrifuge to 1,500 x g at 4 °C and spin the cells for 5 min.
    6. Remove the supernatant without disturbing the pellet and add 1 mL of cold PBS. Mix the cells well.
    7. Transfer the cell mixture (solution) to a new 1.5 mL tube, centrifuge at 1,500 x g at 4 °C, and spin the cells for 5 min.
    8. Check the pellet size (for estimating the volume of the buffer for the next step). Place the samples on ice. Discard the supernatant completely without disturbing the pellet.
    9. Add ice-cold buffer I (10 mM Tris-HCl [pH 7.5], 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and protease inhibitor cocktail), mix the samples well by pipetting up and down, and then, proceed to sonication.
  3. Sonication
    1. Set the sonicator for five cycles, 5 s each, and repeat 3x.
    2. Perform the sonication on ice. Proceed to the ultracentrifuge.
      NOTE: Ice and cold condition preserve the proteins and make the results more reliable.
  4. Ultracentrifugation
    1. Use an ultracentrifuge to separate the cell homogenates into cytoplasmic and membrane fractions. Set the centrifuge to 100,000 x g for 35 min at 4 °C. As shown in Figure 1, check the pellet size.
      NOTE: The membrane fraction is at the very bottom of the tube and the rest is other cytoplasmic components.
    2. Collect the supernatant completely while being careful not to disturb the pellet. The supernatant is the cytosolic fraction. Place the supernatant in a newly labeled tube.
    3. Add 300 µL of dissociation buffer (buffer II) (50 mM Tris-HCl [pH 7.5], 0.15 M NaCl, 1 mM dithiothreitol, 1% SDS, 1 mM EDTA, 1 mM EGTA, and protease inhibitor cocktail) to the pellet (contains the membrane fraction). Mix well by pipetting up and down.
    4. Proceed to protein determination and western blot (immunoblot analysis) sample preparation.
  5. Immunoblotting
    1. Prepare the cell protein extracts from the separated fractions in lysis buffer (20 mM Tris-HCl [pH 7.5], 0.5 mM PMSF, 0.5% non-ionic detergent-40, 100 µM β-glycerol 3-phosphate, and 0.5% protease inhibitor cocktail).
    2. Measure the protein concentration using the Lowry method8 and calculate the volume of the lysis buffer (20 mM Tris-HCl [pH 7.5], 0.5 mM PMSF, 0.5% nondenaturing detergent, octylphenoxypolyethoxyethanol, 100 µM β-glycerol 3-phosphate, and 0.5% protease inhibitor cocktail) to normalize the concentration of protein between the samples.
    3. Heat the samples at 90 °C for 5 min and load 15 - 20 µL of the samples on a 15% SDS-PAGE gel to separate the proteins.
      NOTE: Load 1 µg of protein for each sample. Calculate the volume that needs to be run accordingly.
    4. Transfer the separated proteins to nylon membranes under reducing conditions (500 nM glycine, 50 mM Tris-HCl, and 20% methanol) for 2 h, at room temperature (RT) at 100 V.
      NOTE: To confirm the successful protein transfer, use Ponceau stain or visualize the protein marker on the membrane.
    5. Block the membranes with 5% nonfat dried milk and 1x Tris-buffered saline containing detergent (TBS/0.01% nonionic detergent; TBST) to block nonspecific antibody binding at 4 °C overnight or at RT for 1 h.
    6. Add primary antibodies for immunoblotting analysis and incubate overnight at 4 °C.
      NOTE: In this experiment, Rac1/2/3, cdc42, RhoA, GAPDH, and pan-Cadherin were used at a 1:1,000 dilution in 1% milk in 1x TBST. Pan-Cadherin and GAPDH were used to confirm membrane and cytosolic fraction purity, respectively.
    7. Wash the membranes 3x with a washing buffer with 1x TBST for 20 min.
    8. Incubate the membranes with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody for the respective primary antibodies (for 1 h at RT).
    9. Wash the blots 3x for 20 min and develop them with enhanced chemiluminescence (ECL) detection.

2. Measurement of the RhoA GTP Load Using a Small G-protein Activation Assay

  1. Count 10,000 cells/mL and culture the U251 cells in a 100 mm dish.
  2. When they are 30% confluent, treat the cells with simvastatin as described in step 1.1.2.
  3. Bring the culture plates out of the incubator. Look at the cells under the microscope to confirm confluency. Ensure that the cells are 70% - 80% confluent. Place the Petri dish on ice, aspirate the media, and wash the cells 3x with ice-cold PBS (pH 7.2).
  4. Aspirate the PBS. Tilt the Petri dish on ice for an additional minute to remove all remnants of PBS.
    NOTE: Residual PBS adversely affects this assay.
  5. Lyse the cells in a 700 µL volume of ice-cold lysis buffer containing protease and phosphatase inhibitors.
    NOTE: 700 µL is usually enough for a 100 mm Petri dish. See Table 1 to find the proper volume for each culture vessel.
  6. Harvest the cell lysate with the cell scraper. Incline the culture plate for this technique.
  7. Transfer the lysate to a labeled ice-cold cryotube and keep it on ice.
  8. Mix thoroughly using a vortex. Keep 10 µL of the lysate for the protein assay, to measure the protein concentration in the sample.
  9. Snap-freeze the remaining cell lysate in liquid nitrogen.
    NOTE: Prepare multiple aliquots of cell lysate before snap-freezing them, to avoid repeated freeze/thaw cycles which can lead to the loss of activity of RhoA GTPase.
  10. Transfer the snap-frozen cryotubes to a -80 °C freezer and store the samples for the GTPase-linked immunosorbent assay.
    NOTE: Do not store the samples for longer than 14 days. Work quickly and never leave the samples on ice for longer than 10 min. Never handle all Petri dishes simultaneously.
  11. Measure the protein concentration using the Lowry method8 and calculate the volume of the lysis buffer to normalize the concentration of protein between the samples.
    NOTE: The best concentration is usually 1 mg/mL; however, 0.3 - 2 mg/mL can be detectable.
  12. Prepare a blank control by adding 60 µL of lysis buffer and 60 µL of binding buffer to a microtube.
    NOTE: The blank control has all reagents except the antigen and is used for the subtraction of the background.
  13. Prepare a positive control by adding 12 µL of Rho control protein, 48 µL of lysis buffer, and 60 µL of binding buffer.
    NOTE: The positive control has all reagents plus a confirmed antigen for Rho-A-GTP.
  14. Take the Rho affinity plate out of its bag and place it on ice.
  15. Dissolve the powder in the wells with 100 µL of ice-cold distilled water. Keep the plate on ice.
  16. Thaw the snap-frozen cell lysates in a water bath set to 25 °C.
  17. Add the calculated volume of ice-cold lysis buffer (from step 2.11) to each sample to normalize the protein concentration.
    NOTE: Remove the PBS after washing the cells (using a vacuum tube aspirator) to avoid causing changes in the composition of the lysis buffer. Equalize the sample protein to a concentration between 0.8 and 2 mg/mL for an accurate comparison between samples in GTPase activation assays. Table 2 provides details about the buffer to be used for this assay.
  18. Transfer 60 µL of the normalized ice-cold samples to microtubes and add 60 µL of binding buffer; mix the samples thoroughly and keep them on ice.
  19. Completely remove the water/solutions from the microplate by vigorous flicking, followed by five to seven hard taps on a lab mat.
  20. Add 50 µL of the normalized samples, a blank control, and a positive control to the wells in duplicates.
  21. Place the plate on an orbital shaker for 30 min at 4 °C at 300 rpm.
    NOTE: The shaking step is very important, and it is recommended to use the orbital plate shaker at 300 rpm.
  22. Clear the samples from the plate by flicking and wash them 2x with 200 µL of washing buffer at RT. Vigorously remove the washing buffer from the wells after each wash by flicking, followed by tapping, and keep the plate on the bench at RT.
  23. Add 200 µL of RT antigen-presenting buffer to each well and incubate at RT for 2 min.
  24. Flick out the solution from the wells and wash the wells 3x with 200 µL of washing buffer at RT.
  25. Add 50 µL of freshly prepared 1/250 anti-RhoA primary antibody to each well.
  26. Place the plate on an orbital shaker for 45 min at 300 rpm set to 25 °C. Flick out the solution from the well.
  27. Repeat the washing steps 2x (step 2.24).
  28. Add 50 µL of freshly prepared 1/250 anti-RhoA secondary antibody to each well.
  29. Place the plate on top of an orbital shaker for 45 min at 300 rpm set to 25 °C.
  30. Prepare the HRP detection reagent by mixing equal volumes of reagent A and reagent B.
  31. Flick out the solution from each well and wash the wells 3x with 200 µL of washing buffer at RT.
  32. Add 50 µL of freshly prepared HRP detection reagent to each well.
  33. Read the luminescent signal within 3 - 5 min to obtain the maximum signal and analyze the results using an appropriate software package.
    NOTE: Readings must be taken within 3 - 5 min to obtain the maximum signal. Run a "test plate" to confirm the proper lysis buffer volume is being used for the cell lysates so that the protein concentration is high enough to detect RhoA GTPase activity. (A test plate is a plate of cells used to determine if the protein concentration falls within the acceptable range and also to determine if the volume of the lysis buffer being used is appropriate.) The positive control should read 4- to 10-fold higher than the blank wells if it is in the linear range. If not, then adjust the luminometer by consulting the manufacturer. The settings for the luminometer are given in Table 3.
  34. Enter raw data in the columns where the headings read Sample, Mean, Standard Deviation, rep1, rep2, rep3, and rep4, which is to show the number of replicates being done on each sample.
  35. Under Mean, enter the formula =average(Xn:Yn) where X = the column designator for rep1, Y = the column designator for rep4, and n = the row designator of the row being worked on.
  36. Under Standard Deviation, enter the formula =stdev (Xn:Yn) where X = the column designator for rep1, Y = the column designator for rep4, and n = the row designator of the row being worked on.
  37. Enter the replicate data into rep1, rep2, etc.
  38. After entering the data, use the click-and-drag method to select the Sample, Mean, and Standard Deviation.
  39. Then, in data analysis software, select the function for chart making which looks like a square with a mini bar chart inside.
    NOTE: This brings up the chart making process where it is possible to design charts based on the data entered.
  40. Choose column chart and, for input values, designate the Mean numbers.
    NOTE: The chart for the Mean numbers is first made, and then, the Standard Deviation column for the y-axis error bars is designated. To do this, double-click on the graph bars, select the Y-axis error tab, click the Custom option, and select the area in the worksheet to enter the location of the Standard Deviation data. The difference between the groups that need to be compared can be seen after the creation of the desired charts.

Wyniki

Membrane Fractionation:

Ultracentrifugation was used for the fractionation of membrane and cytosol components. As shown in Figure 1, 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...

Dyskusje

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

Ujawnienia

The authors have nothing to disclose.

Podziękowania

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.

Materiały

NameCompanyCatalog NumberComments
DMEM high GlucoseVWR (Canada)VWRL0101-0500
Fetal Bovine SerumVWR (Canada)CA45001-106
Penicillin/StreptomycinVWR (Canada)97062-806
EDTA (Ethylenediamine tetraacetic acid)VWR (Canada)CA71007-118
EGTA (Ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid)VWR (Canada)CAAAJ60767-AE
DTT (DL-Dithiothreitol)VWR (Canada)CA97061-340
Ammonium PersulfateVWR (Canada)CABDH9214-500G
Tris-HydroxymethylaminomethaneVWR (Canada)CA71009-186
30% Acrylamide/Bis SolutionBiorad (Canada)1610158
TEMEDBiorad (Canada)1610801
Protease Inhibitor cocktailSigma/Aldrich (Canada)P8340-5ML1:75 dilution
Rho-GTPase Antibody Sampler KitCell Signaling (Canada)99681:1000 dilution
Pan-Cadherin antibodyCell Signaling (Canada)40681:1000 dilution
GAPDH antibodySanta Cruz Biotechnology (USA)sc-697781:3000 dilution
RhoA G-LISA Activation Assay (Luminescence format)Cytoskeleton Inc. (USA)BK121Cytoskeleton I. G-LISA Activation Assays Technical Guide. 2016.
RhoA AntibodyCell Signaling2117
ECLAmersham-Pharmacia BiotechRPN2209
Anti-Rabbit IgG (whole molecule) Peroxidase antibodySigmaA6154-1ML
SpectraMax iD5 Multi-Mode Microplate ReaderMolecular Devices 1612071ASpectrophotometer
Nonidet P-40Sigma11332473001non-denaturing detergent, octylphenoxypolyethoxyethanol
DMSOSigmaD8418-50ML
PBSSigmaP5493-1L
Phophatase Inhibitor cocktailSigmaP5726-5ML1:75 Dilution

Odniesienia

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  2. Valencia, A., Chardin, P., Wittinghofer, A., Sander, C. The ras protein family: evolutionary tree and role of conserved amino acids. Biochemistry. 30 (19), 4637-4648 (1991).
  3. Hall, A. Rho family GTPases. Biochemical Society Transactions. 40 (6), 1378-1382 (2012).
  4. Rojas, A. M., Fuentes, G., Rausell, A., Valencia, A. The Ras protein superfamily: evolutionary tree and role of conserved amino acids. The Journal of Cell Biology. 196 (2), 189-201 (2012).
  5. Cherfils, J., Zeghouf, M. Regulation of small GTPases by GEFs, GAPs, and GDIs. Physiological Reviews. 93 (1), 269-309 (2013).
  6. Shojaei, S., et al. Perillyl Alcohol (Monoterpene Alcohol), Limonene. Enzymes. 36, 7-32 (2014).
  7. Ghavami, S., et al. Airway mesenchymal cell death by mevalonate cascade inhibition: integration of autophagy, unfolded protein response and apoptosis focusing on Bcl2 family proteins. Biochimica et Biophysica Acta. 1843 (7), 1259-1271 (2014).
  8. Alizadeh, J., et al. Mevalonate Cascade Inhibition by Simvastatin Induces the Intrinsic Apoptosis Pathway via Depletion of Isoprenoids in Tumor Cells. Scientific Reports. 7, 44841 (2017).
  9. Ghavami, S., et al. Mevalonate cascade regulation of airway mesenchymal cell autophagy and apoptosis: a dual role for p53. PLoS One. 6 (1), e16523 (2011).
  10. Tang, Y., Olufemi, L., Wang, M. T., Nie, D. Role of Rho GTPases in breast cancer. Frontiers in Bioscience: A Journal and Virtual Library. 13, 759-776 (2008).
  11. DerMardirossian, C., Bokoch, G. M. GDIs: central regulatory molecules in Rho GTPase activation. Trends in Cell Biology. 15 (7), 356-363 (2005).
  12. Garcia-Mata, R., Boulter, E., Burridge, K. The 'invisible hand': regulation of RHO GTPases by RHOGDIs. Nature Reviews Molecular Cell Biology. 12 (8), 493-504 (2011).
  13. Etienne-Manneville, S., Hall, A. Rho GTPases in cell biology. Nature. 420 (6916), 629-635 (2002).
  14. Ghavami, S., et al. Geranylgeranyl transferase 1 modulates autophagy and apoptosis in human airway smooth muscle. American Journal of Physiology - Lung Cellular and Molecular Physiology. 302 (4), L420-L428 (2012).
  15. Clark, E. A., Golub, T. R., Lander, E. S., Hynes, R. O. Genomic analysis of metastasis reveals an essential role for RhoC. Nature. 406 (6795), 532-535 (2000).
  16. Ghavami, S., et al. Statin-triggered cell death in primary human lung mesenchymal cells involves p53-PUMA and release of Smac and Omi but not cytochrome c. Biochimica et Biophysica Acta. 1803 (4), 452-467 (2010).
  17. Cordle, A., Koenigsknecht-Talboo, J., Wilkinson, B., Limpert, A., Landreth, G. Mechanisms of statin-mediated inhibition of small G-protein function. Journal of Biological Chemistry. 280 (40), 34202-34209 (2005).
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