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Measurement of Junctional Protein Dynamics Using Fluorescence Recovery After Photobleaching (FRAP)
采用荧光漂白恢复法(FRAP)测定接合蛋白质的动力学   

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Abstract

Fluorescence Recovery After Photobleaching (FRAP) (Lippincott-Schwartz et al., 2003; Reits and Neefjes, 2001) was employed to determine dynamic properties of proteins localized at the ephitelial zonula adherens (ZA) (Kovacs et al., 2011; Otani et al., 2006). The proteins of interest were expressed in cells using a knockdown and reconstitution approach in which endogenous proteins were depleted by RNA interference (RNAi) and replaced by expression of an RNAi-resistant gene fused to GFP (Priya et al., 2013; Smutny et al., 2010; Smutny et al., 2011; Vitriol et al., 2007). By choosing expression levels of GFP-tagged proteins that were comparable to endogenous levels, we minimized transient overexpression artifacts due to overcoming regulatory mechanisms that directly affect protein dynamics (Goodson et al., 2010). Using this approach, junctional E-cadherin-GFP or GFP-Ect2 were subjected to FRAP analysis in small areas corresponding to the ZA using confocal microscopy (Priya et al., 2013; Ratheesh et al., 2012; Gomez et al., 2005; Trenchi et al., 2009). Although in principle this approach is similar in every case, bleaching conditions, acquisition parameters and analysis details might differ depending on the time scale of the recovery process (Lippincott-Schwartz et al., 2003). In this protocol we will describe the experimental procedure to perform FRAP experiments and how to optimize bleaching and acquisition conditions for optimal measurements of protein dynamics at cell-cell junctions.

Keywords: E-cadherin(E -钙黏蛋白), FRAP(FRAP), Cell-cell junctions(细胞-细胞连接), Turnover(周转率), Half life(半条命)

Materials and Reagents

  1. MCF-7 cells, mammary carcinoma epithelial cells derived from metastatic site (ATCC® HTB22 TM)
  2. HEK293T cells
  3. Plasmids
    pLL5.0 lentiviral vector (Figure 1) and packaging plasmids pMDLg/pRRE, pMD2.G (VSV G) and pRSV-Rev. pLL5.0 is a modified version of pLL3.7 and it was generously provided by Jim Bear, Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, NC 27599 (Vitriol et al., 2007; Rubinson et al., 2003)
    pLL5.0 containing both a shRNA against the ORF of human CDH1 (NM_004360) (5′-GGGTTAAGCACAACAGCAA-3′) cloned downstream of the U6 promoter (HpaI and XhoI) (Figure 1) and a mouse E-Cadherin(NM_009864)-GFP fusion construct cloned at SacII and SbfI sites. The E-cadherin-EGFP fusion protein expression was driven by a 5’LTR promoter to facilitate lower expression levels of GFP fusion proteins for imaging (Smutny et al., 2011 )
    pLL5.0 containing both a shRNA against the 3’UTR of human ECT2 (NM_001258315) (5'-GCTGTTTCAAAGTGTGATA-3') and cloned downstream of the U6 promoter (HpaI and XhoI) (Figure 1) in a modified version of pLL5.0. In this modified pLL5.0 the GFP reporter was replaced by the sequence that encompasses both the coding region for GFP and the multiple cloning site of pEGFP-C1 (Clontech) using EcoRI and SbfI sites. These restriction sites were not preserved after this cloning step. Then the human ECT2 coding sequence (NM_001258315) was cloned into the vector using EcoR1 and BamH1 sites (pLL5.0 GFP–shRNA resistant ECT2)


    Figure 1. Schematic of pLL5.0 vector. Sites HpaI and XhoI are used for the subcloning of shRNA sequences desired to knockdown endogenous levels of the protein of interest. The U6 promoter drives the expression of this shRNA sequence. Contrarily, a shRNA resistant version of the same protein can be subcloned downstream of the 5’LTR promoter and fused to GFP. Thus, it is possible to achieve endogenous levels of expression for a fluorescent-tagged protein and preventing effects associated to its overexpression. MCS = Multiple cloning site

  4. Dulbecco’s Modified Eagle’s Medium High glucose with stable L-glutamine (DMEM) (Gibco, catalog number: 11995-073 )
  5. Foetal Bovine Serum (FBS) (Life Technologies, Gibco®, catalog number: 26140079 )
  6. Phosphate buffered saline (PBS) without Ca2+ and Mg2+ (Astral Scientific, catalog number: 09-8912-100 )
  7. 16% Paraformaldehyde (formaldehyde) (PFA) aqueous solution (ProSciTech, catalog number: C004 )
  8. Hank’s balanced salt solution (HBSS) (Sigma-Aldrich, catalog number: H8264 )
  9. In-Fusion cloning kit (Clontech, catalog number: 638909 )
  10. Hank’s Balanced Salt Solution (Sigma-Aldrich, catalogue number: H8264 )
  11. 2.5% Trypsin (10x) (Life Technologies, catalogue number: 15090046 )
    Note: This solution is diluted to 0.25% final concentration with PBS.
  12. Poly(vinylidene difluoride) spin columns (Amicon Ultra Centrifugal filters, UltraCel-100K) (EMD Millipore, catalog number: UFC910024 )
  13. Sodium butyrate (Sigma-Aldrich, catalogue number: B5887 ) (see Recipes)
  14. Hexadimethine bromide (polybrene) (Sigma-Aldrich, catalog number: H9268 ) (see Recipes)
  15. Imaging media (see Recipes)
  16. 4% Paraformaldheyde in PBS (see Recipes)

Equipment

  1. 25 cm2 Nunclon Delta Flasks (Thermo Fisher Scientific, Nunc®, catalog number: 156367 )
  2. 175 cm2 Nunclon Delta Flasks (Thermo Fisher Scientific, Nunc®, catalog number: 159910 )
  3. Laser scanning confocal microscope equipped with acousto-optic tunable filters (AOTF) for bleaching of selected areas and heated chamber (37 °C) for live cell imaging. The microscope must also be equipped with dichroic and emission filter for the use of the 405 and 488 nm laser lines and detection of GFP fluorescence. The experiments shown were performed on LSM 510 Meta or LSM 710 inverted confocal microscopes (ZEISS)
  4. 30 mW Argon (458, 488 and 514 nm laser lines) and 25 mW (405 nm) diode lasers (LASOS Lasertechnik GmbH)
  5. Plug-in FRAP profiler (McMaster University, Canada)
  6. Glass bottom dishes (#1.5) (MatTek, catalog number: P35G-1.5-20-C or Shengyou Biotechnology, catalog number: D29-10-1.5-N)

Software

  1. Image J software
  2. Prism, GraphPad
  3. Matlab, MathWorks

Procedure

  1. Cell preparation
    1. Expression of GFP-tagged proteins in a knockdown background
      We have used this approach in our recent article published in Nature Cell Biology (Ratheesh et al., 2012) to characterize the dynamic properties of the adhesion molecule E-cadherin and the RhoA GEF, Ect2. For the expression of these proteins at endogenous levels, we used the pLL5.0 lentiviral vector (Vitriol et al., 2007; Rubinson et al., 2003). This vector contains two promoters, a U6 promoter that drives the expression of shRNA and a 5’LTR promoter that drives the expression of a shRNA-resistant gene (Figure 1).
    2. Lentivirus preparation and viral transduction
      1. HEK293T cells were cultured in 20 ml DMEM supplemented with 10% FBS at 37 °C and maintained under these condition during the following steps.
      2. Constructs made in the pLL5.0 vector were simultaneously transfected with packaging vectors into HEK-293T cells by CaCl2 precipitation.
      3. 48 h after transfection, cells were treated with sodium butyrate (10 mM final concentration) to increase gene induction.
      4. Virus-like particles (VLPs) were harvested 48–72 h after transfection and concentrated on poly(vinylidene difluoride) spin column as follows:
        1. Collect media of cells and spin down in 50 ml conical tube.
        2. Filter the supernatant into new tubes using 0.2 μm syringe filters.
        3. Add 10 ml filtrate to the poly(vinylidene difluoride) spin column and centrifuge at 3,200 rpm on a bench top centrifuge for 20 min at room temperature. This will reduce the volume of the suspension of VLPs to ~800 μl per tube.
        4. Discard the flow trough and add the remaining supernatant (~10 ml) to the the poly(vinylidene difluoride) spin column and repeat the above step.
        5. Aliquots of virus were subsequently used for titration or stored at -80 °C. Titers were determined as described before (Smutny et al., 2010).
    3. Preparation of the cells for image acquisition
      1. For FRAP experiments, MCF-7 cells were cultured in DMEM supplemented with 10% FBS and infected with lentiviral particles at a multiplicity of infection of 10 per cell on 25 cm2 flasks.
      2. Cells were incubated at 37 °C with the lentivirus in DMEM + FBS and Polybrene (8 μg ml-1) and harvested by trypsinization three days after infection.
      3. Single-cell suspensions were seeded on glass bottom dishes at 80% confluence and allowed to grow for 48 h (or until they reach full confluence) for FRAP experiments.
      4. Prior to image acquisition, cells were washed with imaging media and incubated with 1.5 ml of it for the duration of the experiment.

  2. Image Acquisition
    1. FRAP experiments were performed on a LSM 510 Meta or LSM 710 Zeiss confocal microscope for E-cadherin-GFP or GFP-Ect2, respectively. Microscopes were equipped with a heated stage maintained at 37 °C and a 30 mW Argon laser (458, 488 and 514 nm laser lines). The LSM 710 Zeiss confocal microscope was also equipped with a 405 nm (25 mW) diode laser. Images (pre and post-bleach, Figure 1) were acquired using 60x objective, 1.4 NA oil Plan Apochromat immersion lens at 4x digital magnification with 0.7 μm optical section. A 488 nm laser line of an argon laser (30 mW) was used for fluorescence excitation at 1-3% transmission.
    2. For E-cadherin-GFP dynamics, time-lapse images (416 x 416 pixels, 0.086 μm/pixel) were acquired before and after photobleaching with an interval of 5 seconds per frame for the total time of 280 seconds (Figure 1A). A constant region of interest (ROI) of 2.8 x 1.7 μm with the longer axis parallel to the cell-cell contact was marked for each experiment and E-cadherin-GFP was bleached with 50 iterations of the 488 nm laser with 100% transmission. This resulted in maximum bleach of approximately 70%.
    3. Ect2 dynamics was assessed using GFP-Ect2 co-expressed with Ect2 shRNA by lentiviral infection. A constant circular ROI (1.4 μm diameter) in approximately the center of the cell-cell contact was bleached to ~ 70% with both the 488 and the 405 nm lasers turned on simultaneously at 100% transmission. Time-lapse images of the same region were acquired before (20 frames, 5 seconds) and after (210 frames, 50 seconds) photobleaching with an interval of ~ 250 m per frame (Figure 1B).
    4. For these experiments, cells with slanted contacts were chosen which allowed us to precisely identify and photobleach the ZA.

      Special considerations
      1. For any experimental setup, it is important to consider that the bleaching process and the frequency of acquisition has to match the dynamics of the protein of interest (Lippincott-Schwartz et al., 2003; Weiss, 2004). The above technical details should be first be tested to achieve the optimal conditions for FRAP experiments of specific proteins or for different subcellular compartments. Bleaching and acquisition conditions can be optimized by doing FRAP in fixed cells. We routinely grow cells on glass bottom dishes and fix using 4% PFA in PBS for 15 min at room temperature. After fixation, PFA solution is replaced by imaging media and the FRAP protocols tested on this set of cells. Following this approach, optimization can be achieved in conditions that match the real experimental setup.
        The major aims of these optimization experiments are to:
        1. Determine the best conditions suitable for a fast and efficient photobleaching of molecules in a region of interest that would be used in the real experiments.
        2. Optimize the time-lapse settings for acquisition during pre- and, more importantly, post- bleaching regimes. The main aim is to acquire images without causing photobleaching (< ~5%) of the sample at a given frequency that does not compromise FRAP analysis.
      2. Following the optimization steps, a FRAP test is performed in living cells. There are two important points that needs to be considered that are related to the half time of the observed recovery process (Weiss, 2004). Firstly, if the half time is comparable to the bleaching step, then there is a high chance that recovery is underestimated as bleached molecules can diffuse away from the bleached area during the bleaching step (Weiss, 2004). If so, it is necessary to optimize the bleaching protocol to make this step faster (~< 3 times the half time of recovery). This can be achieved for example, by reducing the area of the region that is wanted to be bleached or, by increasing the laser power and reducing the number of iterations during the bleaching step or, by increasing the number of laser lines activated during the bleaching step or, by reducing the scan speed of the bleaching step at the same time the number of iterations it is also reduced. The conditions mentioned for the bleaching step of E-cadherin and Ect2 are good standard initial conditions to perform FRAP experiments on proteins that exhibits very distinctive dynamics. Secondly, slow post acquisition frames can compromise recovery measurements. As the half time of a FRAP curve is calculated with the information acquired during the first 1.5 half times of the recovery process, confident estimation of FRAP parameters requires that acquisition be fast enough to accurately sample this early period. To satisfy this requirement, increasing scan speed or reducing the area of sampling during pre and postbleaching acquisition can increase the speed of acquisition. This second option was chosen in order to capture the fast dynamics of Ect2 mobility.
      3. After these conditions are set, it is essential to consider that the optimized protocol does not compromise the viability of cells. Normally, UV irradiation causes toxicity, which is evident by changes in the morphology of the cell and membrane blebbing (Frigault et al., 2009). Acquisition of phase contrast or Differential interference contrast (DIC) images before and after FRAP acquisition is a complementary test to assess cell viability. Of note, UV irradiation can cause membrane damage that often results in an unexpectedly high immobile fraction. For this, it has been suggested to perform 2 consecutive FRAP experiments on the same cells and on the same region, in order to determine that recovery occurs even after two consecutive rounds of photobleaching (Lippincott-Schwartz et al., 2003).

  3. Image analysis
    1. E-cadherin Turnover
      Image analysis was performed using Image J software. Noise on images was reduced by applying a median filter of 2 pixels radii. As E-cadherin dynamics at the ZA is relatively slow (in our experience, a FRAP experiment takes ~10 min to plateau), it is inevitable that some cell movements and/or drift occur during image acquisition. If these movements really compromise the measurements, then the experiment is discarded. However, those experiments with slight cell movements can be corrected and/or eliminated by aligning consecutive frames using Turbo-reg (http://bigwww.epfl.ch/thevenaz/turboreg/) plug-in of Image J. After that, FRAP profiles were calculated using a ROI marked at the bleached area and use the plug-in FRAP profiler to obtain fluorescence intensity profiles. Fluorescence intensities in the ROI immediately after bleaching (F(0)) were subtracted from fluorescence intensities at all times (F(t)) and results were then normalized to pre-bleaching values (Eq.1, Figure 2A). Results were then imported into Prism software for statistics analysis. Data from 11 replicates (3 independent experiments) were pooled and fluorescence intensity at time points after the bleaching step were fitted to the equation:
       (Eq.1)
      where F(t), F(-t) and F(0) are the average fluorescence of the ROI at any time, before bleaching and, immediately after bleaching, respectively. Mf is the mobile fraction, t1/2 is the half time of recovery and t is time in seconds. In Prism, this fitting is achieved by using non-linear regression and the exponential one-phase association model using Y0 = 0 and where Mf corresponds to the plateau value. Data then are presented as the average ± SEM and the statistical significance assessed by t-test.


      Figure 2. Examples of E-cadherin-GFP and GFP-Ect2 FRAP experiments. A. Left, Representative images using MCF-7 cells of the subcellular distribution of E-cadherin-GFP and GFP-Ect2 expressed in E-cadherin and Ect2 knockdown backgrounds, respectively. Center, details of acquisition frames during pre (shown) and post bleaching (not shown) stages during a FRAP experiment. Right, Fluorescence recovery plots for E-cadherin-GFP (top graph) and GFP-Ect-2 (bottom graph). Note the difference in time scales. B. Details of non-linear regression of GFP-Ect2 recovery plot using either mono-exponential (Eq.1) or double exponential (Eq.2) functions. This shows that a mono exponential function does not adjust properly to the experimental curve.

    2. Ect2 Turnover
      Image analysis was also performed using Image J software. It is worth to mentioning that an Ect2 FRAP experiment takes ~1 min, therefore no significant drifts or cell movements were observed. To calculate FRAP profiles, a ROI at the bleached GFP-Ect2 area was marked and its average fluorescence determined at every time point using the measure stack plugin in Image J software. Fluorescence intensities were treated as described above for E-cadherin-GFP to obtain recovery plots and data fitted to the double exponential equation (Figure 2B):
      (Eq.2)
      F(t) is the average fluorescence of the ROI, Mf is the mobile fraction, ffast and fslow are weighting factors for fast and slow mobile components, and their respective half times and t is time in seconds. In Prism, this fitting is achieved by using non-linear regression and the exponential two-phase association model using Y0 = 0 and where the plateau value corresponds to Mf.
      For this case, a numerical solution to obtain the t value at which Fluorescence Recovery = 0.5 was applied to obtain the global half time for Ect2 recovery. This was performed in Matlab (MathWorks, Australia) as follows. Values from fitting can be introduced as:

      >> (In the brackets real values are introduced)
      And then calculate the global t1/2 using the FRAPtwo function (see below) and the following sentence:
      >> t1/2 = fzero(@(t) FRAPtwo(Parameters,t),7);
      Data are then presented as the average ± SEM and the statistical significance assessed by t-test.

      The following is the description of the Matlab function used for calculation of t1/2.
      function [ y ] = FRAPtwo(X,t);

      plateau=X(1);
      fractionfast=X(2);
      Kfast=ln(2)/X(3);
      fractionslow=X(4);
      Kslow=ln(2)/X(5);
      y=plateau*fractionfast*(1-exp(-Kfast*t))+plateau*fractionslow*(1-exp(-Kslow*t))-(plateau/2);

      end

Recipes

  1. Sodium butyrate
    A 1 M stock solution of Sodium butyrate is prepared in water
    Filter sterilized
    Stored at 4 °C previous to use
  2. Hexadimethine bromide (polybrene)
    A stock solution of polybrene is made by diluting it into water to a final stock concentration of 8 mg/ml
    Sterilizing by filtering trough a 0.2 μm filter
  3. Imaging media
    Hank’s balanced salt solution supplemented with 10 mM HEPES pH 7.4
    5 mM CaCl2
  4. 4% Paraformaldehyde in PBS
    Prepare by dilution of the stock solution (16% formaldehyde)
    Adjust pH to 7 with HCl or NaOH if necessary using pH indicator papers
    Aliquot dilutions and store at -20 °C

Acknowledgments

This work was supported by the The Kids Cancer Project of The Oncology Children’s Foundation, The University of Queensland Early Career Grant (2012003354) to GAG. RP is supported by UQI (UQ International) Ph.D. Scholarship and ANZ Trustees Ph.D. Scholarship in Medical Research. Confocal microscopy was performed at the ACRF/IMB Cancer Biology Imaging Centre established with the generous support of the Australian Cancer Research Foundation.

References

  1. Frigault, M. M., Lacoste, J., Swift, J. L. and Brown, C. M. (2009). Live-cell microscopy - tips and tools. J Cell Sci 122(Pt 6): 753-767. 
  2. Gomez, G. A. and Daniotti, J. L. (2005). H-Ras dynamically interacts with recycling endosomes in CHO-K1 cells: involvement of Rab5 and Rab11 in the trafficking of H-Ras to this pericentriolar endocytic compartment. J Biol Chem 280(41): 34997-35010.
  3. Goodson, H. V., Dzurisin, J. S. and Wadsworth, P. (2010). Methods for expressing and analyzing GFP-tubulin and GFP-microtubule-associated proteins. Cold Spring Harb Protoc 2010(9): pdb top85. 
  4. Kovacs, E. M., Verma, S., Ali, R. G., Ratheesh, A., Hamilton, N. A., Akhmanova, A. and Yap, A. S. (2011). N-WASP regulates the epithelial junctional actin cytoskeleton through a non-canonical post-nucleation pathway. Nat Cell Biol 13(8): 934-943. 
  5. Lippincott-Schwartz, J., Altan-Bonnet, N. and Patterson, G. H. (2003). Photobleaching and photoactivation: following protein dynamics in living cells. Nat Cell Biol Suppl: S7-14.
  6. Otani, T., Ichii, T., Aono, S. and Takeichi, M. (2006). Cdc42 GEF Tuba regulates the junctional configuration of simple epithelial cells. J Cell Biol 175(1): 135-146.
  7. Priya, R., Yap, A. S. and Gomez, G. A. (2013). E-cadherin supports steady-state Rho signaling at the epithelial zonula adherens. Differentiation. 
  8. Ratheesh, A., Gomez, G. A., Priya, R., Verma, S., Kovacs, E. M., Jiang, K., Brown, N. H., Akhmanova, A., Stehbens, S. J. and Yap, A. S. (2012). Centralspindlin and α-catenin regulate Rho signalling at the epithelial zonula adherens. Nat Cell Biol 14(8): 818-828. 
  9. Reits, E. A. and Neefjes, J. J. (2001). From fixed to FRAP: measuring protein mobility and activity in living cells. Nat Cell Biol 3(6): E145-147.
  10. Rubinson, D. A., Dillon, C. P., Kwiatkowski, A. V., Sievers, C., Yang, L., Kopinja, J., Rooney, D. L., Zhang, M., Ihrig, M. M., McManus, M. T., Gertler, F. B., Scott, M. L. and Van Parijs, L. (2003). A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33(3): 401-406.
  11. Smutny, M., Cox, H. L., Leerberg, J. M., Kovacs, E. M., Conti, M. A., Ferguson, C., Hamilton, N. A., Parton, R. G., Adelstein, R. S. and Yap, A. S. (2010). Myosin II isoforms identify distinct functional modules that support integrity of the epithelial zonula adherens. Nat Cell Biol 12(7): 696-702.
  12. Smutny, M., Wu, S. K., Gomez, G. A., Mangold, S., Yap, A. S. and Hamilton, N. A. (2011). Multicomponent analysis of junctional movements regulated by myosin II isoforms at the epithelial zonula adherens. PLoS One 6(7): e22458.
  13. Trenchi, A., Gomez, G. A. and Daniotti, J. L. (2009). Dual acylation is required for trafficking of growth-associated protein-43 (GAP-43) to endosomal recycling compartment via an Arf6-associated endocytic vesicular pathway. Biochem J 421(3): 357-369. 
  14. Vitriol, E. A., Uetrecht, A. C., Shen, F., Jacobson, K. and Bear, J. E. (2007). Enhanced EGFP-chromophore-assisted laser inactivation using deficient cells rescued with functional EGFP-fusion proteins. Proc Natl Acad Sci U S A 104(16): 6702-6707.
  15. Weiss, M. (2004). Challenges and artifacts in quantitative photobleaching experiments. Traffic 5(9): 662-671.

简介

使用光漂白后的荧光恢复(FRAP)(Lippincott-Schwartz等人,2003; Reits和Neefjes,2001)来确定位于Ephitelial zonula adherens(ZA)处的蛋白质的动力学性质(Kovacs < et al。,2011; Otani et al。,2006)。使用敲除和重建方法在细胞中表达感兴趣的蛋白质,其中内源蛋白质被RNA干扰(RNAi)耗尽,并被与GFP融合的RNAi抗性基因的表达代替(Priya等人。 >,2013; Smutny等人,2010; Smutny等人,2011; Vitriol等人,2007)。通过选择与内源水平相当的GFP标记蛋白的表达水平,我们最小化了由于克服了直接影响蛋白质动力学的调节机制的瞬时过表达人工产物(Goodson等人,2010)。使用这种方法,使用共聚焦显微镜检查,在对应于ZA的小区域中对连接的E-钙粘蛋白-GFP或GFP-Ect2进行FRAP分析(Priya等人,2013; Ratheesh等人。,2012; Gomez等人,2005; Trenchi等人,2009)。虽然原则上这种方法在每种情况下都是类似的,但是漂白条件,获取参数和分析细节可能根据回收过程的时间尺度而不同(Lippincott-Schwartz等人,2003)。在本协议中,我们将描述进行FRAP实验的实验程序,以及如何优化漂白和获取条件,以优化测量细胞 - 细胞连接处的蛋白质动力学。

关键字:E -钙黏蛋白, FRAP, 细胞-细胞连接, 周转率, 半条命

材料和试剂

  1. MCF-7细胞,源自转移部位的乳腺癌上皮细胞(ATCC HTB22 TM )。
  2. HEK293T细胞
  3. 质粒
    pLL5.0慢病毒载体(图1)和包装质粒pMDLg/pRRE,pMD2.G(VSV G)和pRSV-Rev。 pLL5.0是pLL3.7的修饰版本,并且由北卡罗来纳大学,Chapel Hill,NC 27599的细胞和发育生物学系的Jim Bear慷ously提供(Vitriol等人, 2007; Rubinson等人,2003)
    (HpaI和XhoI)(图1)和小鼠E-钙粘着蛋白(NM_009864)的下游的包含针对人CDH1(NM_004360)(5'-GGGTTAAGCACAACAGCAA-3')的ORF的shRNA的pLL5.0- GFP融合构建体克隆在SacII和SbfI位点。 E-钙粘蛋白-EGFP融合蛋白表达由5'LTR启动子驱动以促进用于成像的GFP融合蛋白的较低表达水平(Smutny等人,2011)。
    (NM_001258315)(5'-GCTGTTTCAAAGTGTGATA-3')的3'UTR的shRNA并在pLL5的修饰形式中克隆在U6启动子(HpaI和XhoI)的下游(图1)的pLL5.0。 0。在该修饰的pLL5.0中,GFP报告子被包含GFP的编码区和pEGFP-C1(Clontech)的多克隆位点的序列替换,使用EcoRI和SbfI位点。在该克隆步骤后,这些限制性位点不被保留。然后使用EcoR1和BamH1位点(pLL5.0GFP-shRNA抗性ECT2)将人类ECT2编码序列(NM_001258315)克隆到载体中


    图1.pLL5.0载体的示意图。位点HpaI和XhoI用于亚克隆所需的shRNA序列,以敲除目的蛋白的内源水平。 U6启动子驱动该shRNA序列的表达。相反,相同蛋白的shRNA抗性版本可以亚克隆到5'LTR启动子的下游,并融合到GFP。因此,可以实现荧光标记的蛋白质的内源表达水平和预防与其过表达相关的效应。 MCS =多克隆站点

  4. Dulbecco's Modified Eagle's Medium具有稳定的L-谷氨酰胺(DMEM)的高葡萄糖(Gibco,目录号:11995-073)
  5. 胎牛血清(FBS)(Life Technologies,Gibco ,目录号:26140079)
  6. 没有Ca 2+和Mg 2+的磷酸盐缓冲盐水(PBS)(Astral Scientific,目录号:09-8912-100)
  7. 16%多聚甲醛(甲醛)(PFA)水溶液(ProSciTech,目录号:C004)
  8. Hank's平衡盐溶液(HBSS)(Sigma-Aldrich,目录号:H8264)
  9. In-Fusion克隆试剂盒(Clontech,目录号:638909)
  10. Hank's平衡盐溶液(Sigma-Aldrich,目录号:H8264)
  11. 2.5%胰蛋白酶(10x)(Life Technologies,目录号:15090046) 注意:该溶液用PBS稀释至0.25%最终浓度。
  12. 聚(偏二氟乙烯)离心柱(Amicon Ultra离心过滤器,UltraCel-100K)(EMD Millipore,目录号:UFC910024)
  13. 丁酸钠(Sigma-Aldrich,目录号:B5887)(参见Recipes)
  14. 六溴二苯醚(polybrene)(Sigma-Aldrich,目录号:H9268)(参见配方)
  15. 成像介质(参见配方)
  16. 4%的Paraformaldheyde在PBS(见配方)

设备

  1. 25cm 2 Nunclon Delta Flasks(Thermo Fisher Scientific,Nunc ,目录号:156367)
  2. 175cm 2 Nunclon Delta Flasks(Thermo Fisher Scientific,Nunc ,目录号:159910)
  3. 激光扫描共聚焦显微镜配备声光可调滤波器(AOTF)用于漂白所选区域和加热室(37°C)用于活细胞成像。 显微镜还必须配备二向色和发射滤光器,用于使用405和488nm激光线和检测GFP荧光。 所示的实验在LSM 510 Meta或LSM 710反向共聚焦显微镜(ZEISS)上进行
  4. 30mW氩(458,488和514nm激光线)和25mW(405nm)二极管激光器(LASOS Lasertechnik GmbH)
  5. 插件FRAP分析器(McMaster University,Canada)
  6. 玻璃底皿(#1.5)(MatTek,目录号:P35G-1.5-20-C或Shengyou Biotechnology,目录号:D29-10-1.5-N)

软件

  1. Image J软件
  2. Prism,GraphPad
  3. Matlab,MathWorks

程序

  1. 细胞制备
    1. 在敲低背景中表达GFP标记的蛋白质
      我们在我们最近发表于Nature Cell Biology的文章(Ratheesh等人,2012)中使用这种方法来表征粘附分子E-钙粘蛋白的动力学性质和RhoA GEF,Ect2。为了在内源水平表达这些蛋白质,我们使用pLL5.0慢病毒载体(Vitriol等人,2007; Rubinson等人,2003)。该载体含有两个启动子,驱动shRNA表达的U6启动子和驱动shRNA抗性基因表达的5'LTR启动子(图1)。
    2. 慢病毒制备和病毒转导
      1. 将HEK293T细胞在补充有10%FBS的20ml DMEM中在37℃下培养,并在以下步骤期间保持在这些条件下。
      2. 在pLL5.0载体中制备的构建体通过CaCl 2沉淀用包装载体同时转染到HEK-293T细胞中。
      3. 转染后48小时,用丁酸钠(10mM终浓度)处理细胞以增加基因诱导。
      4. 在转染后48-72小时收获病毒样颗粒(VLP),并如下在聚(偏二氟乙烯)旋转柱上浓缩:
        1. 收集细胞的培养基并在50ml锥形管中旋转
        2. 使用0.2μm注射器过滤器将上清液过滤到新管中
        3. 加入10ml滤液到聚(偏二氟乙烯)离心柱,并在台式离心机上在室温下以3,200rpm离心20分钟。 这将使VLP的悬浮液体积减少到每管约800μl
        4. 弃去流槽,将剩余的上清液(约10ml)加入到聚(偏二氟乙烯)旋转柱中,并重复上述步骤。
        5. 随后将等分的病毒用于滴定或储存在-80℃。 如前所述测定滴度(Smutny等人,2010)。
    3. 制备用于图像采集的细胞
      1. 对于FRAP实验,将MCF-7细胞在补充有10%FBS的DMEM中培养,并在25cm 2烧瓶上以每个细胞10个感染复数感染慢病毒颗粒。
      2. 将细胞在37℃下与慢病毒在DMEM + FBS和聚凝胺(Polybrene)(8μg/ml)中温育,并在感染后三天通过胰蛋白酶消化收获。
      3. 将单细胞悬浮液以80%汇合度接种在玻璃底培养皿上,并允许生长48小时(或直到它们达到完全融合)用于FRAP实验。
      4. 在图像采集之前,用成像介质洗涤细胞,并在实验期间用1.5ml孵育
  2. 图像采集
    1. 在用于E-钙粘蛋白-GFP或GFP-Ect2的LSM 510 Meta或LSM 710 Zeiss共聚焦显微镜上分别进行FRAP实验。显微镜配备有保持在37℃的加热台和30mW氩激光器(458,488和514nm激光线)。 LSM 710 Zeiss共聚焦显微镜还配备有405nm(25mW)二极管激光器。使用60倍物镜,1.4NA油平面Apochromat浸没透镜以4x数字放大倍率,0.7μm光学截面获得图像(漂白前和漂白后,图1)。氩激光器(30mW)的488nm激光线用于1-3%透射的荧光激发
    2. 对于E-钙粘蛋白-GFP动力学,在光漂白之前和之后以每帧5秒的间隔获得延时图像(416×416像素,0.086μm/像素),总时间为280秒(图1A)。对于每个实验,标记2.8×1.7μm的恒定的感兴趣区域(ROI),其中平行于细胞 - 细胞接触的较长轴,并且用具有100%透射的488nm激光的50次重复漂白E-钙粘蛋白-GFP。这导致大约70%的最大漂白。
    3. 使用通过慢病毒感染与Ect2shRNA共表达的GFP-Ect2评估Ect2动力学。在大约细胞 - 细胞接触的中心的恒定圆形ROI(1.4μm直径)漂白至〜70%,同时488和405nm激光在100%透射率同时开启。在(20帧,5秒)之前和之后(210帧,50秒)获得相同区域的延时图像, 间隔〜250 m每帧(图1B)。
    4. 对于这些实验,选择具有倾斜接触的细胞,其允许我们精确地鉴定和光漂白ZA
      特殊注意事项
      1. 对于任何实验设置,重要的是考虑漂白过程和获取的频率必须匹配感兴趣的蛋白质的动力学(Lippincott-Schwartz等人,2003; Weiss,2004) 。应首先测试上述技术细节以实现特定蛋白质或不同亚细胞区室的FRAP实验的最佳条件。漂白和捕获条件可以通过在固定电池中进行FRAP来优化。我们常规地在玻璃底盘上生长细胞并使用PBS中的4%PFA在室温下固定15分钟。固定后,PFA溶液被成像介质替代,并且在该组细胞上测试FRAP方案。按照这种方法,可以在与真实实验设置匹配的条件下实现优化 这些优化实验的主要目的是:
        1. 确定适合在感兴趣区域中快速有效地漂白分子的最佳条件,用于实际实验。
        2. 优化时间推移设置,用于在之前和更重要的是漂白后体系的采集。主要目的是获取图像而不导致光漂白 (<〜5%)的样品在不损害FRAP分析的给定频率下
      2. 在优化步骤之后,在活细胞中进行FRAP测试。有两个重要的点需要考虑,与观察到的恢复过程的一半时间有关(Weiss,2004)。首先,如果半衰期与漂白步骤相当,则漂白分子可能在漂白步骤期间从漂白区域扩散远远低于回收率的可能性(Weiss,2004)。如果是这样,需要优化漂白方案以使该步骤更快(〜小于回收一半时间的3倍)。这可以例如通过减少想要漂白的区域的面积,或通过增加激光功率和减少漂白步骤期间的迭代次数,或通过增加在漂白期间激活的激光线的数量来实现步骤或,通过降低漂白步骤的扫描速度同时进行迭代次数也减少。用于E-钙粘蛋白和Ect2的漂白步骤所提到的条件是对表现出非常独特动力学的蛋白质进行FRAP实验的良好标准初始条件。其次,慢采集帧可能损害恢复测量。由于使用在恢复过程的前1.5个时间期间获取的信息计算FRAP曲线的半时间,因此FRAP参数的自信估计要求采集足够快以准确地采样该早期时间。为了满足这个要求,在漂洗前和漂洗后获取期间增加扫描速度或减少取样面积可以提高采集速度。选择第二个选项是为了捕获Ect2迁移率的快速动力学
      3. 在设置这些条件之后,必须考虑优化的方案不损害细胞的存活力。通常,UV照射引起毒性,这通过细胞形态学和膜起泡的变化是明显的(Frigault等人,2009)。采集FRAP采集之前和之后的相差或差分干涉对比(DIC)图像是评估细胞活力的补充测试。值得注意的是,UV照射可以引起膜损伤,其通常导致意想不到的高固定分数。为此,已经提出在相同细胞上和在相同区域上进行2个连续的FRAP实验,以确定即使在两次连续的光漂白之后恢复也发生(Lippincott-Schwartz等人>,2003)。

  3. 图像分析
    1. 钙粘蛋白营业额
      使用Image J软件进行图像分析。通过应用2个像素半径的中值滤波器减少了图像上的噪声。由于在ZA的E-钙粘蛋白动力学相对缓慢(在我们的经验中,FRAP实验花费〜10分钟到平台),在图像采集期间不可避免地发生一些细胞运动和/或漂移。如果这些运动真的损害测量,则放弃实验。然而,通过使用Turbo-reg()可以校正和/或消除那些具有轻微细胞运动的实验http://bigwww.epfl.ch/thevenaz/turboreg/)插件。之后,使用在漂白区域标记的ROI计算FRAP谱,并使用插件FRAP分析器获得荧光强度分布。从所有时间的荧光强度中减去漂白后立即的ROI中的荧光强度(F em(0))( t ),然后将结果标准化为预漂白值(Eq.1,图2A)。然后将结果导入Prism软件用于统计分析。汇集来自11个重复的数据(3次独立实验),并且将漂白步骤后的时间点的荧光强度拟合为等式:
        (Eq.1)
      其中 (t), ( - t) )是分别在漂白之前和漂白后任何时间的ROI的平均荧光。 Mf 是移动部分, t 1/2 是恢复的一半时间,t是以秒为单位的时间。在Prism中,通过使用非线性回归和使用Y sub = 0的指数一相关联模型来实现这种拟合,并且其中E ref对应于平稳值。然后将数据表示为平均值±SEM,并通过t检验评估统计显着性

      图2.E-钙粘着蛋白-GFP和GFP-Ect2 FRAP实验的实施例 ,左图,使用MCF-7细胞的E-钙粘蛋白亚细胞分布的代表性图像-GFP和GFP-Ect2分别在E-钙粘蛋白和Ect2敲除背景中表达。 中心,在FRAP实验期间在pre(显示)和后漂白(未显示)阶段的采集帧的细节。 右,E-钙粘蛋白-GFP(上图)和GFP-Ect-2(下图)的荧光恢复曲线。注意时间刻度的差异。 B.使用单指数(Eq.1)或双指数(Eq.2)函数的GFP-Ect2恢复曲线的非线性回归的细节。这表明单指数函数不能适当地调整到实验曲线。

    2. Ect2周转
      还使用Image J软件进行图像分析。值得一提的是,Ect2 FRAP实验需要〜1分钟,因此没有观察到显着的漂移或细胞移动。为了计算FRAP谱,在漂白的GFP-Ect2区域的ROI被标记,并且在每个时间点使用图像J软件中的测量堆栈插件确定其平均荧光。如上所述处理荧光强度用于E-钙粘蛋白-GFP以获得拟合到双指数方程(图2B)的恢复曲线和数据:
      (公式2)
      F inf(t)是ROI的平均荧光, Mf 是移动分数, > 和 f 是快速和慢速移动组件的加权因子,它们各自的一半时间,而 t 是以秒为单位的时间。在Prism中,这种拟合通过使用非线性回归和使用Y sub = 0的指数两相关联模型来实现,并且其中平台值对应于M f。 br /> 对于这种情况,应用获得荧光恢复= 0.5时的t值的数值解以获得Ect2恢复的总半衰期。这在Matlab(MathWorks,Australia)中进行如下。拟合的值可以引入为:

      >> (括号中为实际值)
      然后使用FRAPtwo函数(见下文)和下面的语句计算全局 t 1/2 1/2 = fzero(@(t)FRAPtwo(Parameters,t),7);
      然后将数据表示为平均值±SEM,并通过t检验评估统计学显着性
      以下是用于计算 t 1/2 的Matlab函数的描述。
      function [y] = FRAPtwo(X,t);

      高原= X(1);
      fractionfast = X(2);
      Kfast = ln(2)/X(3);
      fractionslow = X(4);
      Kslow = ln(2)/X(5);
      y =平台*分数快速*(1-exp(-Kfast * t))+平台*分数low *(1-exp(-Kslow * t)) -
      结束

食谱

  1. 丁酸钠
    在水中制备丁酸钠的1M储备溶液 过滤灭菌
    在使用前保存在4°C
  2. 己二胺溴化物(polybrene)
    聚凝胺的储备溶液通过将其稀释到水中至最终储存浓度为8mg/ml 通过0.2μm过滤器过滤灭菌
  3. 映像媒体
    Hank's平衡盐溶液,补充有10mM HEPES pH7.4 5mM CaCl 2
  4. 4%多聚甲醛的PBS溶液
    通过稀释储备溶液(16%甲醛)制备 用HCl或NaOH调节pH至7,如有必要,使用pH指示剂
    等分试样稀释并储存在-20°C

致谢

这项工作是由儿童癌症项目的肿瘤学儿童基金会,昆士兰大学早期职业资助(2012003354)支持GAG。 RP由UQI(UQ国际)博士支持。奖学金和澳新银行受托人博士医学研究奖学金。在澳大利亚癌症研究基金会的慷ous支持下建立的ACRF/IMB癌症生物学成像中心进行共聚焦显微镜检查。

参考文献

  1. Frigault,M.M.,Lacoste,J.,Swift,J.L.and Brown,C.M。(2009)。 活细胞显微镜 - 提示和工具 J Cell Sci < em> 122(Pt 6):753-767。 
  2. Gomez,G.A。和Daniotti,J.L。(2005)。 H-Ras与CHO-K1细胞中的回收内体动态相互作用:Rab5和Rab11参与 贩运H-Ras到这个pericentriolar endocytic隔室。 J Biol Chem 280(41):34997-35010。
  3. Goodson,H.V.,Dzurisin,J.S.and Wadsworth,P。(2010)。 表达和分析GFP-微管蛋白和GFP-微管相关蛋白的方法。 em> Cold Spring Harb Protoc 2010(9):pdb top85。 
  4. Kovacs,E.M.,Verma,S.,Ali,R.G.,Ratheesh,A.,Hamilton,N.A.,Akhmanova,A.and Yap,A.S。(2011)。 N-WASP通过非规范后成核途径调节上皮连接肌动蛋白细胞骨架。 a> Nat Cell Biol 13(8):934-943。 
  5. Lippincott-Schwartz,J.,Altan-Bonnet,N。和Patterson,G.H。(2003)。 光漂白和光活化:活细胞中的蛋白质动力学。增补: S7-14。
  6. Otani,T.,Ichii,T.,Aono,S.and Takeichi,M。(2006)。 Cdc42 GEF Tuba调节简单上皮细胞的连接配置 Biol.175(1):135-146。
  7. Priya,R.,Yap,A.S。和Gomez,G.A。(2013)。 E-钙粘蛋白支持在上皮粘膜粘附的稳态Rho信号。 区分。 
  8. Ratheesh,A.,Gomez,G.A.,Priya,R.,Verma,S.,Kovacs,E.M.,Jiang,K.,Brown,N.H.,Akhmanova,A.,Stehbens,S.J.and Yap, Centralspindlin和α-catenin调节上皮粘膜上的Rho信号。 Nat Cell Biol 14(8):818-828。 
  9. Reits,E.A。和Neefjes,J.J。(2001)。 从固定到FRAP:测量活细胞中的蛋白质流动性和活性。 Nat Cell Biol 3(6):E145-147
  10. 这些研究结果表明,这些研究结果表明,这些研究结果表明,这些研究结果表明,这些研究结果表明, ML和Van Parijs,L。(2003)。 基于慢病毒的系统通过RNA功能性沉默原代哺乳动物细胞,干细胞和转基因小鼠中的基因 Nat Genet 33(3):401-406。
  11. Smutny,M.,Cox,H.L.,Leerberg,J.M.,Kovacs,E.M.,Conti,M.A.,Ferguson,C.,Hamilton,N.A.,Parton,R.G.,Adelstein,R.S.and Yap,A.S.(2010)。 肌球蛋白II同种型识别支持上皮粘膜粘附蛋白完整性的不同功能模块。 em> Nat Cell Biol 12(7):696-702
  12. Smutny,M.,Wu,S.K.,Gomez,G.A.,Mangold,S.,Yap,A.S。和Hamilton,N.A。(2011)。 由肌球蛋白II同种型在上皮粘膜附着处调节的连接运动的多组分分析。 em> PLoS One 6(7):e22458。
  13. Trenchi,A.,Gomez,G.A。和Daniotti,J.L。(2009)。 需要双重酰化以将生长相关蛋白43(GAP-43)转运至内体循环通过Arf6相关的内吞性囊泡途径。生物化学杂志421(3):357-369。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Priya, R. and Gomez, G. A. (2013). Measurement of Junctional Protein Dynamics Using Fluorescence Recovery After Photobleaching (FRAP). Bio-protocol 3(20): e937. DOI: 10.21769/BioProtoc.937.
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