Measurement of Junctional Protein Dynamics Using Fluorescence Recovery After Photobleaching (FRAP)

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