This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.
Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the ‘free’ surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed ‘dominant negative’, whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a ‘dominant active’ state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39°C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31°C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.
1. Isolation of Plasmid DNA
2. Culture of MDCK Cells
3. Microinjection Procedure and Post-injection Incubations
4. Surface Staining for Immunofluorescence Analysis
Note, in order to avoid bleaching of the GFP signal of VSVGts045-GFP, protect the specimens from light by covering with aluminum foil during all subsequent procedures.
5. Representative Results
For examples on how the co-expression of small GTPases interferes with VSVG sorting kindly refer to published articles for either apical missorting 3, 4 or inhibition of surface delivery 7.
Figure 1. In the mock-injection, i.e. an injection of only the plasmid encoding VSVGts045-GFP, the protein is delivered to the basolateral surface of well-polarized MDCK cells as judged by surface staining (in red, Figure 1 A). Note that not all of the VSVGts045-GFP is delivered to the basolateral membrane during the chase at 31°C as evidenced by the extensive intracellular signal for the total protein (in green, Figures 1 A and B). In cells that are not well-polarized VSVGts045-GFP will be delivered also to the apical membrane (Figure 1 B). If your control specimens look like the cell in Figure 1 B, you cannot trust your data and have to repeat the experiment with better-polarized cells. Scale bars are 5 μm.
The most critical steps for a successful microinjection experiment are the quality and purity of the DNA and the polarity of your cells. Without polarized cells, your injection control will already have mistargeted VSVG and the experiment cannot be used. If the DNA is of poor quality, the DNA may clog the injection needle leading to poor or no expression of the desired protein at all. Also, it is advisable to use expression plasmids that are known to lead to high expression levels such as pRKV.
If you desire to test for sorting of other cargo proteins, the temperature shift protocols can be modified. For example, to express low-density lipoprotein receptors, we perform the microinjection at 37°C followed by a 1 h incubation at 37°C, 4 h at 20°C (to arrest the cargo in the TGN) and 2 h at 37°C in the presence of 0.1 mg/ml cycloheximide to chase the protein to the surface. For some proteins such as Fc receptors, the incubation time at 20°C may be lowered to 2 h dependent on how fast your cargo protein is synthesized and travels through the secretory pathway. For a detailed description of incubations that we used for different cargo proteins see Nokeset al., 2008 7.
Finally, this protocol can not only be used for the analysis of small GTPases, but in theory for all proteins or protein fragments that you suspect may have a function in (polarized) surface delivery.
The authors have nothing to disclose.
This work was funded by a grant from the National Institutes of Health (GM070736) to H. Fölsch. S.F. Ang was supported by an A*STAR Graduate Scholarship award, and R.S. Kang was supported by the Cellular and Molecular Basis of Disease Training Program (GM8061)
Name of reagent | Company | Catalogue number |
Axiovert 200 Microscope with heated stage | Carl Zeiss Inc | Custom order |
Injectman NI2 Femtojet micromanipulator | Eppendorf | Custom order |
Femtotips II (Microinjection needles) | Eppendorf | 930000043 |
Microloader tips | Eppendorf | 930001007 |
Clear 12-mm transwell filter supports | Corning Costar | 3460 |
Endotoxin-free plasmid maxiprep kit | Sigma-Aldrich | NA0400 |