Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Summary

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Abstract

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.

Protocol

1. Isolation of Plasmid DNA Use a Sigma-Aldrich endotoxin-free maxiprep kit to prepare endotoxin free DNA according to the manufacturer’s protocol. This kit works for us, because it reliably removes any endotoxins from DNA preparations. Endotoxins that are injected with the DNA into cell nuclei will lead to cell death. Add 100 μl phenol/chlorofom/isoamylalcohol (25:24:1) to the isolated DNA, vortex and spin for 1 min at 13,000 rpm in an Eppendorf microcentrifuge. Transfer the upper water phase i…

Discussion

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.

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Materials

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