Plasma membrane receptors, transporters, and ion channel molecules are often found as oligomeric structures that participate in signaling cascades essential for cell survival. Different states of protein oligomerization may play a role in functional control and allosteric regulation. Stochastic GFP-photobleaching (SGP) has emerged as an affordable and simple method to determine the stoichiometry of proteins at the plasma membrane. This non-invasive optical approach can be useful for total internal reflection of fluorescence microscopy (TIRFM), where signal-to-noise ratio is very high at the plasma membrane. Here, we report an alternative methodology implemented on a standard laser scanning confocal microscope (LSCM). The simplicity of our method will allow for its implementation in any epifluorescence microscope of choice.
The formation of oligomeric complexes between subunits of ion channels, transporters, and plasma membrane receptors often determines the activation state or modifies the biophysical properties of component proteins within these complexes. Multimerization (i.e., changes in quaternary architecture) plays a fundamental role in cellular flow of information and signal transduction (1-3). The elucidation of plasma membrane protein stoichiometry is an important goal. Unfortunately, discerning the oligomerization state of membrane proteins often requires tissue disruption and indirect biochemical methods. One problem with the biochemical approach is that changes in protein stoichiometry may be subtle or the dynamics could be fast enough to make some states “invisible” to detection.
For these reasons, non-invasive optical methods such as FRET (4, 5), bimolecular complementation (6), spatial fluorescence intensity fluctuation analysis (7), analysis of fluorescence intensity either at TIRF plane (8) or during recovery after photobleaching (9), and stochastic GFP-photobleaching (10) can be highly informative.
Here we describe a method for stochastic GFP photobleaching on a laser scanning confocal microscope to determine plasma membrane protein stoichiometry. The background signal can be limited through the use of a membrane sheet preparation method, making single molecule studies possible.
With a restricted optical section (less than 200 nm) and low-background imaging, TIRF microscopy is the method of choice to observe membrane proteins (Figure 1A; 11). For this purpose, membrane proteins are often fused with a GFP-tag, which can be seen in a discrete punctate pattern. One solution for determining the number of subunits present in each fluorescent spot is to use the single-molecule photobleaching technique first implemented by Iino et al. (2001). This method relies on the fact that GFP bleaching is stochastic and molecules bleach independently (10, 12).
Because each channel complex contains as many GFP molecules as it does subunits, single-molecule photobleaching of GFP fluorescence allows a direct count of the number of protein subunits present in any given bright spot. This technique was recently used to determine the stoichiometry of E-cadherin, L-type Ca2+ channels, AQP4, CFTR, CNG channels, Hv1, TRPM8, Orai 1 and 2, and GluK kainate receptors (10, 12, 13, 14, 15, 16, 17, 18, 19).
Although useful, the technique has two main difficulties. First, membrane proteins are mobile elements that can diffuse laterally or be associated with vesicular trafficking. For this reason, it is difficult to observe multiple bleaching events from the same particle without stopping the movement of the particle, which can be accomplished by lowering the temperature (18) or by incubation with glucose-free medium (19). The second drawback is the need for a TIRF microscope to perform the experiments. In comparison with the preparation of planar lipid bilayers (PLB), a method of choice for the reconstitution of ion channels (20, 21), the preparation of membrane sheets preserves the native environment of the protein of interest.
Materials and methods
Cell culture, transfection, and preparation of the sample
HEK293T cells were cultured in DMEM LG supplemented with 5% fetal bovine serum on 22 mm round coverslips or 100mm culture plates. Twenty-four hours after seeding, the cells were transfected with cDNA encoding the protein Hv1-GFP using Metafectene Pro (Biontex Laboratories, Planegg, Germany) in a 1:3 ratio. After 24 h of transfection the cells were washed twice in ice imaging buffer (HBS pH 7,4), then subjected to 3 sonication pulses of 40% power, 1 s each (Cole Palmer 4710 ultrasonics homogenizer; Cole Palmer, Vernon Hills, IL), directly on the culture plate, then washed twice in Ringer's solution before mounting the coverslip on a microscopy chamber.
Microscopy was performed on an Olympus IX81/Fluoview 1000 confocal microscope (Olympus Corporation, Tokyo, Japan), equipped with a 488nm Argon laser, a DM 405/488 excitation filter and a BA 505–605 emission filter. Images of 70x70 μM regions in 12 bit format with a 10 Hz scanning frequency were collected with a 60x oil NA 1.3 objective and 100 μM confocal aperture. TIRF imaging was performed using a though-the-objective TIRF microscope. A thin evanescence field next to the glass-water interface was generated using a 473-nm diode pump laser (LaserGlow Technologies, Toronto, Canada). The laser was focused onto a 3.5μm optical fiber and transmitted via the rear illumination port of an Olympus IX71 microscope. A digitally synchronized shutter (Vincent Associates, Rochester, NY) controlled exposure times through a high-numerical aperture objective (N.A. 1.49, 60X, oil; Olympus). Fluorescence transmission was acquired at 10Hz using micromanager (Vale Lab, UCSF, US) and a CCD camera (ORCA 12ER; Hamamatsu Photonics, Bridgewater, NJ).
Single particle analysis
The identification and quantification of Hv1-GFP particles was performed automatically using the free software ImageJ (http://rsb.info.nih.gov/ij/). Briefly, a binary mask was generated and used to identify discrete particles through the ImageJ plugin Find Maxima; then a 3x3 pixel ROI was quantified for each particle by using the ImageJ plugin Time Series 2.0 Analyzer. Numerical data was plotted and filtered through an FFT filter implemented in Origin 8.0. The graphs obtained were normalized as follows: