This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.
This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.
A new approach was introduced recently, to image whole cells with labeled proteins in liquid state using STEM1-3, or correlative fluorescence microscopy and STEM4-7. This methodology is capable of studying the spatial distribution of membrane proteins and protein complexes including their stoichiometry at the single molecule level in the intact plasma membranes of whole cells. Here, we describe a protocol involving a two-step specific labeling of receptor proteins with fluorescent quantum dots (QDs)8, and correlative light microscopy and ESEM-STEM. As example, we studied the EGFR, a membrane protein that belongs to the protein kinase superfamily. Upon ligand binding, the receptor activates and then forms a dimer with another activated EGFR; the subsequent signal cascade drives cell proliferation9. Mutations leading to abnormal EGFR expression or activity play a central role in many types of cancer10. Studying the spatial arrangement of this membrane receptor in whole cells provides important context information about its function and possible changes thereof11-14. But it remains challenging to probe the distribution of EGFR monomers, dimers, and clusters directly on a (sub-)cellular level with biochemical- or conventional microscopy methods15. Our method is capable of visualizing EGFRs with a spatial resolution of a few nanometers such that the locations of proteins in a complex can be determined. Most importantly, the obtained information about the stoichiometric distribution relates to the native situation of the protein in the intact cell.
In order to achieve a short distance between the label and the receptor, EGFR was directly labeled via its ligand EGF, using a biotin-streptavidin bond to a QD. This label can be detected both with fluorescence- and electron microscopy. We have used this label for correlative imaging of COS7 cells in liquid enclosed in a microfluidic chamber previously1,16,17. However, on account of a multiple of streptavidin molecules per QD, this label may induce receptor clustering, leading to a low labeling efficiency or rather expensive experiments, and it is not possible to conclude on the stoichiometry of the protein under investigation. Label induced receptor clustering can be avoided altogether by employing the two-step labeling procedure presented here, resulting in a probe comprising biotinylated EGF to which only one streptavidin-quantum dot (STR-QD) is coupled. The cells are first incubated with biotinylated EGF, and then fixed. The fixation step determines the time point at which protein processes are stopped (depending on the speed of fixation). STR-QD is applied thereafter as second step of the labeling protocol. Clustering does not take place since dynamic membrane motion of the proteins is hindered once the proteins are fixed. Moreover, the STR-QD solution, which is the most expensive component of the experiment, can be applied in an optimized low concentration, and this second step of labeling is not time-crucial, i.e., the QD can be coupled in several minutes.
To study the stoichiometry of EGFR as distributed in whole cells, cellular samples were prepared on silicon microchips18. After applying the two-step QD labeling, and the recording of fluorescence microscopy images, the cells were rinsed with pure water, and imaged in hydrated state using ESEM-STEM19. The resulting correlative images provide information about the cellular EGFR distribution, and the stoichiometry in their natural context of hydrated cells. A similar method was used to study HER2 proteins in breast cancer cells in a recent study7.
Studying the spatial distribution and stoichiometry of the membrane proteins, such as the EGFR, in whole cells provides important context information about its function and possible changes thereof11-14. It is challenging to study the distribution of monomers, dimers, and clusters directly on a subcellular level using commonly used biochemical methods, for which information is lost about the localization of the protein in the cell or differences between cells15. The labeling and microscopy methods described here allow the visualization of protein complexes on a length scale of a few nanometers so that its stoichiometry can be studied within the context of individual, intact cells4-7. This is not possible with (super resolution) light microscopy23 because its resolution is insufficient to distinguish molecules engaged in a protein complex. Förster Resonance Energy Transfer (FRET) is an ensemble averaging technique24, and does not necessarily detect the dimers and higher order clusters as a consequence of the short range of the energy transfer25. Proximity ligation assays26 do not actually measure distances and are thus not capable of distinguishing between a cellular region with a high protein density, inevitably leading to a number of detected proximities by random chance, from a cellular region with a similar protein density but containing protein complexes exhibiting distinct distances between the labels. The required high resolution to visualize the constituents of protein complexes is achieved by transmission electron microscopy (TEM). However, conventional TEM of whole cells is limited by the requirement of thin samples/sections slicing through the plasma membrane27, or plasma membrane ripping or breaking off28,29 resulting in randomly formed small pieces of membrane. The plasma membrane is thus not imaged as a whole, leading to a lack of possibly crucial cellular context information.
Other experimental challenges for the study of protein stoichiometry in cells arise from the applied protein labeling. Commonly used immunolabeling bears the difficulty that one-to-one stoichiometry between receptor and label can only be achieved with a monovalent probe; this is not the case when primary and secondary antibodies are required. To obtain information about the protein stoichiometry, it is required that a probe binds uniquely to one epitope on the receptor and has one binding site for a fluorescent probe or a high atomic number nanoparticle on the other side. Secondly, the precision achievable with antibody-labeling is restricted on account of their large size30 to 30 nm, so that a discrimination between neighboring single proteins, homodimers, or larger clusters is not feasible, at least not for receptors of the EGFR family. Much smaller specific labels are known in literature and can be applied even for intracellular labeling31 but those are not commonly used. The length of the whole label (EGF-biotin-streptavidin-QD) is of a similar dimension as the EGFR, and sufficiently small to be able to detect the dimer. Moreover, the described two-step labeling procedure avoids labeling induced receptor clustering.
Our method is not very difficult, not much more than fluorescence microscopy of labeled proteins. However, an experiment needs to be carried out with great care, since the number of steps adds up and an error in one of the steps may lead to a complete failure in the experiment. Handling the microchips is not more tedious than the handling of TEM grids but some training empty chips is recommended. ESEM-STEM of whole hydrated cells is probably the most difficult aspect, and requires at least a skilled operator and several days of practice in order to reach a resolution around 3 nm as needed to visualize the QDs. Radiation damage of the specimen under investigation presents a risk. The pressure and the temperature should be carefully watched to maintain a water layer. Moreover, the thickness of the water layer may vary between cells. It is advisable to monitor the presence of the water layer using the gaseous electron detector above the specimen, as described elsewhere6. Cellular regions should only be imaged once or twice to avoid damage.
A key limitation of the method is that high-resolution information about the ultrastructure is absent. Other techniques, for example, cryo TEM, are needed to study the protein structure, and the cellular ultrastructure15. Secondly, studying the dynamic interplay of a multiple of proteins in live cell in time-lapse microscope is not feasible on account of radiation damage, and requires state-of-the-art light microscopy23. Currently, our method is not capable of determining the absolute level of dimers since the labeling efficiency is unknown but we expect to add such data in future. It is nevertheless possible to tell if dimers are present or not. From the literature is it known, that even a labeling efficiency around 15% is enough to detect dimers with sufficient statistical significance32.
It is readily possible to study other membrane-bound receptors via their ligand, via biotinylated Fab fragments of specific antibodies, or via other small linkers7 using the described two-step labeling protocol. Since we have already demonstrated that two different colors (sizes) of QDs can be used, and gold nanoparticle labeling was used in prior work6, it seems feasible to label multiple protein species in the same experiment. The key challenge for the study of the stoichiometry of multiple protein species is ensuring a similar labeling efficiency for both species or at least a normalization of the obtained relative stoichiometry on the respective labeling efficiencies. Yet, the two different QDs used in this study do not seem to differ much in labeling efficiency (see Figures 2 and 4). The described method involving two-step labeling with QDs, and correlative fluorescence microscopy and ESEM-STEM presents a viable method to study the complex interplay of membrane proteins in the intact plasma membranes of whole cells.
The authors have nothing to disclose.
We thank E. Arzt for his support through INM, M. Koch for help with the ESEM, and DENS Solutions for providing the microchips. Research in part supported by the Leibniz Competition 2013.
Inverted fluorescence microscope | Leica | AF6000 + DMI6000B | |
ESEM | FEI Company | Quanta 400 FEG | |
Chemicals | |||
DMEM (1x) + GlutaMax + glucose + pyruvate | Gibco | 31966-021 | |
DPBS (1x) – Calcium chloride – Magnesium chlorid | Gibco | 14190-144 | |
Fetal bovine serum, Performance Plus (FBS) | Gibco | 16000-036 | |
Cellstripper | Mediatech, Inc. | 25-056 Cl | |
Water, chromasolv Plus for HPLC | Sigma-Aldrich | 34877-2.5L | |
PBS Roti-Stock 10x PBS | Carl Roth | 1058.1 | |
Ethanol, Rotisolv HPLC grade | Carl Roth | P076.2 | |
Albumin Fraction V, biotin-free (BSA) | Carl Roth | O163.2 | |
Gelatin, from cold water fish skin | Sigma-Aldrich | G7041-500G | |
Glycine | Carl Roth | T873.1 | |
shoul | Molecular Probes | E3477 | |
Sodium teraborate decahydrate | Sigma-Aldrich | S9640-500G | |
Boric acid | Sigma-Aldrich | B6768-500G | |
Qdot 655 streptavidin conjugate | Molecular Probes | Q10121MP | |
Qdot 800 streptavidin conjugate | Molecular Probes | Q10173MP | |
Materials | |||
Silicon microchips with silicon nitride membranes | DENS Solutions | Custom made | |
of 50 nm thickness and dimensions of 50 µm x 0.40 mm |