Subcellular Protein Localization With High Resolution: The Next Step in Proteomics

Genomics and proteomics have determined the molecular sequences and structures of a large number of proteins. The ultimate goal of understanding the function, or dysfunction, of a specific protein requires detailed knowledge of its localization within the cells as well as its interactions with other proteins and the pathways in which it participates. Localizing protein activity within cellular structures can provide important insights into all of these. Confocal microscopy is widely used in cell biology and provides useful information about protein dynamics, but its resolution is insufficient for precise localization studies. Transmission electron microscopy (TEM) offers orders of magnitude improvement in resolution and, when combined with advanced labeling techniques using ultrasmall gold particles, can localize proteins at the deep subcellular level on a near nanometer scale. Recent advances in electron microscope technology, particularly in operation and application software, have made the instruments significantly easier to use and more accessible to the wide range of laboratories involved in proteomics and cell biology research.

Bridging the gap

Electron microscopy (EM) has the ability to bridge the resolution gap between the atomic-scale resolution techniques required for primary structure analysis and the micron-scale resolution typical of light microscopy. This will prove invaluable as proteomics research moves to the analysis of structure–function relationships, interaction pathways, and the role of protein function in the intracellular organization.

Biologists have always confronted the same challenges in their efforts to elucidate structure, the twin needs for sufficient contrast and resolution. The flood of molecular-scale information that has resulted from recent progress in genomics and proteomics has put renewed emphasis on the high-resolution imaging available only by electron microscopy.

Biological materials are notorious for their low contrast in both light and electron microscopy. Historically, scientists have tackled this problem with increasingly sophisticated staining and labeling techniques. Microscopists have developed a broad range of techniques that preferentially stain different cellular components. For example, hematoxylin, which is attracted to negatively charged molecules, is used to locate DNA, RNA, and acidic proteins with a light microscope. In a TEM, contrast depends on the mass thickness, and stains are typically salts of heavy metals such as uranium and lead. Staining remains an important way to reveal higher level structure, but it lacks the specificity required to distinguish particular proteins or other macromolecules.

Immunological labeling techniques can identify specific molecules or even fractions of molecules and localize them with nanoscale precision relative to surrounding cellular structures. In light microscopy, labeling focuses on attaching fluorescent markers that offer very high sensitivity, but remain spatially limited by the resolving power of the microscope. In TEM, the markers are usually gold particles that offer high contrast against a biological background composed primarily of lighter elements. Conventional gold particles have diameters of several nanometers, which limits their ability to penetrate deeply or reach within some cellular structures. As described below, ultrasmall gold particles address many of the shortcomings of traditional, larger gold labels. Another class of TEM labeling techniques involves the use of an enzyme label that reveals the location of its target by catalyzing a local reaction that creates high-contrast reaction products. Enzyme labels offer high sensitivity since a single label molecule can create many marker molecules, but their spatial resolution is limited by diffusion of the markers.

Ultrasmall gold particles

Ultrasmall gold particles (USG), less than 1 nm in diameter, offer better sensitivity and penetration than larger gold particles, and higher spatial resolution than enzyme labels. Silver enhancement techniques, which add silver to the USG particle after it has found its target, increase the label’s visibility. Sequential silver enhancement permits the simultaneous labeling of more than one target species for co-localization studies.

Prior to the introduction of USG, most ultrastructural localization studies were confined to identifying the nature of the labeled element. In contrast, USG can precisely locate the labeled element within its structural context. For example, in neuroscience, USG can pinpoint the location of a receptor protein to its functional site on the neuronal membrane. Other examples, discussed below, demonstrate the resolution, penetration, and antigen accessibility of the USG technique.

Localization resolution

Figure 2 - Dopamine transporter labeled (N-terminus as in Figure 1) with silver-enhanced USG particles. The labels (black dots, some indicated by arrows) are clearly localized on the cytoplasmic side of the axon terminal (at) membrane.

Figure 1 - Dopamine transporter labeled with an enzyme-based (peroxidase) method. The primary antibody is specific to the N-terminus of the DAT molecule. The reaction products (dark regions) fill the axon terminal, making it impossible to localize the protein on any finer scale.

The dopamine transporter (DAT) is a protein in the central nervous system responsible for removing dopamine from the extracellular space after synaptic transmission. In striatum, it is found in the terminals of dopaminergic axons. Enzyme-based labels have insufficient spatial resolution to localize the protein within the terminal. In Figures 1 and 2, DAT was labeled with a primary monoclonal antibody specific to its N-terminus. The primary antibody was subsequently detected with an enzymatic peroxidase label. The peroxidase reaction products appear to fill the entire terminal (Figure 1). However, when the primary antibody was labeled with USG (Figure 2), the silver-enhanced particles were clearly associated with the cytoplasmic side of the terminal membranes. Only the combination of EM and USG offers sufficient resolution to localize proteins with respect to structures such as intracellular membranes and organelles.

Figure 3 - USG reagent penetration. Labeled DAT is apparent to a depth of 8 μm.

Reagent penetration

Conventional gold particles, with diameters of several nanometers, have demonstrated a limited ability to penetrate the sample. The lack of penetration is difficult to account for based on particle size alone. One explanation proposes that the effective radius of the particle is increased by several nanometers by a surrounding cluster of water molecules. USG are perhaps small enough to prevent the formation of the water jacket, giving them an ability to penetrate that is disproportionate to the decrease in particle size alone. The sample in Figure 3 is from a 50-μm-thick section that was labeled for DAT with USG prior to embedding. The thick section was then cut into thin sections perpendicular to the original section, such that the edge of the thin section originated in the surface of the thick section. Labeled DAT can be seen to a depth of 8 μm. In another study that labeled parvalbumin with the same method, labels could be seen up to 12 μm below the section surface.