A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed Nanogold-labeled molecules

Abstract

We have developed methods to locate individual ligands that can be used for electron microscopy studies of dynamic events during endocytosis and subsequent intracellular trafficking. The methods are based on enlargement of 1.4 nm Nanogold attached to an endocytosed ligand. Nanogold, a small label that does not induce misdirection of ligand-receptor complexes, is ideal for labeling ligands endocytosed by live cells, but is too small to be routinely located in cells by electron microscopy. Traditional pre-embedding enhancement protocols to enlarge Nanogold are not compatible with high pressure freezing/freeze substitution fixation (HPF/FSF), the most accurate method to preserve ultrastructure and dynamic events during trafficking. We have developed an improved enhancement procedure for chemically fixed samples that reduced auto-nucleation, and a new pre-embedding gold enlarging technique for HPF/FSF samples that preserved contrast and ultrastructure and can be used for high-resolution tomography. We evaluated our methods using labeled Fc as a ligand for the neonatal Fc receptor. Attachment of Nanogold to Fc did not interfere with receptor binding or uptake, and gold-labeled Fc could be specifically enlarged to allow identification in 2D projections and in tomograms. These methods should be broadly applicable to many endocytosis and transcytosis studies.

Figure 1

Preparation of Au-Fc. (a) Ribbon diagram of the structure of Fc (pdb code 1I1c) with a gold sphere (drawn to scale) representing a 1.4 nm monomaleimido Nanogold covalently attached to a reduced cysteine in the Fc hinge region. One Nanogold is depicted, based on the calculated Nanogold/Fc ratios obtained in most labeling reactions (0.8 - 1.1). The Nanogold cluster bound in a region that is distant from the FcRn binding site (indicated on each Fc chain with a bracket). (b) S75 Superdex gel filtration profile following incubation of reduced Fc with 1.4 nm Nanogold. (c) 10% SDS-PAGE analysis of non-reduced and unboiled unlabeled Fc (lane 1) and Au-Fc (lane 2). The majority of Fc protein migrated at a higher apparent molecular weight in the Au-Fc sample, demonstrating covalent attachment of 1.4 nm Nanogold. (d) Confocal images (∼5 μm below the apical surface) of FcRn-expressing MDCK cells after Au-Fc uptake (bar = 10 μm). Filter-grown monolayers were incubated with ∼1 μM Au-Fc for one hour at pH 6 and processed for immunofluorescence using antibodies against FcRn (green; left panel) and Fc (red; middle panel) as described in the Supplementary Methods. The merged image (right panel) shows regions of colocalization as yellow. The nearly equimolar ratio of gold to protein in our Au-Fc samples (see Methods) suggested that most or all of the Fc detected by immunofluorescence contained gold. Untransfected MDCK cells showed only background levels of fluorescence when subjected to the same incubation and staining protocols (data not shown).

Figure 2

Gold-enhanced, chemically-fixed intestinal segments. 13-day-old neonatal rats were fed ∼2 μM Au-Fc or a control solution not containing Au-Fc prior to extraction, fixation and gold enhancement of duodenal samples. (a) Gold enhancement using GoldEnhance-EM 2113 (Nanoprobes, Inc.) following the manufacturer’s protocol. (b) Gold enhancement using a modified protocol (Methods). The control sample derived using the modified protocol showed a significant decrease of background color development, which resulted from auto-nucleation, compared with the control sample prepared using the original protocol. (c) Projection EM image derived from a 120 nm section of the control sample shown in Panel a (bar = 200 nm). Randomly-distributed 5-10 nm background gold particles were found in mitochondria and regions of the cell that did not engage in FcRn-mediated endocytosis.

Figure 3

Chemically-fixed, gold-enhanced samples of intestinal segments prepared using the modified enhancing protocol. (a) Specificity of gold enhancement in 70 nm sections derived from the distal small intestine (bar = 1000 nm). A region of the ileum (left) showed enhanced gold particles in giant lysosomes, consistent with fluid phase uptake of Au-Fc. By contrast, gold particles were not found in secretion vesicles in an adjacent goblet cell (right), demonstrating a lack of background resulting from auto-nucleation events. (b) Specificity of Au-Fc uptake and gold enhancement in sections derived from the proximal small intestine (bar = 500 nm). The section on the right was derived from the enhanced segment shown in Figure 2b. Gold particles were located in coated vesicles near the basolateral region of the cell (left; section thickness = 200 nm), on the surface of microvilli, in coated vesicles, and in the extracellular space near the apical surface of a cell (right; section thickness = 150 nm). The section on the left was gold enhanced in the absence of added gum arabic, resulting in specific enhancement of 1.4 nm Au-Fc, but a non-uniform particle size. The addition of gum arabic to the section on the right resulted in a more uniform (∼20 nm) size of enhanced gold particle.

Figure 4

Projection images of HPF/FSF samples of intestinal samples after silver enhancement/gold-toning/gold enhancement of 1.4 nm Au-Fc. (a) 150 nm section from the neonatal jejunum showing gold particles attached to inner surfaces of tubular vesicles, consistent with Au-Fc bound to FcRn (bar = 300 nm). (b) 150 nm section from the neonatal duodenum showing gold particles attached to the inner surface of a multivesicular body (bar = 300 nm). (c) 70 nm section from the neonatal duodenum showing gold particles after release into the extracellular space from the basolateral membrane (bar = 500 nm). (d) 120 nm section from the neonatal ileum showing enlarged gold particles distributed only inside apical vesicles (bar = 500 nm). (e) 120 nm section from the neonatal jejunum showing auto-nucleation resulting in small (∼5 nm) background gold particles distributed randomly inside and outside of vesicles (bar = 500 nm). Auto-nucleation in this case might have resulted from over-exposure to NaBH₄ and/or too long of a developing time.

Figure 5

Digital slices (1.6 nm) derived from tomograms of HPF/FSF samples of jejunal samples after silver enhancement/gold-toning/gold enhancement of 1.4 nm Au-Fc (bar = 300 nm). Dual axis tilt series were recorded at 6500x from 180nm sections at 120 kV on a Tecnai T12 microscope. Panels a-d show enhanced gold particles near the membranes of tubular and spherical vesicles. Panel d shows enhanced Au-Fc that was released into the lateral intercellular space between adjacent intestinal cells.