Dataset Information

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.

SUBMITTER: He W

PROVIDER: S-EPMC2076746 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

He Wanzhong W Kivork Christine C Machinani Suman S Morphew Mary K MK Gail Anna M AM Tesar Devin B DB Tiangco Noreen E NE McIntosh J Richard JR Bjorkman Pamela J PJ

Journal of structural biology 20070725 1

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. Traditi ...[more]

PMID: 17723309

Similar Datasets

Project description:BackgroundWolbachia ?-proteobacteria are essential for growth, reproduction and survival for many filarial nematode parasites of medical and veterinary importance. Endobacteria were discovered in filarial parasites by transmission electron microscopy in the 1970's using chemically fixed specimens. Despite improvements of fixation and electron microscopy techniques during the last decades, methods to study the Wolbachia/filaria interaction on the ultrastructural level remained unchanged and the mechanisms for exchange of materials and for motility of endobacteria are not known.Methodology/principal findingWe used high pressure freezing/freeze substitution to improve fixation of Brugia malayi and its endosymbiont, and this led to improved visualization of different morphological forms of Wolbachia. The three concentric, bilayer membranes that surround the endobacterial cytoplasm were well preserved. Vesicles with identical membrane structures were identified close to the endobacteria, and multiple bacteria were sometimes enclosed within a single outer membrane. Immunogold electron microscopy using a monoclonal antibody directed against Wolbachia surface protein-1 labeled the membranes that enclose Wolbachia and Wolbachia-associated vesicles. High densities of Wolbachia were observed in the lateral chords of L4 larvae, immature, and mature adult worms. Extracellular Wolbachia were sometimes present in the pseudocoelomic cavity near the developing female reproductive organs. Wolbachia-associated actin tails were not observed. Wolbachia motility may be explained by their residence within vacuoles, as they may co-opt the host cell's secretory pathway to move within and between cells.Conclusions/significanceHigh pressure freezing/freeze substitution significantly improved the preservation of filarial tissues for electron microscopy to reveal membranes and sub cellular structures that could be crucial for exchange of materials between Wolbachia and its host.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets