13 research outputs found
A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed nanogold-labeled molecules
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
Three-dimensional cellular architecture of the flagellar pocket and associated cytoskeleton in trypanosomes revealed by electron microscope tomography
This study uses electron tomography linked to a variety of other EM methods
to provide an integrated view of the flagellar pocket and basal body area of
the African trypanosome procyclic trypomastigote. We reveal the pocket as an
asymmetric membranous `balloon' with two boundary structures. One of these
– the collar – defines the flagellum exit point. The other defines
the entry point of the flagellum into the pocket and consists of both an
internal transitional fibre array and an external membrane collarette. A novel
set of nine radial fibres is described in the basal body proximal zone. The
pocket asymmetry is invariably correlated with the position of the probasal
body and Golgi. The neck region, just distal to the flagellum exit site, is a
specialised area of membrane associated with the start of the flagellum
attachment zone and signifies the point where a special set of four
microtubules, nucleated close to the basal bodies, joins the subpellicular
array. The neck region is also associated with the single Golgi apparatus of
the cell. The flagellar exit point interrupts the subpellicular microtubule
array with discrete endings of microtubules at the posterior side. Overall,
our studies reveal a highly organised, yet dynamic, area of cytoplasm and will
be informative in understanding its function
Crystal morphology of MV-1 magnetite
Intracellular magnetite (Fe_3O_4) crystals produced by magnetotactic bacteria strain MV-1 are in the single-domain size range, and are chemically pure. We have previously suggested that they exhibit an unusual crystal habit described as truncated hexa-octahedral. Such a crystal morphology has not been demonstrated for any inorganic population of magnetite, nor would it be expected, given considerations of symmetry and free energy. By inference, this morphology is a physical signature of a biological origin. Here we report data from transmission electron microscope (TEM) tomography of such crystals isolated from magnetotactic bacteria, which confirm the unusual geometry, originally proposed from classical TEM tilt imaging