Studying cellular architecture in three dimensions with improved resolution: Ta replicas revisited

Abstract

Metal replicas have been used for surface analysis of biological structures with a variety of spatial resolutions. Platinum (Pt) has been the metal of choice because it provides very stable replicas and images of high contrast. Some other metals, such as tantalum (Ta) have been reported to provide better resolution on isolated macromolecular complexes and cellular structures. Our goal is to study the gain in detail with Ta and to evaluate if it provides enough detail and resolution to assist in the study of complex volumes of intact cellular structures obtained by methods that reach molecular resolution. To this purpose Pt and Ta replicas of cellular structures and viruses have been studied by transmission electron microscopy (TEM). Replicas of Ta show new details on the surface of two types of isolated viral particles such as 100 nm bunyaviruses and large, > 300 nm, vaccinia virus (VV). Inside cells, the structural pieces that build VV immature particles are visualized only in Ta replicas. Looking for smaller intracellular complexes, new details are also seen in nuclear pores from Ta replicas. Additional masses, most likely representing the cargo during transport, are distinguished in some of the pores. Visualization of proteins in plasma membranes strongly suggests that detail and resolution of Ta replicas are similar to those estimated for 3D maps currently obtained by electron tomography of viruses and cells.

Mechanism of an Amphipathic α-Helical Peptide’s Antiviral Activity Involves Size-Dependent Virus Particle Lysis

“…Infectivity assays support this prediction, namely, that treatment with AH peptide dramatically reduced the infectivity of HCV particles (Figure , panel a and Supplementary Figure S2). In contrast, analogous experiments performed with vaccinia virus (average particle diameter of 360 nm ) indicates that AH peptide does not significantly decrease this virus’ infection potential (Figure , panel a). Similar results have recently been reported by Cheng et al , where a larger number of viruses were treated with AH peptide, although no mechanism was provided to account for the varying effects of the AH peptide on the various viruses.…”

Three-dimensional structure of Rubella virus factories

“…Freeze-etching of BHK-21 cells transfected with RUBrep/GFP/neo replicon was done as described in Cabezas and Risco (2006) Tantalum/Wolfram (Ta/W) was used instead of Platinum/carbon (Pt/C) to make the metal replicas of exposed surfaces because it is known that Ta forms thinner layers of fine grain showing a detectable increase in resolution of a variety of biological structures (Cabezas and Risco, 2006). Briefly, cells where fixed with 1% glutaraldehyde in PBS, cryo-protected with glycerol and fast-frozen in liquid ethane.…”

“…Random thin-sectioning and electron microscopy (EM) has missed many of the fine features in RUBV factories such as connections between recruited organelles and the relationships between different membranous compartments inside CPVs and with the surrounding cytosol. The main technical options for 3D characterization of cells include reconstruction from serial sections, which is particularly useful for very large structures such as whole eukaryotic cells (Fiala, 2005;Fontana et al, 2008;Romao et al, 2008), metal replication after freeze-fracture or freezeetching that provides planar views of the internal organization of membranes (Cabezas and Risco, 2006;Risco and Pinto da Silva, 1998;Severs, 2007;Severs and Robenek, 2008), and electron tomography (ET), a process involving rotation of the specimen in the electron beam, capturing images at incremental rotations, and using the images for 3D reconstruction. Cellular electron tomography (ET) is a powerful technique that can provide molecular resolution of cell ultra-structure if combined with "close-to-native" preservation of biological samples (Lucić et al, 2005).…”

A primer on resolving the nanoscale structure of the plasma membrane with light and electron microscopy

“…Platinum replica EM is still the gold standard for visualizing the dense protein-based structure of the plasma membrane and even deep into the cell with freeze-fracture techniques ( Meier and Beckmann, 2018 ). Other exotic metals or carbon itself might produce even better resolution ( Cabezas and Risco, 2006 ; Krystofiak et al, 2019 ). Furthermore, when metals are evaporated on the sample from one side, the metal shadow cast by the height of the sample produces a contrast effect that makes the replicas appear 3-D ( Williams and Wyckoff, 1946 ).…”