3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography

Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Effects of cell motility and morphology on the rheology of algae suspensions

Algae have been proposed as a source of biofuels and high value chemical products, but if this potential is to be fully realised, it is crucial to understand the factors affecting the suspension rheology. Suspensions of three algae species, Tetraselmis chuii, Chlorella sp. and Phaeodactylum tricornutum, were sheared in a rotational rheometer in order to characterise their rheology and examine the effects of cell concentration, motility and morphology. The volume fraction ranged from 0.05 to 0.2, and the shear rate from 20 to 200 s−1. The rheology measurements are fitted to the Herschel-Bulkley model, and the intrinsic viscosity is estimated using both Einstein’s equation and the Krieger-Dougherty model, which are found to perform well for low concentrations. The intrinsic viscosity of T. chuii suspensions is shown not to be constant, but decreases with strain rate, indicating that the suspension viscosity is less sensitive to the cell concentration at high strain rates. The rate of decline is constant for strain rates below approximately 100 s−1, after which it continues to decline linearly, but at a slower rate. It is speculated that this transition at 100 s−1 is related to the appearance of flocculation at low strain rates. The effect of the cell motility on the rheology of T. chuii suspensions is investigated by comparing the rheology of motile and passive cells. The shear-thinning behaviour is absent and the effective viscosity is considerably lower for the passive cell suspensions, indicating that the motility of the T. chuii cells causes them to align to resist the flow. In contrast, the Chlorella sp. suspensions exhibit shear-thickening behaviour, which has not previously been reported. Finally, the influence of the effective aspect ratio on the cell suspensions is examined by comparing the intrinsic viscosity of all three species. The algal species with the largest aspect ratio, P. tricornutum, has the largest intrinsic viscosity, while the smallest aspect ratio strain, Chlorella sp., has the smallest viscosity. However, it is shown that the increase in viscosity of motile compared to non-motile T. chuii suspensions cannot be attributed to a change in the effective aspect ratio of individual cells due to the motion of the flagella alone