Seeking Membranes: Positive-Strand RNA Virus Replication Complexes

How much do we really understand about how +RNA viruses usurp and transform the intracellular architecture of host cells when they replicate

Autophagy: A Forty-Year Search for a Missing Membrane Source

Autophagy is central to diverse biological processes in eukaryotes including animal development and cellular survival, and also to neurodegenerative diseases, but the origin of the membranes that make up autophagic vesicles is unknown

Bridging fluorescence microscopy and electron microscopy

Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major breakthrough in fluorescence microscopy in biology is the ability to follow specific targets on or in living cells, revealing dynamic localization and/or function of target molecules. One of the inherent limitations of fluorescence microscopy is the resolution. Several efforts are undertaken to overcome this limit. The traditional and most well-known way to achieve higher resolution imaging is by electron microscopy. Moreover, electron microscopy reveals organelles, membranes, macromolecules, and thus aids in the understanding of cellular complexity and localization of molecules of interest in relation to other structures. With the new probe development, a solid bridge between fluorescence microscopy and electron microscopy is being built, even leading to correlative imaging. This connection provides several benefits, both scientifically as well as practically. Here, I summarize recent developments in bridging microscopy

3-D Ultrastructure of O. tauri: Electron Cryotomography of an Entire Eukaryotic Cell

The hallmark of eukaryotic cells is their segregation of key biological functions into discrete, membrane-bound organelles. Creating accurate models of their ultrastructural complexity has been difficult in part because of the limited resolution of light microscopy and the artifact-prone nature of conventional electron microscopy. Here we explored the potential of the emerging technology electron cryotomography to produce three-dimensional images of an entire eukaryotic cell in a near-native state. Ostreococcus tauri was chosen as the specimen because as a unicellular picoplankton with just one copy of each organelle, it is the smallest known eukaryote and was therefore likely to yield the highest resolution images. Whole cells were imaged at various stages of the cell cycle, yielding 3-D reconstructions of complete chloroplasts, mitochondria, endoplasmic reticula, Golgi bodies, peroxisomes, microtubules, and putative ribosome distributions in-situ. Surprisingly, the nucleus was seen to open long before mitosis, and while one microtubule (or two in some predivisional cells) was consistently present, no mitotic spindle was ever observed, prompting speculation that a single microtubule might be sufficient to segregate multiple chromosomes

Contrast-tuneable microscopy for single-shot real-time imaging

A novel real image in-line laser holography enables a tuneable image contrast, edge sharpness, and visualization of sub-wavelength structures, using a simple pair of filters and large-diameter lenses that can incorporate higher-order scattered light. Demonstrated also are the accuracy in object sizing and the ease of imaging along the focal depth, based on a single-shot imaging via holographic principle. In addition, the use of broad, collimated laser beam for irradiation has led to a wider field of view, making it particularly useful for an extensive monitoring of, and sweeping search for, cells and microbial colonies and for the real-time imaging of cancer-cell dynamics

Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes

<p>Abstract</p> <p>Background</p> <p>Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered.</p> <p>Results</p> <p>Here, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination) of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching.</p> <p>Conclusions</p> <p>We demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B), medium (gap junction channel protein connexin 43), and high (α-tubulin; clathrin light chain) dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion methods, this technique promises to further increase the general usability of photoconvertible PAFPs to track the dynamic movement of cells and proteins over time.</p

Viral detection by electron microscopy: past, present and future.

International audienceViruses are very small and most of them can be seen only by TEM (transmission electron microscopy). TEM has therefore made a major contribution to virology, including the discovery of many viruses, the diagnosis of various viral infections and fundamental investigations of virus-host cell interactions. However, TEM has gradually been replaced by more sensitive methods, such as the PCR. In research, new imaging techniques for fluorescence light microscopy have supplanted TEM, making it possible to study live cells and dynamic interactions between viruses and the cellular machinery. Nevertheless, TEM remains essential for certain aspects of virology. It is very useful for the initial identification of unknown viral agents in particular outbreaks, and is recommended by regulatory agencies for investigation of the viral safety of biological products and/or the cells used to produce them. In research, only TEM has a resolution sufficiently high for discrimination between aggregated viral proteins and structured viral particles. Recent examples of different viral assembly models illustrate the value of TEM for improving our understanding of virus-cell interactions

Labeling of Multiple HIV-1 Proteins with the Biarsenical-Tetracysteine System

Due to its small size and versatility, the biarsenical-tetracysteine system is an attractive way to label viral proteins for live cell imaging. This study describes the genetic labeling of the human immunodeficiency virus type 1 (HIV-1) structural proteins (matrix, capsid and nucleocapsid), enzymes (protease, reverse transcriptase, RNAse H and integrase) and envelope glycoprotein 120 with a tetracysteine tag in the context of a full-length virus. We measure the impact of these modifications on the natural virus infection and, most importantly, present the first infectious HIV-1 construct containing a fluorescently-labeled nucleocapsid protein. Furthermore, due to the high background levels normally associated with the labeling of tetracysteine-tagged proteins we have also optimized a metabolic labeling system that produces infectious virus containing the natural envelope glycoproteins and specifically labeled tetracysteine-tagged proteins that can easily be detected after virus infection of T-lymphocytes. This approach can be adapted to other viral systems for the visualization of the interplay between virus and host cell during infection