48 research outputs found
Structural studies demonstrating a bacteriophage-like replication cycle of the eukaryote-infecting \u3ci\u3eParamecium bursaria\u3c/i\u3e chlorella virus-1
A fundamental stage in viral infection is the internalization of viral genomes in host cells. Although extensively studied, the mechanisms and factors responsible for the genome internalization process remain poorly understood. Here we report our observations, derived from diverse imaging methods on genome internalization of the large dsDNA Paramecium bursaria chlorella virus-1 (PBCV-1). Our studies reveal that early infection stages of this eukaryotic- infecting virus occurs by a bacteriophage-like pathway, whereby PBCV-1 generates a hole in the host cell wall and ejects its dsDNA genome in a linear, base-pair-by-base-pair process, through a membrane tunnel generated by the fusion of the virus internal membrane with the host membrane. Furthermore, our results imply that PBCV-1 DNA condensation that occurs shortly after infection probably plays a role in genome internalization, as hypothesized for the infection of some bacteriophages. The subsequent perforation of the host photosynthetic membranes presumably enables trafficking of viral genomes towards host nuclei. Previous studies established that at late infection stages PBCV-1 generates cytoplasmic organelles, termed viral factories, where viral assembly takes place, a feature characteristic of many large dsDNA viruses that infect eukaryotic organisms. PBCV-1 thus appears to combine a bacteriophage-like mechanism during early infection stages with a eukaryotic-like infection pathway in its late replication cycle.
Author summary -- Although extensively studied, the mechanisms responsible for internalization of viral genomes into their host cells remain unclear. A particularly interesting case of genome release and internalization is provided by the large Paramecium bursaria chlorella virus-1 (PBCV-1), which infects unicellular eukaryotic photosynthetic chlorella cells. In order to release its long dsDNA genome and to enable its translocation to the host nucleus, PBCV-1 must overcome multiple hurdles, including a thick host cell wall and multilayered chloroplast membranes that surround the host cytoplasm. Our observations indicate that these obstacles are dealt with perforations of the host wall, the host cellular membrane, and the host photosynthetic membranes by viral-encoded proteins. Furthermore, our results highlight a bacteriophage-like nature of early PBCV-1 infection stages, thus implying that this virus uniquely combines bacteriophage-like and eukaryotic-like pathways to accomplish its replication cycle
On-site secretory vesicle delivery drives filamentous growth in the fungal pathogen Candida albicans
International audienceCandida albicans is an opportunistic fungal pathogen that colonises the skin as well as genital and intestinal mucosa of most healthy individuals. The ability of C. albicans to switch between different morphological states, for example, from an ellipsoid yeast form to a highly polarised, hyphal form, contributes to its success as a pathogen. In highly polarised tip-growing cells such as neurons, pollen tubes, and filamentous fungi, delivery of membrane and cargo to the filament apex is achieved by long-range delivery of secretory vesicles tethered to motors moving along cytoskeletal cables that extend towards the growing tip. To investigate whether such a mechanism is also critical for C. albicans filamentous growth, we studied the dynamics and organisation of the C. albicans secretory pathway using live cell imaging and three-dimensional electron microscopy. We demonstrate that the secretory pathway is organised in distinct domains, including endoplasmic reticulum membrane sheets that extend along the length of the hyphal filament, a sub-apical zone exhibiting distinct membrane structures and dynamics and a Spitzenkörper comprised of uniformly sized secretory vesicles. Our results indicate that the organisation of the secretory pathway in C. albicans likely facilitates short-range "on-site" secretory vesicle delivery, in contrast to filamentous fungi and many highly polarised cells
Virusâhost interactions: insights from the replication cycle of the large \u3ci\u3eParamecium bursaria\u3c/i\u3e chlorella virus
The increasing interest in cytoplasmic factories generated by eukaryotic-infecting viruses stems from the realization that these highly ordered assemblies may contribute fundamental novel insights to the functional significance of order in cellular biology. Here, we report the formation process and structural features of the cytoplasmic factories of the large dsDNA virus Paramecium bursaria chlorella virus 1 (PBCV-1). By combining diverse imaging techniques, including scanning transmission electron microscopy tomography and focused ion beam technologies, we show that the architecture and mode of formation of PBCV-1 factories are significantly different from those generated by their evolutionary relatives Vaccinia and Mimivirus. Specifically, PBCV-1 factories consist of a network of single membrane bilayers acting as capsid templates in the central region, and viral genomes spread throughout the host cytoplasm but excluded from the membranecontaining sites. In sharp contrast, factories generated by Mimivirus have viral genomes in their core, with membrane biogenesis region located at their periphery. Yet, all viral factories appear to share structural features that are essential for their function. In addition, our studies support the notion that PBCV-1 infection, which was recently reported to result in significant pathological outcomes in humans andmice, proceeds througha bacteriophage -like infection pathway
Defect-Free Carbon Nanotube Coils
Carbon nanotubes are promising building blocks for various nanoelectronic
components. A highly desirable geometry for such applications is a coil.
However, coiled nanotube structures reported so far were inherently defective
or had no free ends accessible for contacting. Here we demonstrate the
spontaneous self-coiling of single-wall carbon nanotubes into defect-free coils
of up to more than 70 turns with identical diameter and chirality, and free
ends. We characterize the structure, formation mechanism, and electrical
properties of these coils by different microscopies, molecular dynamics
simulations, Raman spectroscopy, and electrical and magnetic measurements. The
coils are highly conductive, as expected for defect-free carbon nanotubes, but
adjacent nanotube segments in the coil are more highly coupled than in regular
bundles of single-wall carbon nanotubes, owing to their perfect crystal
momentum matching, which enables tunneling between the turns. Although this
behavior does not yet enable the performance of these nanotube coils as
inductive devices, it does point a clear path for their realization. Hence,
this study represents a major step toward the production of many different
nanotube coil devices, including inductors, electromagnets, transformers, and
dynamos
The Tomato MIXTA-like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly
In-situ fiducial markers for 3D correlative cryo- fluorescence and FIB-SEM imaging
Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), which offer high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. However, correlative light and electron microscopy (CLEM) workflows combining cryo- fluorescence microscopy (cryo-FM) and FIB-SEM are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in-situ fiducial markers for correlating cryo- FM and FIB-SEM datasets, and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization and mineral deposits in cells
Structural studies demonstrating a bacteriophage-like replication cycle of the eukaryote-infecting \u3ci\u3eParamecium bursaria\u3c/i\u3e chlorella virus-1
A fundamental stage in viral infection is the internalization of viral genomes in host cells. Although extensively studied, the mechanisms and factors responsible for the genome internalization process remain poorly understood. Here we report our observations, derived from diverse imaging methods on genome internalization of the large dsDNA Paramecium bursaria chlorella virus-1 (PBCV-1). Our studies reveal that early infection stages of this eukaryotic- infecting virus occurs by a bacteriophage-like pathway, whereby PBCV-1 generates a hole in the host cell wall and ejects its dsDNA genome in a linear, base-pair-by-base-pair process, through a membrane tunnel generated by the fusion of the virus internal membrane with the host membrane. Furthermore, our results imply that PBCV-1 DNA condensation that occurs shortly after infection probably plays a role in genome internalization, as hypothesized for the infection of some bacteriophages. The subsequent perforation of the host photosynthetic membranes presumably enables trafficking of viral genomes towards host nuclei. Previous studies established that at late infection stages PBCV-1 generates cytoplasmic organelles, termed viral factories, where viral assembly takes place, a feature characteristic of many large dsDNA viruses that infect eukaryotic organisms. PBCV-1 thus appears to combine a bacteriophage-like mechanism during early infection stages with a eukaryotic-like infection pathway in its late replication cycle.
Author summary -- Although extensively studied, the mechanisms responsible for internalization of viral genomes into their host cells remain unclear. A particularly interesting case of genome release and internalization is provided by the large Paramecium bursaria chlorella virus-1 (PBCV-1), which infects unicellular eukaryotic photosynthetic chlorella cells. In order to release its long dsDNA genome and to enable its translocation to the host nucleus, PBCV-1 must overcome multiple hurdles, including a thick host cell wall and multilayered chloroplast membranes that surround the host cytoplasm. Our observations indicate that these obstacles are dealt with perforations of the host wall, the host cellular membrane, and the host photosynthetic membranes by viral-encoded proteins. Furthermore, our results highlight a bacteriophage-like nature of early PBCV-1 infection stages, thus implying that this virus uniquely combines bacteriophage-like and eukaryotic-like pathways to accomplish its replication cycle
Sample preparation and image registration for correlative cryo-FM and cryo-FIB-SEM of plunge-frozen mammalian cells
International audienceWe recently demonstrated how lipid droplets can serve as in situ fiducials for correlating cryo-fluorescence microscopy (cryo-FM) and cryo-focused ion beam scanning electron microscopy (cryo-FIB-SEM) datasets of mammalian cells grown on grids. Here we describe a step-by-step protocol for correlative cryo-FM and cryo-FIB-SEM, starting from sample preparation of C2C12 cell line, followed by imaging with cryo-FM and cryo-FIB-SEM. Finally, we detail how to perform the 3D-correlation with sub-micron accuracy. For complete details on the use and execution of this profile, please refer to Scher et al. (2021)
Three-dimensional correlative microscopy of the Drosophila female reproductive tract reveals modes of communication in seminal receptacle sperm storage
Abstract In many taxa, females store sperm in specialized storage organs. Most insect sperm storage organs have a tubular structure, typically consisting of a central lumen surrounded by epithelial cells. These specialized tubules perform the essential tasks of transporting sperm through the female reproductive tract and supporting long-term sperm survival and function. Little is known about the way in which female sperm storage organs provide an environment conducive to sperm survival. We address this using a combined light microscopy, micro computed tomography (microCT), and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) approach for high-resolution correlative three-dimensional imaging to advance our understanding of sperm-female interactions in Drosophila melanogaster. Using this multimodal approach, we were able to scan the lower female reproductive tract and distal portion of the seminal receptacle at low magnification, and to subsequently zoom in for further analysis on an ultrastructural level. Our findings highlight aspects of the way in which the seminal receptacle keeps sperm viable in the lumen, and set the stage for further studies. The methods developed are suitable not only for Drosophila but also for other organisms with soft, delicate tissues