In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular Plasmodium falciparum parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10-30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress. malaria | egress | electron tomography | soft X-ray microscopy | electron energy loss spectroscopy T he major cause of severe human malaria is Plasmodium falciparum, and its asexual blood cycle is the source of all clinical disease (1). Egress is an important step in the blood life cycle, as it allows daughter merozoites produced by intracellular parasite replication to escape and invade new erythrocytes, thereby continuing and amplifying the infection. Merozoites develop within a parasitophorous vacuole (PV), a membrane-bound compartment that forms during invasion (2-4), so the daughter parasites have two compartments to escape (5, 6).Blood-stage malaria parasites replicate by schizogony, in which several rounds of nuclear division form a multinucleated syncytium called a schizont. Individual merozoites are then produced by an unusual form of cytokinesis called budding or segmentation, which involves invagination of the single plasma membrane of the schizont. Minutes before egress, the segmented schizont suddenly transforms from an irregular to a relatively symmetrical structure with the merozoites arranged around the central digestive vacuole (5). This process, referred to as "flower formation" or rounding up, is usually accompanied by noticeable swelling of the PV and apparent shrinkage of the host cell (4, 5, 7-9). The first membrane to rupture at egress is the parasitophorous vacuole membrane (PVM) (5,6,8). When the PV does not occupy the entire infected cell, the individual merozoites can be seen to be expelled into the blood cell cytosol seconds before they escape fr...
Cryo-soft X-ray tomography (cryo-SXT) is a powerful imaging technique that can extract ultrastructural information from whole, unstained mammalian cells as close to the living state as possible. Subcellular organelles including the nucleus, the Golgi apparatus and mitochondria have been identified by morphology alone, due to the similarity in contrast to transmission electron micrographs. In this study, we used cryo-SXT to image endosomes and autophagosomes, organelles that are particularly susceptible to chemical fixation artefacts during sample preparation for electron microscopy. We used two approaches to identify these compartments. For early and recycling endosomes, which are accessible to externally-loaded markers, we used an anti-transferrin receptor antibody conjugated to 10 nm gold particles. For autophagosomes, which are not accessible to externally-applied markers, we developed a correlative cryo-fluorescence and cryo-SXT workflow (cryo-CLXM) to localise GFP-LC3 and RFP-Atg9. We used a stand-alone cryo-fluorescence stage in the home laboratory to localise the cloned fluorophores, followed by cryo-soft X-ray tomography at the synchrotron to analyse cellular ultrastructure. We mapped the 3D ultrastructure of the endocytic and autophagic structures, and discovered clusters of omegasomes arising from ‘hotspots’ on the ER. Thus, immunogold markers and cryo-CLXM can be used to analyse cellular processes that are inaccessible using other imaging modalities.
Cryo-soft X-ray tomography is an imaging technique that addresses the need for mesoscale imaging of cellular ultrastructure of relatively thick samples without the need for staining or chemical modification. It allows the imaging of cellular ultrastructure to a resolution of 25–40 nm and can be used in correlation with other imaging modalities, such as electron tomography and fluorescence microscopy, to further enhance the information content derived from biological samples. An overview of the technique, discussion of sample suitability and information about sample preparation, data collection and data analysis is presented here. Recent developments and future outlook are also discussed.
With modern detectors and synchrotron sources, it is now routine to collect complete data sets in 10–30 min. To make the most efficient use of these resources, it is desirable to automate the collection and processing of the diffraction data, ideally to a level at which multiple data sets can be acquired without any intervention. A scheme is described to allow fully automated data collection and processing. The design is modular, so that it can easily be interfaced with different beamline‐control programs and different data‐processing programs. An expert system provides a communication path between the data‐processing software and the beamline‐control software and takes decisions about the data collection based on project information provided by the user and experimental data provided by the data‐processing program.
Segmentation of biological volumes is a crucial step needed to fully analyse their scientific content. Not having access to convenient tools with which to segment or annotate the data means many biological volumes remain under-utilised. Automatic segmentation of biological volumes is still a very challenging research field, and current methods usually require a large amount of manually-produced training data to deliver a high-quality segmentation. However, the complex appearance of cellular features and the high variance from one sample to another, along with the time-consuming work of manually labelling complete volumes, makes the required training data very scarce or non-existent. Thus, fully automatic approaches are often infeasible for many practical applications. With the aim of unifying the segmentation power of automatic approaches with the user expertise and ability to manually annotate biological samples, we present a new workbench named SuRVoS (Super-Region Volume Segmentation). Within this software, a volume to be segmented is first partitioned into hierarchical segmentation layers (named Super-Regions) and is then interactively segmented with the user's knowledge input in the form of training annotations. SuRVoS first learns from and then extends user inputs to the rest of the volume, while using Super-Regions for quicker and easier segmentation than when using a voxel grid. These benefits are especially noticeable on noisy, low-dose, biological datasets.
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