Detailing organelle division and segregation in Plasmodium falciparum.

The malaria causing parasite, Plasmodium falciparum, replicates through a tightly orchestrated process termed schizogony, where approximately 32 daughter parasites are formed in a single infected red blood cell and thousands of daughter cells in mosquito or liver stages. One-per-cell organelles, such as the mitochondrion and apicoplast, need to be properly divided and segregated to ensure a complete set of organelles per daughter parasites. Although this is highly essential, details about the processes and mechanisms involved remain unknown. We developed a new reporter parasite line that allows visualization of the mitochondrion in blood and mosquito stages. Using high-resolution 3D-imaging, we found that the mitochondrion orients in a cartwheel structure, prior to stepwise, non-geometric division during the last stage of schizogony. Analysis of focused ion beam scanning electron microscopy (FIB-SEM) data confirmed these mitochondrial division stages. Furthermore, these data allowed us to elucidate apicoplast division steps, highlighted its close association with the mitochondrion, and showed putative roles of the centriolar plaques (CPs) in apicoplast segregation. These observations form the foundation for a new detailed mechanistic model of mitochondrial and apicoplast division and segregation during P. falciparum schizogony and pave the way for future studies into the proteins and protein complexes involved in organelle division and segregation.

Comparative Clustering (CompaCt) of eukaryote complexomes identifies novel interactions and sheds light on protein complex evolution.

Complexome profiling allows large-scale, untargeted, and comprehensive characterization of protein complexes in a biological sample using a combined approach of separating intact protein complexes e.g., by native gel electrophoresis, followed by mass spectrometric analysis of the proteins in the resulting fractions. Over the last decade, its application has resulted in a large collection of complexome profiling datasets. While computational methods have been developed for the analysis of individual datasets, methods for large-scale comparative analysis of complexomes from multiple species are lacking. Here, we present Comparative Clustering (CompaCt), that performs fully automated integrative analysis of complexome profiling data from multiple species, enabling systematic characterization and comparison of complexomes. CompaCt implements a novel method for leveraging orthology in comparative analysis to allow systematic identification of conserved as well as taxon-specific elements of the analyzed complexomes. We applied this method to a collection of 53 complexome profiles spanning the major branches of the eukaryotes. We demonstrate the ability of CompaCt to robustly identify the composition of protein complexes, and show that integrated analysis of multiple datasets improves characterization of complexes from specific complexome profiles when compared to separate analyses. We identified novel candidate interactors and complexes in a number of species from previously analyzed datasets, like the emp24, the V-ATPase and mitochondrial ATP synthase complexes. Lastly, we demonstrate the utility of CompaCt for the automated large-scale characterization of the complexome of the mosquito Anopheles stephensi shedding light on the evolution of metazoan protein complexes. CompaCt is available from https://github.com/cmbi/compact-bio.

Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum.

Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.

CEDAR, an online resource for the reporting and exploration of complexome profiling data.

Complexome profiling is an emerging 'omics' approach that systematically interrogates the composition of protein complexes (the complexome) of a sample, by combining biochemical separation of native protein complexes with mass-spectrometry based quantitation proteomics. The resulting fractionation profiles hold comprehensive information on the abundance and composition of the complexome, and have a high potential for reuse by experimental and computational researchers. However, the lack of a central resource that provides access to these data, reported with adequate descriptions and an analysis tool, has limited their reuse. Therefore, we established the ComplexomE profiling DAta Resource (CEDAR, www3.cmbi.umcn.nl/cedar/), an openly accessible database for depositing and exploring mass spectrometry data from complexome profiling studies. Compatibility and reusability of the data is ensured by a standardized data and reporting format containing the "minimum information required for a complexome profiling experiment" (MIACE). The data can be accessed through a user-friendly web interface, as well as programmatically using the REST API portal. Additionally, all complexome profiles available on CEDAR can be inspected directly on the website with the profile viewer tool that allows the detection of correlated profiles and inference of potential complexes. In conclusion, CEDAR is a unique, growing and invaluable resource for the study of protein complex composition and dynamics across biological systems.

Complexome Profiling-Exploring Mitochondrial Protein Complexes in Health and Disease.

Complexome profiling (CP) is a state-of-the-art approach that combines separation of native proteins by electrophoresis, size exclusion chromatography or density gradient centrifugation with tandem mass spectrometry identification and quantification. Resulting data are computationally clustered to visualize the inventory, abundance and arrangement of multiprotein complexes in a biological sample. Since its formal introduction a decade ago, this method has been mostly applied to explore not only the composition and abundance of mitochondrial oxidative phosphorylation (OXPHOS) complexes in several species but also to identify novel protein interactors involved in their assembly, maintenance and functions. Besides, complexome profiling has been utilized to study the dynamics of OXPHOS complexes, as well as the impact of an increasing number of mutations leading to mitochondrial disorders or rearrangements of the whole mitochondrial complexome. Here, we summarize the major findings obtained by this approach; emphasize its advantages and current limitations; discuss multiple examples on how this tool could be applied to further investigate pathophysiological mechanisms and comment on the latest advances and opportunity areas to keep developing this methodology.

The baculovirus Ac108 protein is a per os infectivity factor and a component of the ODV entry complex.

Wild-type ODVs (Wt) have an intact ODV entry complex in their envelope and are orally infectious towards insect larvae (left panel). In the absence of Ac108 (mut ac108), the stable core is still present but nevertheless fails to form an entry complex, affecting the ODV oral infectivity (right panel). The components of the core complex are depicted in yellow and the loosely associated components are depicted in red. PIF7 is depicted in green as its affinity with the complex is currently not known.Baculoviruses orally infect insect larvae when they consume viral occlusion bodies (OBs). OBs consist of a crystalline protein matrix in which the infectious virus particles, the occlusion-derived viruses (ODVs), are embedded. The protein matrix dissolves in the alkaline environment of the insect's midgut lumen. The liberated ODVs can then infect midgut endothelial cells through the action of at least nine different ODV-envelope proteins, called per os infectivity factors (PIFs). These PIF proteins mediate ODV oral infectivity, but are not involved in the systemic spread of the infection by budded viruses (BVs). Eight of the known PIFs form a multimeric complex, named the ODV entry complex. In this study, we show for Autographa californica multiple nucleopolyhedrovirus that mutation of the ac108ORF abolishes the ODV oral infectivity, while production and infectivity of the BVs remains unaffected. Furthermore, repair of the ac108 mutant completely recovered oral infectivity. With an HA-tagged repair mutant, we were able to demonstrate by Western analysis that the Ac108 protein is a constituent of the ODV entry complex, where the formation was abolished in the absence of this protein. Based on these results, we conclude that ac108 encodes a per os infectivity factor (PIF9) that is also an essential constituent of the ODV entry complex.