Comparative 3D ultrastructure ofPlasmodium falciparumgametocytes

Abstract

Despite the enormous significance of malaria parasites for global health, some basic features of their ultrastructure remain obscure. In this study, we apply high-resolution volumetric electron microscopy to examine and compare the ultrastructure of Plasmodium falciparum gametocytes of both genders and in different stages of development as well as the more intensively studied asexual blood stages revisiting previously described phenomena in 3D. In doing so, we challenge the widely accepted notion of a single mitochondrion by demonstrating the presence of multiple mitochondria in gametocytes. We also provide evidence for a gametocyte-specific cytostome variant. Furthermore, we generate, among other organelles, the first 3D reconstructions of endoplasmic reticulum (ER), Golgi apparatus, and exported structures in gametocytes. Assessing interconnectivity between organelles, we find frequent structural appositions between the nucleus, mitochondria, and apicoplast. We provide evidence that the ER is a promiscuous interactor with numerous organelles and the trilaminar membrane of the gametocyte. Public availability of these volumetric electron microscopy resources of wild-type asexual and sexual blood-stage malaria parasites will facilitate reinterrogation of this global dataset with different research questions and expertise. Taken together, we reconstruct the 3D ultrastructure of P. falciparum gametocytes in high detail and shed light on the unique organellar biology of these deadly parasites.

Detailing organelle division and segregation inPlasmodium falciparum

“…While expansion microscopy is currently not feasible with MitoRed, addition of a linear epitope tag would make this marker compatible with the required denaturation step. In line with our earlier observations, we demonstrated multiple mitochondria in gametocyte stages 19 . As discussed by Evers et al, there are several possible reasons for the emergence of multiple mitochondria in gametocytes, such as adaptation to a metabolically varied environment, distribution mechanism of mtDNA, or management of reactive oxygen species.…”

“…Parasites were stained for α tubulin to distinguish male and female gametocytes in stage IV and V. In stage II and III gametocytes, the mitochondrion appears as a small knot that increases slightly in size when gametocytes become more mature (Figure 2A). This is consistent with our FIB-SEM data 19 . Evers et al also showed that gametocytes have multiple mitochondria already from early gametocyte development onwards.…”

“…During schizogony, the mitochondrion and apicoplast show different moments of close association. Prior to apicoplast division, the mitochondrion and apicoplast have several apposition sites, which have also been described by Evers et al 19 (Figure S8A and S8B). It remains unclear if these close associations represent true membrane contact sites that facilitate the exchange of metabolites or lipids between the organelles, or if these are merely random due to the limited space in the parasite.…”

“…Surprisingly, both MitoTracker dyes showed a discontinuous, punctuated staining pattern (Figure 1A). FIB-SEM studies have confirmed the prevailing notion that the mitochondrion is a single, branched network during these schizont stages 19 . While this observation may arise from crosslinking of the MitoTracker dyes to specific proteins and aggregations thereof resulting from the fixation process, we concluded that the punctuated staining pattern is likely an artifact and consequently limits our ability to dissect and visualize the process mitochondrial fission.…”

Plasmodium falciparum ZIP1 Is a Zinc-Selective Transporter with Stage-Dependent Targeting to the Apicoplast and Plasma Membrane in Erythrocytic Parasites

“…Clear Pf ZIP1 signals were detected at the gametocyte plasma membrane (Figure A). Male and female gametocytes were differentiated using α-tubulin as the marker. , Pf ZIP1 was at the plasma membrane, adjacent to prominent tubulin signals at the periphery of male gametocytes through stage III to V (Figure B). Pf ZIP1 was localized to the parasite plasma membrane in both male and female gametocytes.…”