Expansion Microscopy Reveals Plasmodium falciparum Blood-Stage Parasites Undergo Anaphase with A Chromatin Bridge in the Absence of Mini-Chromosome Maintenance Complex Binding Protein

Abstract

The malaria parasite Plasmodium falciparum undergoes closed mitosis, which occurs within an intact nuclear envelope, and differs significantly from its human host. Mitosis is underpinned by the dynamics of microtubules and the nuclear envelope. To date, our ability to study P. falciparum mitosis by microscopy has been hindered by the small size of the P. falciparum nuclei. Ultrastructure expansion microscopy (U-ExM) has recently been developed for P. falciparum, allowing the visualization of mitosis at the individual nucleus level. Using U-ExM, three intranuclear microtubule structures are observed: hemispindles, mitotic spindles, and interpolar spindles. A previous study demonstrated that the mini-chromosome maintenance complex binding-protein (MCMBP) depletion caused abnormal nuclear morphology and microtubule defects. To investigate the role of microtubules following MCMBP depletion and study the nuclear envelope in these parasites, we developed the first nuclear stain enabled by U-ExM in P. falciparum. MCMBP-deficient parasites show aberrant hemispindles and mitotic spindles. Moreover, anaphase chromatin bridges and individual nuclei containing multiple microtubule structures were observed following MCMBP knockdown. Collectively, this study refines our understanding of MCMBP-deficient parasites and highlights the utility of U-ExM coupled with a nuclear envelope stain for studying mitosis in P. falciparum.

Expansion microscopy of Plasmodium gametocytes reveals the molecular architecture of a bipartite microtubule organisation centre coordinating mitosis with axoneme assembly

“…In addition, we found Pan-ExM to be experimentally a lot more demanding than U-ExM. Altogether these observations indicate that the combination of U-ExM with bulk proteome labelling represents an accessible approach to study the ultrastructural context of Plasmodium cells with a conventional confocal microscope and will nicely complement electron microscopy or live-cell imaging studies [ 46 , 47 ].…”

IMC10 and LMF1 mediate membrane contact between the mitochondrion and the inner membrane complex in Toxoplasma gondii

“…Ultrastructure Expansion Microscopy (U-ExM) was performed as described (Liffner and Absalon, 2021) with the following modification: the parasites were seeded in an HFF monolayer grown on glass coverslips in 24 well plates. Primary antibodies used are rabbit anti-HA (C29F4 Cell Signalling 1:100 dilution) mouse anti-Myc (9B11 Cell Signalling 1:500).…”

The dually localized EF-hand domain-containing protein TgEFP1 regulates the lytic cycle of Toxoplasma gondii

“…Host cells were seeded on 15 mm coverslips in a 24-well plate, infected with parasites for 18 hours, and fixed. After fixation, ultrastructure expansion microscopy protocol was used to expand and image samples as outlined in a previous study [18]. Level of expansion was determined by measuring the diameter of the gel after expansion and comparing it to the diameter of the coverslip.…”

“…As to more precisely observe the localization and dynamics of TgEFP1 in intracellular parasites, we employed Expansion Microscopy (U-ExM), which allows for higher resolution of cellular structures and protein localization [18]. Intracellular parasites were treated according to the U-ExM protocol to expand samples [18] and stained for TgEFP1-HA and TgGRA5 using antibodies and for protein density using NHS-ester.…”

“…As to more precisely observe the localization and dynamics of TgEFP1 in intracellular parasites, we employed Expansion Microscopy (U-ExM), which allows for higher resolution of cellular structures and protein localization [18]. Intracellular parasites were treated according to the U-ExM protocol to expand samples [18] and stained for TgEFP1-HA and TgGRA5 using antibodies and for protein density using NHS-ester. Following expansion the size of the samples increased five-fold, which revealed a high level of detail in not only the localization of TgEFP1-HA but also the dynamic fragmentation and biogenesis of the compartment within which it localizes (Fig.…”