An extended DNA-free intranuclear compartment organizes centrosomal microtubules in Plasmodium falciparum

Abstract

Rapid proliferation of Plasmodium falciparum parasites in human red blood cells is the cause of malaria and is underpinned by an unconventional cell division mode, called schizogony. Contrary to model organisms, P. falciparum replicates by multiple rounds of closed and asynchronous nuclear divisions that are not interrupted by cytokinesis. Organization and dynamics of the critical nuclear division factors are, however, poorly understood. Centriolar plaques, the centrosomes of P. falciparum, are important regulators of division and serve as microtubule organizing centers. Early microscopy studies reveal an acentriolar, amorphous structure although its detailed organization remains elusive. Intranuclear microtubules mediate chromosome segregation, but the small size of parasite nuclei has precluded detailed analysis of their arrangement by classical fluorescence microscopy. We apply our recently developed STED super-resolution microscopy protocol and live cell imaging to describe the reconfiguration of microtubules during schizogony. Analysis of centrin, nuclear pore, and microtubule positioning reveals a bipartite organization of the centriolar plaque. While centrin is extranuclear, we confirm by correlative light and electron tomography that microtubules are nucleated in a previously unknown and extended intranuclear compartment, which is devoid of chromatin. This study enables us to build a working model of the organization of an unconventional centrosome and better understand the diversity of eukaryotic cell division modes.

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

“…1C-E). On the nuclear side this might correspond to the future acentriolar plaques that stain negative for DNA, as observed in asexual blood stages [14]. Upon transition to stage V, the microtubule network progressively disassembled and the tips sequentially became more rounded.…”

“…The structural organisation of the acentriolar plaque has recently been refined in asexually replicating blood stages [14,41] that undergo endomitosis but without axoneme nucleation. In these stages, the acentriolar plaque also shows a bipartite organisation with an extranuclear region containing centrin that is linked through a nuclear pore to an intranuclear chromatin-free region harbouring microtubule nucleation sites.…”

“…Available data on the structural organisation and the biogenesis of the centriolar plaque is very limited. In asexual stages, the centriolar plaque shows a bipartite organization, with an extranuclear region containing centrin and an intranuclear DNA-free region harbouring microtubule nucleation sites [14]. It appears as an electron-dense area that neither shows a centriole nor any other known distinct structures such as the yeast spindle pole body, the Dyctiostelium nucleus associated body or the diatom microtubule centre.…”

How Many Is Enough? - Challenges of Multinucleated Cell Division in Malaria Parasites

“…A factor that more overtly limits nuclear number is the centrosome (Arnot et al, 2011). Each round of division precludes centrosome duplication to form the poles between which chromosomes will be segregated (Simon et al, 2021). Transient localization of the aurora kinase PfArk1 to the centrosome just prior to its duplication has been described (Reininger et al, 2011).…”

Structural and Functional Insights into the Microtubule Organizing Centers of <em>Toxoplasma gondii</em> and <em>Plasmodium spp</em>.

“…However, recent insights from UExM has revealed that the CP is in fact extranuclear, and is not embedded in the nuclear envelope at any point during mitosis. UExM, STED microscopy, and correlative light electron microscopy (CLEM) has further revealed that nucleation of microtubules resides within a chromatin free subcompartment within the nucleus, distal to the location of Centrin, a CP component, and proximal to a nuclear pore marker (Nup313) (Simon et al, 2021). This study has clearly established that whereas the CP is at the poles of the mitotic spindle, the CP is extranuclear, and MT nucleation capacity resides at a protein-dense region internal to the nucleus, but devoid of chromatin (Fig.…”