Published On: Tue, Jan 29th, 2019

Plasmodium centrin PbCEN-4 localizes to the putative MTOC and is dispensable for malaria parasite proliferation [RESEARCH ARTICLE]

INTRODUCTION

Plasmodium species, the causative agents of malaria, undergo two types of atypical closed mitotic cell division during their complex life cycles. The first is during schizogony within hepatocytes and erythrocytes in the vertebrate host and sporogony within the oocyst attached to the mosquito midgut. The second is an endoreduplicative process during the sexual stage, where a rapid threefold DNA replication within 8–10 min results in the formation of eight male gametes within the mosquito midgut (Sinden, 1991; Arnot et al., 2011; Gerald et al., 2011; Guttery et al., 2012). Closed mitosis in the Plasmodium parasite is characterized by multiple rounds of DNA replication and asynchronous nuclear division, without dissolution of the nuclear membrane. This karyokinesis proceeds in the absence of concomitant cytokinesis, forming a multinucleate syncytium, and cytokinesis only occurs once the multiple rounds of nuclear division are complete. In erythrocytes, the final round of DNA replication and segregation is synchronous and coordinated with final daughter merozoite assembly (Arnot et al., 2011).

In several eukaryotes, particularly humans, plants and yeast, the mechanisms controlling nuclear and cell division, and the molecules involved are well studied. Progression through mitosis requires many proteins, including centrins, cyclins, protein kinases, phosphatases and the microtubule organizing centre (MTOC) (Sluder, 2005; Harashima et al., 2013). Previous studies in Plasmodium have identified key genes whose products regulate the different phases of the life cycle and the distinctive transitions between them. In particular, systematic gene knockout screens have highlighted the importance of several protein kinases and phosphatases as likely essential for erythrocytic schizogony and for sexual stage development (Tewari et al., 2010; Solyakov et al., 2011; Guttery et al., 2014; Gomes et al., 2015). Most recently, Pfcrk4 and Pfcyclin1 have been shown to be essential for P. falciparum asexual blood stage proliferation (Ganter et al., 2017; Robbins et al., 2017) with Pfcyclin1 involved in cytokinesis (Robbins et al., 2017). However many cell cycle regulators are absent from the parasite, including classical cyclins, various anaphase-promoting complex (APC) molecules, CDC25 and CDC14 (Guttery et al., 2014; Roques et al., 2015; Wall et al., 2018).

The MTOC is the centrosome in mammalian cells, the spindle pole body (SPB) embedded in the nuclear membrane in yeast, and the basal body in flagellated and ciliated cells (Zhang and He, 2012; Seybold and Schiebel, 2013; Bornens and Gönczy, 2014). In Plasmodium, as in budding yeast, mitosis is closed and occurs without dissolution of the nuclear envelope. The putative MTOC (as it will be named here) resembles the yeast SPB, serving as the anchor for the mitotic spindles, and has also been referred to as the ‘centriolar plaque’, despite clear centriolar structures not having been observed (Arnot et al., 2011; Gerald et al., 2011). During the cell cycle, the MTOC has to be duplicated and separated to allow correct progression through mitosis, and centrin proteins are among the main orchestrators of these events (Azimzadeh and Bornens, 2007; Mardin and Schiebel, 2012).

Centrins (also known as caltractrins) belong to a family of calmodulin-like calcium-binding phosphoproteins containing four EF-hand domains (Wright et al., 1985; Salisbury, 1995; Zhang and He, 2012). Centrin was initially identified at the centrioles in the green alga Chlamydomonas reinhardtii (Wright et al., 1985), and is associated with the MTOC (Bornens and Gönczy, 2014) in the pericentriolar material (PCM), as well as at the distal lumen of centrioles (Wright et al., 1985; Baron et al., 1992; Paoletti et al., 1996). Four centrins have been described in mammals (Huang et al., 1988) and grouped into those belonging to a subfamily related to budding yeast CDC31 (centrin 3) or those with most homology to the C. reinhardtii centrin (centrin 1, 2 and 4) (Middendorp et al., 1997, 2000).

There have been some functional studies of centrins in protozoa. In the ciliate Tetrahymena, CEN-1 and -2 function in basal body orientation, maintenance, and separation (Vonderfecht et al., 2012). In the flagellated Kinetoplastida group, e.g. Leishmania donovani, CEN-1 is involved in the duplication of basal bodies in amastigote stages, and in Trypanosoma brucei, deletion of cen-1, -2 and -3 affected organelle segregation and potentially inhibited cytokinesis (Selvapandiyan et al., 2012). In Apicomplexa, Toxoplasma gondii has three centrins, which have been localized to an MTOC-like structure during cell division (Hu et al., 2006; Hu, 2008). Four centrin-like proteins have been identified in P. falciparum (PfCEN-1 to -4) and phylogenetic analysis showed that CEN-2 and -4 are members of an alveolate-specific subgroup (Mahajan et al., 2008). Transcription and protein expression profiles demonstrated that these centrins are differentially expressed at different stages of the life cycle (Hall et al., 2005; Mahajan et al., 2008). By microscopy and co-localization with specific MTOC markers, PfCEN-2 and PfCEN-3 were observed to be associated with the putative MTOC, close to the nucleus in the sporozoite and in asexual stages for PfCEN-2 and PfCEN-3, respectively (Mahajan et al., 2008).

Here, using live cell imaging, we characterize the dynamics of an alveolate-specific Plasmodium centrin (CEN-4) in the rodent malaria parasite P. berghei, to examine atypical mitotic division in all three phases of asexual replication. PbCEN-4 shows a distinct temporal profile – it is cytoplasmic preceding replication, but then locates to discrete foci that correspond to the putative MTOC structure during nuclear division. CEN-4 forms a part of a multi-centrin protein complex. However, the deletion of cen-4 has no effect on parasite proliferation and this redundancy is not due to compensation by the increased expression of one of the other centrin genes.

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