A guide to apicomplexan cell cycle: endodyogeny and schizogony

Abstract

This review focuses on recent advances in the field of cell division of eukaryotic pathogens of the Apicomplexa phylum. This group of pathogens is known for its diverse modes of cell division. We cover two topics, an organization and a regulation of the apicomplexan cell cycles, using models of Toxoplasma gondii tachyzoites and Plasmodium falciparum merozoites of the red blood stage. The review emphasizes differences and similarities between the conventional cell cycle of the parasite’s host cells and apicomplexan cell cycles, and between binary and multinuclear divisions of Apicomplexa. Although many factors play a role in cell cycle regulation, we concentrated on the central cell cycle regulators, cyclin-dependent kinase complexes, and their immediate effectors.

Conventional Cell Cycle

The cell cycle is a fundamental process of life, governing when and how the cell replicates itself. Alongside other changes that arose throughout evolution, the cell cycle program has undergone significant changes. To some extent, the current repertoire of cell cycle programs is a reflection of the variety in biological life. Generally, the cell cycle has many rules, some of which are strict, and some can be overruled without significant detriment. The strict fundamental rules are in place to ensure that genetic information is preserved and safely inherited in descendants. Take, for example, “DNA replication always precedes DNA segregation,” which, in other words, indicates that the order of cell cycle phases (see in Glossary) is absolute (Fig. 1A). The duplication and segregation of chromosomes are executed in four sequential cell cycle phases (Box 1) [1, 2]. Each phase has a precise set of tasks, whose timely completion is regulated by the checkpoints that operate at critical transitions (Box 1). Preparations for DNA replication take place during the G₁ period (Box 1), and chromosomes are duplicated during S-phase (Box 1). The G₂ period is devoted to correcting mistakes made during DNA replication and preparing for the segregation of duplicated chromosomes, which then takes place during mitosis (Box 1). Nuclear division, or karyokinesis, and the separation of daughter cells, or cytokinesis, both happen at the end of mitosis (Box 1).

Figure 1. Conventional and apicomplexan cell cycle organization.

(A) Circular representation of the conventional (on the left) and apicomplexan (on the right) cell cycles. The schematics show the location of the major checkpoints. The arrows indicate shortcuts of the cell cycle variants. Note that the illustrations depict a simplified apicomplexan cell cycle, and the actual organization is shown in panel B. (B) Cell cycle organization of the binary endodyogeny and multinuclear schizogony. The upper panel represents T. gondii endodyogeny. The alignment of the linear cell cycle progression and corresponding morphological transitions of the tachyzoite are shown. Red stop signs represent cell cycle checkpoints. Daughter cell scaffold (DCS) includes the inner membrane, apical and basal complexes, and subpellicular microtubules of the future daughter bud. The original work on the bud scaffold biogenesis presented at the 3rd virtual Toxoplasmosis symposium (2023) vividly described changing morphology of the growing buds as “open and “closed umbrella transitions”. The bottom panel shows schematics of the P. falciparum schizogony. The cell cycle phases are aligned with the morphotypes shown at the bottom. The first appearance of the daughter basal complexes is indicated.

Box 1. Conventional Cell Cycle Phases and Checkpoints.

Cells are programmed to perform cell division in the ordered series of phases: G₁, S, G₂, and mitosis. The critical phase transitions are safeguarded by three major checkpoints that ensure specific requirements are met before the cell advances to the next step.

G₁ phase:

This is the first Gap period during which the cell accelerates protein biosynthesis, bulks up the building blocks of DNA required in the next phase, and expands organelles, leading to increased cell size. Restriction checkpoint controls progression through the G₁ phase. The cell size, nutrient availability, and growth factors are measured and sensed, allowing the cell to decide to divide.

S phase:

In the Synthesis phase, the entire cell genome is duplicated, resulting in the formation of two identical copies of each chromosome. The microtubule-organizing centers of eukaryotic cells, the centrosomes, are also duplicated in this phase.

G₂ phase:

This is the second Gap period, where the cell replenishes its resources and proteins essential for entry into mitosis. Key processes include the synthesis of microtubule components required for bipolar spindle assembly and the surveillance of DNA integrity to ensure any damage is repaired before division. The completion of these processes is regulated by CDK1/Cyclin A complex at the G₂/M checkpoint, also known as the entry into mitosis.

Mitosis:

The primary goal of mitosis is to properly align and segregate duplicated chromosomes (mitosis) into two daughter cells by the process called cytokinesis. This is a multistep phase composed of prophase, metaphase, anaphase, and telophase. The eukaryotic cells divide by open mitosis, dissolving the nuclear membrane in prophase. During metaphase, chromosomes are aligned on the equatorial plate and separated by the mitotic spindle in anaphase. This most critical step of mitosis is tightly regulated by the CDK1/Cyclin complex at the spindle assembly checkpoint (SAC), also known as the exit from mitosis. The SAC checkpoint controls the sister chromatids’ attachment to spindle fibers before separation. In the final stage of mitosis, a telophase, the nucleus is divided (karyokinesis), resulting in two nuclei being separated into two daughter cells (cytokinesis).

Cell division relies on duplication, a template-based mechanism that results in the replication of genetic information [3]. Duplicated chromosomes are segregated into separate nuclei to be packaged into two identical daughter cells. This describes the program that controls binary cell division, known as the conventional cell cycle, which directs division in the most evolved species and parasites’ hosts, mammals (Box 1) (Fig. 1A). The G₂ period is particularly critical for binary division, providing not only a safety net in the form of a break between chromosome duplication (S-phase) and segregation (mitosis), but also enacts the fundamental rule of binary division: “once and only once DNA replication per division cycle” [4, 5]. Nevertheless, certain types of cells in multicellular organisms break this rule and undergo multinuclear cell division [6–8]. One such example is endoreplication, a case in which cells are locked into continuous DNA replication cycles is widely used by anthropods, plants, and mammals [7, 8]. Another variation is endomitosis, where cells exit the cell cycle at different stages of mitosis and reenter S-phase [9, 10]. The most notable examples of which include platelet production in mammals and the hyphae growth of filamentous fungi [10, 11]. Both the endoreplication and endomitosis cell cycles permanently repress cytokinesis, since in the opistokonta clade, they are part of the terminal differentiation pathway.

Apicomplexan cell cycle

Apicomplexan parasites are a large group of unicellular eukaryotes comprised of five phylogenetically separate groups: eugregarines, cryptosporidia, haemosporida, piroplasmorida, and coccidia [12]. Aside from their widely known role as human and animal pathogens, apicomplexans are also known for the implementation of vastly variable division modes, which were identified and classified into five major modes by the research groups of Dr. Gubbels and Dr. Duraisingh [13, 14]. Two progeny originate from a single parasite by endodyogeny and binary fission, while a parasite that produces more than two progenies uses multinuclear divisions such as endopolygeny I (with karyokinesis), endopolygeny II (without karyokinesis), and schizogony (Box 2). Because chromosomes are clearly segregated in all these cases, the multinuclear divisions of apicomplexans would fall under the umbrella of endomitotic cell cycles (Fig. 1A). However, unlike in the multinuclear division that can take place in the parasite host cells, apicomplexans only postpone cytokinesis until the target number of nuclear cycles is reached. Such multinuclear division is employed by the vast majority of apicomplexans, whereas only a fraction of coccidians and piroplasmids produce two cells from one parent, which suggests that multinuclear division is the primary mode of cell division in apicomplexans.

Box 2. Apicomplexa Cell Division Modes.

Apicomplexan parasites employ a variety of cell division modes, giving them flexibility and adaptability to diverse host environments. Apicomplexans vary by the number of progenies produced, the timing of nuclear division (synchronous versus asynchronous), and where the daughters are formed (internal versus external budding).

Endodyogeny (endo=internal; dyo=two; geny=production): It is a form of binary cell division wherein two daughters are formed within the mother cell through internal budding. Two offspring consume the mother cell in the process. There is an extended period when mother’s and daughter’s inner membrane complexes (IMCs) coexist within the same cell. Toxoplasma gondii uses endodyogeny to propagate in the intermediate hosts in the form of a tachyzoite or a bradyzoite.

Binary fission is the process by which two daughter cells are produced externally. Binary fission is used by Babesia and Theileria species.

Endopolygeny I (endo=internal; poly=many; geny=production) is a type of cell division wherein a cell undergoes several rounds of DNA replication (S-phase) and mitosis without cytokinesis. The intermediate multinucleated cell is formed prior to synchronized internal budding. The karyokinesis is performed at the end of every S/mitosis round. Toxoplasma gondii performs endopolygeny I in the gut of the definitive host, cats.

Endopolygeny II is used by Sarcocystis neurona. Multiple rounds of DNA replication occur without nuclear divisions, resulting in a polyploid nucleus that is simultaneously separated and segregated into internal daughter cells in the last round of cell division.

Schizogony (schizo=split; gonia=generation): The majority of apicomplexan parasites, including Plasmodium species, replicate by schizogony. It is a cell division mode in which a parasite performs multiple rounds of DNA replication and mitosis without cytokinesis. The final round of division is synchronized with the assembly of the daughters of the mother exterior.

Composite cell cycle phase: Contrary to the conventional cell cycle, the apicomplexan cell cycle is more complex. A recent study confirmed multiple overlapping cell cycle phases in T. gondii endodyogeny. In addition to the G₁ phase (50% of the cell division time), the five intervals of the “composite cell cycle” phase are S (19%), S/G₂/C (3%), S/M/C (9%), M/C (18%), and C/G₁ (<1%).

Over the last decade, the apicomplexan research community has produced many groundbreaking discoveries regarding the mechanics of apicomplexan cell division. Some key findings and contributions include but are not limited to: insights into nuclear and DNA replication dynamics [15–19], the organization of specialized structures like centrosomes and kinetochores [18, 20–26], and the biogenesis of intricate cytoskeletal landmarks such as the conoid, basal, and inner membrane complexes [27–42]. We have also learned a great deal about the cell cycle of other life forms of apicomplexan parasites, such as the mosquito and liver stages of Plasmodium spp. [43–51]. However, we must leave the in-depth discussions of these outstanding studies for another time, instead narrowing our focus on the discoveries that are related to the cell cycle organization and regulation used by asexual stages of apicomplexan parasites.

Toxoplasma endodyogeny

The binary division of Toxoplasma gondii has received considerable attention, but only recently its cell cycle organization has been characterized in greater detail (Fig. 1B) [16, 21, 22]. The T. gondii asexual stages, tachyzoite and bradyzoite, divide by endodyogeny, forming two daughter cells inside the mother cell (Box 2 and 3). In the original work by Dr. Striepen and Dr. White groups, three cell cycle phases of endodyogeny were detected: G₁, S-phase, and concurrent mitosis and cytokinesis (referred to as budding) [52]. Technological advances in the last decade have led to the identification of the G₂/M transition, implying that the G₂ period does exist in the tachyzoite cell cycle (Box 1) [22, 53]. Although it was now known that T. gondii has a complete set of cell cycle phases, recent analyses using an adaptation of the fluorescent ubiquitination-based cell cycle indicator (FUCCI) revealed these phases are organized in a highly unusual manner [16, 54]. Only the G₁ period appeared essentially unchanged, while S, G₂, mitosis, and cytokinesis exhibited significant temporal overlap, and often all three phases were observed running concurrently [16] (Key Figure). Due to difficulties in distinguishing these overlapping phases, this period of endodyogeny cell cycle was named a composite cell cycle phase (Box 2). The FUCCI analyses brought to light a striking discovery that, similar to budding yeasts, Toxoplasma tachyzoites begin mitosis before chromosome replication is completed [55]. This phenomenon can be observed by visualizing tachyzoite nuclear architecture, dynamic changes in mitotic markers and tracking the temporal progression of DNA replication. The Toxoplasma centromeres are bundled and replicated first, which in theory would make these duplicated centromeres available for segregation (mitosis) early in S-phase [16, 56]. Although an overlap between S-phase and budding had been anticipated, the recent discovery of early budding markers by the groups of Dr. Bradley and Dr. Gubbels made it possible to pinpoint the timing of budding initiation, which occurs in the second half of S-phase (Fig. 1B) [32, 57, 58]. There is still much to uncover and new studies that shed light on the place and time of Toxoplasma microtubule nucleation, provided valuable information on the timeline of tachyzoite cytokinesis [27, 31]. Notably, the extent of the composite cell cycle phase complicates the creation of a synchronization model for endodyogeny. Blocking one of the processes within the composite phase often has little or no effect on the concurrent cell cycle pathways that continue to progress leading to abnormal physiological and morphological states [16, 22]. Currently, the best option for synchronization tachyzoite cultures is the cell cycle arrest in the G₁ phase, which also presents challenges due to the limited number of internal G₁ controls.

Box 3. T. gondii and Plasmodium spp. Morphotypes and Infectious Stages.

Toxoplasma gondii requires two hosts to complete its life cycle. Cats are the definitive hosts wherein the parasites carry out the sexual stages, while the asexual reproduction occurs in the intermediate hosts like humans, rodents, or any warm-blooded animals.

Tachyzoites (tachos=speed) are the rapidly growing, motile, crescent-shaped infectious form of parasites responsible for acute toxoplasmosis. They have a centrally located nucleus and replicate via repeated endodyogeny within a parasitophorous vacuole until egress.

Bradyzoites (brady=slow) are the slow-dividing, cyst-forming form of parasites responsible for chronic toxoplasmosis. Similar to tachyzoites, bradyzoites are crescent-shaped, but their nucleus is located towards the posterior end.

Plasmodium parasites also require two hosts, a vertebrate host and an insect vector, to complete their life cycle. Sexual stages are executed in the insects, and asexual stages are carried out in the liver and blood of the vertebrate hosts, including humans. The blood stage morphotypes are ring, trophozoites, schizonts, and merozoites.

Ring is the earliest phase of the intraerythrocytic developmental cycle, where the parasite forms the parasitophorous vacuole through host cell membrane invagination. A delicate, ring-like appearance with a chromatin dot within the red blood cell characterizes this stage.

Trophozoite: The ring stage parasite matures into a larger and irregular form of the parasite that has a single nucleus. During this stage, parasites consume hemoglobin and release hemozoin as a by-product.

Schizont is a multinucleated stage formed by several asynchronous rounds of genome replication and mitosis with karyokinesis (nuclear division).

Merozoites are formed from the late schizonts after the last round of genome replication and segregation synchronized with cytokinesis, called segmentation in Plasmodium spp. Merozoites rupture the RBCs and continue the infection, increasing the parasite burden.

Key Figure. Comparative organization of the conventional and T. gondii tachyzoite cell cycle organization.

Circular representation of the conventional (on the top) and Toxoplasma endodyogeny (on the bottom) cell cycles. The schematics illustrate the locations of the major checkpoints and central regulators that control cell cycle stage transitions. Note the sequential flow of the cell cycle phases in the conventional model and substantial overlap of S, G₂, mitosis, and cytokinesis in the composite cell cycle phase of the Toxoplasma endodyogeny.

Plasmodium schizogony

Multinuclear division of apicomplexans had been studied using the asexual stages of the malaria parasites Plasmodium spp. [59, 60]. The Plasmodium asexual cell cycle, or schizogony, is often described in morphological terms, which has made it challenging to assign conventional cell cycle processes. In this review, we strive to align these morphologies with the cell cycle transitions of schizogony (Fig. 1B). There is a general consensus that the “ring” stage corresponds to early G₁ of the conventional cell cycle (Box 3) [60, 61]. In contrast, the cell cycle physiology of the “trophozoite,” which is characterized by its expanded size and a single nucleus, remains unclear (Box 3). The fact that the resolved bipolar spindle was detected in single-nucleated cells confirms that the trophozoite stage corresponds not only to the second half of the G₁ phase, but also to the first S-phase and mitosis (prior to telophase, where karyokinesis takes place) in multinuclear cycles [18]. The “schizont” emerges after the first nuclear division, and this term refers to parasites with two or more nuclei; therefore, schizonts can be assigned to S phase as well as mitotic parasites undergoing rounds of endomitosis (Box 3) (Fig. 1B). Due to the asynchrony of schizogony, the same schizont contains multiple nuclei in either S-phase or in mitosis, suggesting the concurrent run of several cell cycles that are locally regulated [17].

It has previously been established that the G₁ phase of the red blood stage in P. falciparum takes about 20-24 hours [62]. In recent studies of Dr. Merrick, Dr. Ganter and Dr. Gomes groups examining the dynamics of genome replication in P. falciparum, the timing of bromodeoxyuridine incorporation, PfPCNA1 activity and mapping the origins of replication showed that each round of DNA replication (S-phase) is completed within one hour [19, 29, 60]. Note that DNA replication of the Toxoplasma tachyzoite takes 1.5 hours, which may be attributed to its genome size, which is larger than the Plasmodium genomes [16]. Dr. Ganter’s group also established that an endomitotic round consists of DNA replication (S-phase), nuclear division (mitosis), and a post-nuclear division period; the last of which varies in its duration and is the primary cause of asynchrony in Plasmodium schizogony [17]. This unnamed post-nuclear division phase likely corresponds to late mitosis in the conventional cell cycle, which is used to license the origins of replication (Fig. 1B) [63]. The same study also revealed that a reduction in the rates of the final nuclear divisions ultimately leads to synchronous egress of merozoites (Box 3) [17].

In a recent study of nuclear mechanics, the Absalon group timed the biogenesis of the basal ring, which is one of the first components of the daughter cytoskeleton and is a marker of active cytokinesis [18]. P. falciparum typically completes 4-5 nuclear divisions and forms an average 20-22 merozoites from a single mother cell, which is a reflection of the asynchronous cell cycles. So, the fact that basal rings are detected in schizonts with 3-5 nuclei indicates that Plasmodium parasites first perform 2-3 cycles of genome replication and segregation without cytokinesis, and that the last few cycles are coupled to, or concurrent with, cytokinesis (Fig. 1B) [18]. This observation raised an important question for future studies: what triggers cytokinesis in the middle of apicomplexan endomitosis (see Outstanding Questions)? Establishing the temporal dynamics of apical polar rings and inner membrane complex (IMC) assembly, similar to studies of the bud initiation in Toxoplasma, would help to address this question [27, 31, 32]. It is tempting to suggest that assembly of the daughter cell cytoskeleton, which is an energy-consuming task, is one of the reasons why schizonts slow down their endomitotic cycles coupled to cytokinesis (Fig. 1B) [17]. Given that the division of P. falciparum red blood stages can be synchronized, and that the parasite’s internal structures can be resolved by ultra-expansion microscopy, it would be of great interest to reconstruct the timeline of schizogony from the first duplication event of the centrosomal plaque (G₁/S, 24h pi) to the last synchronized nuclear division and surface budding (M/C, 48h pi) (Box 3).

Comparing endodyogeny and schizogony reveals surprisingly similar cell cycle organization, as in both division modes, the G₁ phase occupies approximately half of the total cell cycle duration, and DNA replication and mitosis occur concurrently with cytokinesis. While it is still unclear whether or not schizogony can formally be assigned a G₂ phase, each endomitotic round completes with two nuclei, and Plasmodium parasites likely repress genome reduplication after S-phase, which is a major G₂ task (see Outstanding Questions).

Regulation of the apicomplexan cell cycles

Progression through the cell cycle is regulated by stage-specific complexes of cyclin-dependent kinases (CDKs) and their cognate cyclins (Fig. 2) [64]. Cyclins function as catalytic activators of CDK activity, and several CDK-cyclin complexes are involved in the control of the conventional cell cycle. To start, the G₁ phase is promoted by the CDK4/6-Cyclin D complexes [64]. CDK2 plays multiple roles, regulating entry into S-phase when in complex with Cyclin A, and operating the G₂/M checkpoint when in complex with Cyclin E [64]. The spindle assembly checkpoint (SAC) activates the anaphase-promoting complex (APC/C), which is dependent on the CDK1-Cyclin B complex (Box 1) [64]. These CDK-cyclin complexes activate transcriptional factors, typically those of the E2F family, which initiate stage-specific waves of gene expression, ensuring the sequential progression of cell cycle phases [64]. While CIP/KIP family proteins act as allosteric inhibitors of the G₁, S, and G₂ CDKs, CDC25 phosphatase removes inhibitory phosphorylation of the mitotic CDK [64, 65]. Upon completion of a cell cycle phase, the large proteolytic machineries, SCF and APC/C complexes, target CDK activators and cyclins for degradation [66]. These groups of factors represent the central regulators of the cell cycle.

Figure 2. Central machinery of the conventional and apicomplexan cell cycles.

The changes in the central cell cycle machineries of the conventional (top), endodyogeny (middle), and schizogony (bottom) are shown as cells progress through the cell cycle. Solid arrows represent confirmed relationships, and dotted arrows show putative connections between factors. Similarly, the identified cell cycle factors are encircled with a solid line; the hypothetical counterparts are encircled with dotted lines, and their names are dimmed. At critical cell cycle transitions and checkpoints, a common mechanism acts where certain CDK-cyclin complexes activate their corresponding E2F transcriptional factors to promote stage-specific gene expression. The E2F factors can act directly or indirectly to activate the CDK/Cyclin complexes of the next stage to ensure that each phase of the cell cycle can progress without interruption. E2Fs often bind promoters and upregulate the transcription of dynamically expressed cyclins, while the pocket proteins of the CIP/KIP family and the phosphatase CDC25 inhibit constitutively expressed CDKs outside the stages in which they act. The mitotic CDK/Cyclin complex induces reorganization of the mitotic apparatus and activates the E3 ligase APC/C, which leads to the degradation of cell cycle regulators and termination of the cell cycle program.

The complexity and diversity of the apicomplexan division modes require equally complex and diverse cell cycle machinery. Queries through apicomplexan genomes show that much of the traditional cell cycle machinery had either been severely altered throughout the evolution of Apicomplexa or is entirely absent [21, 22, 67–69]. For example, apicomplexans lack CIP/KIP CDK inhibitors, cell cycle phosphatase CDC25, and pocket proteins (DP1, Retinoblastoma protein) that regulate E2F transcriptional factors (Fig. 2) [69]. Searching by sequence similarity for cell cycle CDKs and cyclins also fails to retrieve orthologs of CDK1/2, CDK4/6 kinases, and Cyclins A, B, E, and D [67]. Such roadblocks had prevented the discovery of checkpoints in the apicomplexan cell cycle, leaving their existence under debate until recently [60, 67, 69–72].

Over the last decade, systematic analyses of CDK-related kinases (Crks) and cyclins in Toxoplasma enabled the identification and verification of functional G₂/M, SAC, and Restriction checkpoints in endodyogeny (Box 1) (Fig. 1B) [21, 22, 67, 73]. It has been demonstrated that diverse Toxoplasma Crks form complexes with atypical cyclins, confirming that Crk cyclin-binding properties are conserved. The G₂/M checkpoint is now known to be regulated by TgCrk4-TgCyc4 [22]. A successful mitotic transition from metaphase to anaphase, the hallmark of the SAC in eukaryotes, requires activity of the TgCrk6-TgCyc1 complex [21], while TgCrk2 likely acts with multiple cyclins to regulate the Restriction checkpoint (Fig. 2) [67, 73]. The continued interest in the Apicomplexa-specific family of DNA-binding factors AP2s has produced several critical studies that support TgAP2s’ potential role as cell cycle transcriptional factors: for example, TgAP2XII-8 (G₁) [74], TgAP2IX-5 (G₁/S) [75], TgAP2XII-2 (S) [76], TgAP2X-9 (mitosis) [22], and TgAP2X-7 (G₁/C) [77] (Fig. 2). Additionally, TgAP2XII-9, TgAP2XI-5, TgAP2X-5, and TgAP2X-4 may be required for progression through the composite cell cycle phase that encompasses S, G₂, and mitosis [78–81]. The full list of Toxoplasma AP2s can be found in a recently published review [82]. The studies primarily focus on downstream effectors of AP2s, but, regrettably, did not detect any central cell cycle regulators among them, suggesting an indirect communication between AP2s and Crks-cyclin complexes. Only TgAP2X-9 was found to be directly regulated by the G₂/M kinase TgCrk4 and, yet, remains to be confirmed as a cell cycle transcriptional factor [22]. Several Toxoplasma protein phosphatases are required for tachyzoite cell division, primarily for cytokinesis, but none seem to substitute the role of the missing CDC25 [83–86]. Further studies are needed to determine both if and how these phosphatases communicate with Crks and cyclins. These latest findings began to fill in the gaps of the Toxoplasma cell cycle network, which is still sparse. For one, we do not know how the activity of multiple Crks is coordinated, since most of them are statically expressed. We also do not know how cyclins, the dynamic components of the complex, are degraded and whether Toxoplasma SCF and APC/C play any role in this process. Most importantly, we have very little information on how Crks regulate stage-specific gene expression (see Outstanding Questions).

Our knowledge of the regulation of multinuclear divisions in Apicomplexa is even more limited. The hypothesis of apicomplexan cell cycles, such as schizogony, lacking checkpoints had been entertained for a while, and was based on the lack of conserved regulators and the seemingly chaotic mode of cell division [60]. While schizogony checkpoints have not been identified to date, it is difficult to proclaim that they are entirely absent, as some form of machinery is likely to exist, given the vital importance of checkpoint functions. The initiation of DNA replication (G₁/S), prevention of unlawful DNA re-replication and premature DNA segregation (G₂/M), and secure attachment of the duplicated chromosomes to spindle fibers (SAC) all occur during schizogony and must be regulated for the successful production of parasite progeny. Recent studies show that Plasmodium spp. has a metabolic G₁ checkpoint with characteristics of the Restriction checkpoint, which is likely under the control of the non-CDK kinases PfPK2 and PfCDPK4 [62, 87]. It supports that in a dearth of conservative regulators, the strict order of schizogony events in a conserved cell cycle landscape can be maintained by new players [88, 89].

Several Plasmodium Cdk-related kinases have been studied, but only two are implicated in the regulation of the cell cycle of Plasmodium asexual stages [90–92]. This is complicated by the fact that Plasmodium species can show different requirements for CRKs; for example, while CRK5 is dispensable for red blood stage merozoites in P. falciparum, it is essential for the same stage in P. berghei [90, 93]. The second kinase, P. falciparum CRK4, has received the most attention. A detailed analysis of PfCRK4-deficient parasites showed that PfCRK4 primarily regulates entry into S-phase [91], and a more recent study by Dr. Ganter’s group concluded that PfCRK4 also plays a role in mitosis, making this kinase a perfect candidate as a factor that links mitosis to the next S-phase in endomitotic cycles (Fig. 2) [92]. Interestingly, PfCRK4 displays certain features that are shared by Toxoplasma G₂/M kinase TgCrk4. For one, PfCRK4 is localized near the microtubule-nucleating gamma-tubulin complex, and TgCrk4 phosphorylates a subunit of the gamma-tubulin complex that is critical for spindle assembly [22]. In another example, depletion of TgCrk4 affects the separation of the duplicated centrosomes, which presents as a single centrosome, and PfCRK4-deficient parasites are found to be arrested with a single centriolar plaque. Super-resolution microscopy may help determine whether this centriolar plaque is a single structure or duplicated but joined. Finally, T. gondii tachyzoites lacking TgCrk4 could not form a bipolar spindle, which is reminiscent of the hemispindle state of PfCRK4-deficient Plasmodium parasites [22, 92]. The fact that PfCRK4 controls entry into S-phase and displays commonalities with the Toxoplasma G₂/M kinase evoke the thought that PfCRK4 may be a major regulator of the endomitotic cycles of schizogony. Whether schizogony uses canonical G₁/S, G₂/M, and SAC checkpoints remains unresolved (Fig. 2) (see Outstanding Questions). The role of AP2 factors as downstream CRK effectors and transcriptional cell cycle regulators is likely universal among apicomplexans. The most promising candidates for regulating schizogony include PfAP2-LT, PfAP2-I, PfAP2-P, and PF3D7_0613800, which are essential for P. falciparum survival in the blood stage [62, 94–96]. However, further studies are needed to establish the networks that connect the cell cycle kinases with AP2 transcriptional factors.

The incorporation of cytokinesis in the middle of apicomplexan division cycles, a parasite-specific process in the assembly of infectious daughters, suggests the use of new regulatory strategies (Fig. 1B). Indeed, studies show that apicomplexans have adapted conserved Crks, cyclins or phosphatases to control the assembly of the daughter cell cytoskeleton. Specifically, TgCrk1-TgCycL is related to the conserved eukaryotic CDK11-Cyclin L complex and controls the assembly of Toxoplasma internal buds [67, 97] and is likely regulated by the plant-like protein phosphatase TgPPKL [86]. Plasmodium cyclin PfCyc1, related to eukaryotic Cyclin H, is required for proper segmentation in P. falciparum [98].

The binary division is often referred to as the “simplest” division mode in Apicomplexa. Contrary to this, experimental evidence suggests that endodyogeny involves more factors, such as CRKs and cyclins, to maintain the cell cycle than schizogony. Is there a possibility that binary division evolved as a repression of the endomitotic cycles of multinuclear division? The future studies of the other apicomplexan species would help to address this question (see Outstanding Questions).

Concluding Remarks

The last decade has seen many remarkable studies that have advanced our understanding of apicomplexan cell cycles. The creation of DNA replication probes, such as ToxoFUCCI^S and endogenous PfPCNA1 tagging, has greatly helped to dissect the organization of binary and multinuclear cell cycles [16, 17, 60]. The new and improved maps reveal a rare architecture of multiple overlapping cell cycle phases, which necessitate complex regulatory machinery. Ultra-expansion microscopy has provided much-needed deep resolution of mitotic structures, which, in combination with conditional cell cycle models, offer a powerful tool to uncover the regulation of Apicomplexa cell cycles [16, 18, 21, 22, 53, 60]. We were able to confirm the three major checkpoints in Toxoplasma endodyogeny, which, while conserved, operate using previously unknown machinery [21, 22, 73]. Three separate Crk-cyclin complexes regulate the Restriction, G₂/M, and SAC checkpoints of endodyogeny, but it appears that a single PfCRK4 may regulate progression through the endomitotic cycles of Plasmodium schizogony [91, 92]. There is still much to learn about apicomplexan cell cycles, both in their mechanics and their regulation. Currently, only two types of apicomplexan cell cycles have been examined in detail, and unveiling more information on more apicomplexan models could provide valuable insights to better understand the rules and exceptions of the cell cycle across eukaryotes (see Outstanding Questions).

Glossary

APC/C

Anaphase Promoting Complex or a Cyclosome is a large ubiquitin ligase complex that triggers metaphase to anaphase transition by specifically targeting mitotic Cyclin B and Securin for 26S proteasome degradation

Apical polar rings

a hallmark organelle of apicomplexans. It organizes the subpellicular microtubules, forming the parasite’s “ribcage” and involved in coordination of the function of the apical organelles such as rhoptries and micronemes

Basal ring

the contractile ring, located at the basal end of the parasite, is required for proper cytokinesis

Binary division

refers to a process of cell division that creates two daughter cells from a single mother cell

Budding

It is the process of final assembly of new daughter cells either internally or externally

CDKs

Cyclin-Dependent Kinases are regulatory enzymes that are activated by cyclins. Activated CDKs phosphorylate other proteins and regulate many cellular functions, including cell cycle progression

Cell cycle phases

The distinct physiological stages that a cell goes through to produce a progeny. There are four major phases: G₁, S, G₂, and mitosis

Centromere

Am epigenetically defined locus on the chromosome responsible for attachment to kinetochores and spindle fibers during mitosis

Centrosomal plaque

a specialized, bipartite structure embedded in the nuclear membrane of Plasmodium parasites and functions as a microtubule-organizing center for the bipolar spindle

CRKs

Cdk-related kinases are functionally similar to CDKs, but their dependence on cyclin activation has not been experimentally proven yet

Cyclins

A group of cell cycle regulatory proteins expressed in an oscillatory pattern. They bind and activate their partner CDKs

Endomitosis

A type of multinuclear cell division where a cell exits the cell cycle in mitosis and re-enters S-phase. The cytokinesis is either postponed (example: apicomplexans) or omitted (example: terminally differentiated tissues of mammalian cells and some plants)

Endoreplication

A type of multinuclear cell division where a cell performs rounds of S-phases without entering mitosis or cytokinesis, yielding a polyploid nucleus (example: Drosophila salivary glands)

Karyokinesis

A step of cell division where the nucleus divides into two, ensuring that each daughter cell receives an identical set of genetic material. Karyokinesis is executed during the last phase of mitosis, telophase, prior to cytokinesis

Multinuclear Cell Division

refers to a cell process leading to the formation of multinucleated cells. It results in the production of multiple progenies from a single cell division (e.g., apicomplexans) or enhanced cellular function (e.g., mammalian cells)

Origin of replication

the region of the chromosome where the DNA replication starts. Each eukaryotic chromosome has multiple origins of replication that are orderly licensed (mitosis and G₁) and fired (S-phase)

SCF

Skp1-Cullin-F-box protein complex is an E3 ligase that primarily regulates the G₁/S transition of the cell cycle by degrading S-phase cyclins and CDKs

Segmentation

It is the last step in Plasmodium’s schizogony during which the schizont undergoes synchronized cytokinesis, resulting in the release of merozoites