Fussing About Fission: Defining Variety Among Mainstream and Exotic Apicomplexan Cell Division Modes

Abstract

Cellular reproduction defines life, yet our textbook-level understanding of cell division is limited to a small number of model organisms centered around humans. The horizon on cell division variants is expanded here by advancing insights on the fascinating cell division modes found in the Apicomplexa, a key group of protozoan parasites. The Apicomplexa display remarkable variation in offspring number, whether karyokinesis follows each S/M-phase or not, and whether daughter cells bud in the cytoplasm or bud from the cortex. We find that the terminology used to describe the various manifestations of asexual apicomplexan cell division emphasizes either the number of offspring or site of budding, which are not directly comparable features and has led to confusion in the literature. Division modes have been primarily studied in two human pathogenic Apicomplexa, malaria-causing Plasmodium spp. and Toxoplasma gondii, a major cause of opportunistic infections. Plasmodium spp. divide asexually by schizogony, producing multiple daughters per division round through a cortical budding process, though at several life-cycle nuclear amplifications stages, are not followed by karyokinesis. T. gondii divides by endodyogeny producing two internally budding daughters per division round. Here we add to this diversity in replication mechanisms by considering the cattle parasite Babesia bigemina and the pig parasite Cystoisospora suis. B. bigemina produces two daughters per division round by a "binary fission" mechanism whereas C. suis produces daughters through both endodyogeny and multiple internal budding known as endopolygeny. In addition, we provide new data from the causative agent of equine protozoal myeloencephalitis (EPM), Sarcocystis neurona, which also undergoes endopolygeny but differs from C. suis by maintaining a single multiploid nucleus. Overall, we operationally define two principally different division modes: internal budding found in cyst-forming Coccidia (comprising endodyogeny and two forms of endopolygeny) and external budding found in the other parasites studied (comprising the two forms of schizogony, binary fission and multiple fission). Progressive insights into the principles defining the molecular and cellular requirements for internal vs. external budding, as well as variations encountered in sexual stages are discussed. The evolutionary pressures and mechanisms underlying apicomplexan cell division diversification carries relevance across Eukaryota.

Keywords: apicomplexa; binary fission; cell cycle; cell division; endodyogeny; endopolygeny; karyokinesis; schizogony.

Figure 1

Select apicomplexan phylogeny and division modes. 18S ribosomal RNA based phylogeny of species whose division modes have been studied. Cryptosporidium spp. were used as outgroup. Bars on the right indicate different naming and biological relationships, with asexual division modes in blue and red. “in vacuole” and “in cytoplasm” indicate whether asexual replication occurs in a parasitophorous vacuole, or whether the parasite escapes from the vacuole and resides in the cytoplasm of the host cell for its replication. Note that only the acute stage merozoites of S. neurona replicates by endopolygeny in the cytoplasm, whereas metrocytes preceding the bradyzoites as well as the bradyzoites divide by endodyogeny within a vacuole supporting a proteoglycan cyst wall. Furthermore, tissue cysts for C. suis have not been described and this lacking ability is likely a secondary loss. In addition, for several Plasmodium spp. (Simonetti, 1996), as well as Babesia and Theileria spp. (Jalovecka et al., 2018) it has been shown that sporozoite formation progresses without karyokinesis to produce large polyploid nuclei while budding is from the cortex. We note that Plasmodium sporozoites infect hepatocytes wherein they divide by schizogony and manipulate the hepatocytes to expand their size, whereas Theileria sporozoites infect white blood cells, replicate by schizogony (Shaw and Tilney, 1992) and trigger white blood expansion as well as division (i.e., transformation, which basically is leukemia (Luder et al., ; Chakraborty et al., 2017), which contrasts with Babesia sporozoites as they directly infect red blood cells. “epg with kk” means “endopolygeny with karyokinesis,” in case of select Babesia spp., “k.a.” means “kinete amplification”.

Figure 2

Apicomplexan asexual cell division by schizogony. (A) Schematic representation of progressive cell division steps during “classic” schizogony. The mother's cytoskeleton (present in 1) is disassembled following successful host cell invasion resulting in a pleomorphic cell (2) before onset of mitosis and karyokinesis, which can be asynchronous (3). Daughter cells bud from the cortex (4/5), positioned by the centrosome anchored at the plasma membrane (4). (B) Schematic representation of phases in the cell division during schizogony with limited karyokinesis as seen during Heamosporidian sprorogenesis. Karyokinesis is inconsistently following each S/M-phase leading to a nuclei population with varying levels of ploidity. Note that nuclear cycles within the same nucleoplasm are similar (1), whereas budding is synchronous for all nuclei and linked to a final round of S/M (2). Note that the number of offspring in both forms of schizogony can reach into the 1000s, which is not represented in the schematics. Chromosome condensation does not occur and the chromosomes are only drawn to convey the principle of spindle pole attachment. Mother cytoplasm represented in gray, daughter cytoplasms in pink.

Figure 3

Binary fission by Babesia spp. (A–D) Babesia bigemina iRBC stages. (A) Immunofluorescence using MAb α-tubulin 12G10 (green) and TgCentrin1(red) polyclonal antibody. Centrin staining is not observed in interphase (G1) while diffuse and weak during cell division (cyt.). (B) Immunofluorescence using MAb α-tubulin 12G10 (green) and microtubule (+)-end binding protein TgEB1 polyclonal antibody (red) demonstrates that the cortical microtubules are relatively short and do not reach the nucleus. (C) Immunofluorescence using polyclonal guinea pig BbIMC1a antiserum (see Figure S1 for validation) and MAb α-tubulin 12G10 shows the IMC is present during G/M, outlines budding daughters (cyt.), and extends along the length of the mature G1 parasites. *Marks a recently invaded parasites wherein the cytoskeleton is completing disassembly; we observed many trophozoites without detectable PbIMC1a. (D) Transmission electron microscopy of progressive iRBC developmental stages (1 → 6). Panel 1 is a disassembling merozoite already escaped from the vacuole displaying typical pattern of six numbered remnants of the disassembling mother IMC. Panel 2 represents early daughter formation (“Mickey Mouse”) with the nucleus being separated into the daughter buds. Panels 3–5 represent different sections though developing daughters where showing parallel assembly (in contrast to polar assembly) consistent with the angle under which the daughter buds assemble. Panel 6 displays two just divided daughters under the typical angle. N, nucleus; SB, spherical body. Yellow marks IMC; orange arrows mark the typical angle and direction of daughter buds. Scale bars represent 500 nm. (E) Babesia divergens basal complex upon completion of cell division. Scale bar represents 500 nm. (F) Schematic of apicomplexan asexual cell division by binary fission, which mechanistically represents a binary form of schizogony. Note that the mother's cytoskeleton is largely disassembled following successful host cell invasion resulting in a pleomorphic cell (2), but that fragments of the IMC are remaining throughout the division stages (2–4).

Figure 4

Apicomplexan asexual cell division by endodyogeny. (A–E) Show progressive steps of cell division as observed for T. gondii tachyzoites. Two daughters bud internally, while the mother's cytoskeleton is maintained and only destabilized just before emergence of nearly mature daughters (E). A dark blue SFA fiber anchors the centrosomes in the apex of the daughter buds.

Figure 5

Endopolygeny with karyokinesis during merogony of Cystoisospora suis. (A–D) Parasites at different stages of endopolygeny. Acetylated (Ac) α-tubulin (red) marks the mother cells conoid [arrow heads in (A,B)], subpellicular microtubules, the spindle poles associated with nuclei undergoing mitosis, and the daughter conoids and subpellicular microtubules (C,D). Antiserum generated against TgNuf2 (green) marks the clustered kinetochores at mitotic spindles. (A) Eight parasites at different stages in the division cycle. Green numbers indicate the number of spindle poles seen per mother cell, which is expanding non-geometrically in five of the parasites indicating asynchronous nuclear cycles. (B) Five parasites at different stages of endopolygeny are shown. The parasite on the bottom has five nuclei: nuclei marked “M” are in early stages of mitosis as two spindle poles flanking a single 2N kinetochore cluster are seen; the circled nuclei represent just completed mitosis as the kinetochore clusters are relatively weak intensity (1N vs. 2N) and associated with only a single spindle pole. (C,D) Two examples of cells at the synchronous daughter budding stage, which both produce 12 daughters as enumbered by the number of kinetochore clusters, again, consistent with non-geometric expansion of the nuclei. (E) Schematic of apicomplexan asexual cell division by endopolygeny with karyokinesis. Note that the mitotic cycles of the nuclei in the same cytoplasm are not synchronous (2) resulting in non-geometrically expanded daughter numbers (4,5). Also note that the mother's cytoskeleton is maintained throughout the meront (technically “schizont”) stages and is only destabilized just before emergence of nearly mature daughters (5).

Figure 6

Endopolygeny without nuclear fission by Sarcocystis neurona in the intermediate host. (A) Staining of progressive cell division cycle stages (1–4) with α-tubulin (green) marking the subpellicular microtubules of the mother cytoskeleton (arrow heads), the mitotic spindles (2b, right parasite; 3) and daughter merozoites subpellicular microtubules (4). Centrin staining (red) marks the centrosomes, which due to z-stack selection are not visible for all spindles/parasites. Scale bar applies across panels. (B) Overexpression of a Toxoplasma YFP-IMC15 fusion protein (green) highlights the mother cell's cortical IMC sutures in both panels, whereas in further progressed panel 2 the bright internal spots mark the centrosomes poised for budding. Arrowhead marks the apical end of the mother parasite. (B) Modified from Dubey et al. (2017). Scale bar applies across panels. (C) Schematic of apicomplexan asexual cell division by endopolygeny without karyokinesis. Note the polyploid nuclei undergoing synchronized cycles of M-phase and mitosis resulting in geometrically expanded daughter cell numbers. Also note that the mother's cytoskeleton is maintained throughout the schizont stages (2–4) and is only destabilized just before emergence of nearly mature daughters (5).