Hepatitis B virus (HBV) capsid assembly is traditionally thought to occur predominantly in the cytoplasm, where the virus gains access to the virion egress pathway. To better define sites of HBV capsid assembly, we carried out single cell imaging of HBV Core protein (Cp) subcellular trafficking over time under conditions supporting genome packaging and reverse transcription in Huh7 hepatocellular carcinoma cells. Time-course analyses including live cell imaging of fluorescently tagged Cp derivatives showed Cp to accumulate in the nucleus at early time points (~24 h), followed by a marked re-distribution to the cytoplasm at 48 to 72 h. Nucleus-associated Cp was confirmed to be capsid and/or high-order assemblages using a novel dual label immunofluorescence strategy. Nuclear-to-cytoplasmic re-localization of Cp occurred predominantly during nuclear envelope breakdown in conjunction with cell division, followed by strong cytoplasmic retention of Cp. Blocking cell division resulted in strong nuclear entrapment of high-order assemblages. A Cp mutant, Cp-V124W, predicted to exhibit enhanced assembly kinetics, also first trafficked to the nucleus to accumulate at nucleoli, consistent with the hypothesis that Cp's transit to the nucleus is a strong and constitutive process. Taken together, these results provide support for the nucleus as an early-stage site of HBV capsid assembly, and provide the first dynamic evidence of cytoplasmic retention after cell division as a mechanism underpinning capsid nucleus-to-cytoplasm relocalization. IMPORTANCE Hepatitis B virus (HBV) is an enveloped, reverse-transcribing DNA virus that is a major cause of liver disease and hepatocellular carcinoma. Subcellular trafficking events underpinning HBV capsid assembly and virion egress remain poorly characterized. Here, we developed a combination of fixed and long-term (>24 h) live cell imaging technologies to study the single cell trafficking dynamics of the HBV Core Protein (Cp). We demonstrate that Cp first accumulates in the nucleus, and forms high-order structures consistent with capsids, with the predominant route of nuclear egress being relocalization to the cytoplasm during cell division in conjunction with nuclear membrane breakdown. Single cell video microscopy demonstrated unequivocally that Cp's localization to the nucleus is constitutive. This study represents a pioneering application of live cell imaging to study HBV subcellular transport, and demonstrates links between HBV Cp and the cell cycle.

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.

Rac1 regulates a wide variety of cellular processes. The polybasic region of the Rac1 C terminus functions both as a plasma membrane-targeting motif and a nuclear localization sequence (NLS). We show that a triproline N-terminal to the polybasic region contributes to the NLS, which is cryptic in the sense that it is strongly inhibited by geranylgeranylation of the adjacent cysteine. Subcellular fractionation demonstrated endogenous Rac1 in the nucleus and Triton X-114 partition revealed that this pool is prenylated. Cell cycle-blocking agents, synchronization of cells stably expressing low levels of GFP-Rac1, and time-lapse microscopy of asynchronous cells revealed Rac1 accumulation in the nucleus in late G2 and exclusion in early G1. Although constitutively active Rac1 restricted to the cytoplasm inhibited cell division, activated Rac1 expressed constitutively in the nucleus increased the mitotic rate. These results show that Rac1 cycles in and out of the nucleus during the cell cycle and thereby plays a role in promoting cell division.

Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.

The medial preoptic area of the adult sheep contains an ovine sexually dimorphic nucleus (oSDN) that is larger and has more neurons in males than in females. In the lamb fetus, the nascent oSDN occupies the central division of the medial preoptic nucleus (MPNc) and consists of a cluster of cells that is organized by the action of testosterone during gestational days 60-90 of a 147 day term pregnancy. The current study sought to determine whether programmed cell death contributes to the emergence of the oSDN. Male and female lamb fetuses were euthanized at different ages spanning the period during which the oSDN is organized. The expression of the pro- and anti-apoptotic genes bcl-2 and bax, respectively, was measured by quantitative RT-PCR to assess possible sex differences in neuron vulnerability to programmed cell death. The appearance of DNA-fragmentation was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and used to estimate the occurrence of apoptotic cell death. We found that bcl-2 and bax mRNA expression in the medial preoptic area of the developing lamb fetus decreased during the last half of the 147-day gestation. The ratio of bcl-2/bax gene expression was highest at gestational day 85 but was equivalent between males and females. TUNEL staining in the MPNc was very low and although it decreased significantly with age, it was not significantly different between sexes. These results using two different methods to assess cell death indicate that a sex difference in the incidence of cell death is not a primary mechanism leading to sexual differentiation of the oSDN.

All 302 neurons in the C. elegans hermaphrodite arise through asymmetric division of neuroblasts. During embryogenesis, the C. elegans ham-1 gene is required for several asymmetric neuroblast divisions in lineages that generate both neural and apoptotic cells. By antibody staining, endogenous HAM-1 is found exclusively at the cell cortex in many cells during embryogenesis and is asymmetrically localized in dividing cells. Here we show that in transgenic embryos expressing a functional GFP::HAM-1 fusion protein, GFP expression is also detected in the nucleus, in addition to the cell cortex. Consistent with the nuclear localization is the presence of a putative DNA binding winged-helix domain within the N-terminus of HAM-1. Through a deletion analysis we determined that the C-terminus of the protein is required for nuclear localization and we identified two nuclear localization sequences (NLSs). A subcellular fractionation experiment from wild type embryos, followed by Western blotting, revealed that endogenous HAM-1 is primarily found in the nucleus. Our analysis also showed that the N-terminus is necessary for cortical localization. While ham-1 function is essential for asymmetric division in the lineage that generates the PLM mechanosensory neuron, we showed that cortical localization may not required. Thus, our results suggest that there is a nuclear function for HAM-1 in regulating asymmetric neuroblast division and that the requirement for cortical localization may be lineage dependent.

Previous cell cycle studies have been based on cell-nuclear proliferation only. Eukaryotic cells, however, have double membranes-bound organelles, such as the cell nucleus, mitochondrion, plastids and single-membrane-bound organelles such as ER, the Golgi body, vacuoles (lysosomes) and microbodies. Organelle proliferations, which are very important for cell functions, are poorly understood. To clarify this, we performed a microarray analysis during the cell cycle of Cyanidioschyzon merolae. C. merolae cells contain a minimum set of organelles that divide synchronously. The nuclear, mitochondrial and plastid genomes were completely sequenced. The results showed that, of 158 genes induced during the S or G2-M phase, 93 were known and contained genes related to mitochondrial division, ftsZ1-1, ftsz1-2 and mda1, and plastid division, ftsZ2-1, ftsZ2-2 and cmdnm2. Moreover, three genes, involved in vesicle trafficking between the single-membrane organelles such as vps29 and the Rab family protein, were identified and might be related to partitioning of single-membrane-bound organelles. In other genes, 46 were hypothetical and 19 were hypothetical conserved. The possibility of finding novel organelle division genes from hypothetical and hypothetical conserved genes in the S and G2-M expression groups is discussed.

The nucleus accumbens (NAc) is the key area of the reward circuit, but its heterogeneity has been poorly studied. Using single-cell RNA sequencing, we revealed a subcluster of GABAergic neurons characterized by cell division cycle 20 (Cdc20) mRNA expression in the NAc of adult rats. We studied the coexpression of Cdc20 and Gad1 mRNA in the NAc neurons of adult rats and assessed Cdc20 protein expression in the NAc during rat development. Moreover, we microinjected AAV2/9-hSyn-Cdc20 with or without the dual-AAV system into the bilateral NAc for sparse labeling to observe changes in the synaptic morphology of mature neurons and assessed rat behaviors in open field and elevated plus maze tests. Furthermore, we performed the experiments with a Cdc20 inhibitor, Cdc20 over-expression AAV vector, and Cdc20 conditional knockout primary striatal neurons to understand the ubiquitination-dependent degradation of fragile X mental retardation protein (FMRP) in vitro and in vivo. We confirmed the mRNA expression of Cdc20 in the NAc GABAergic neurons of adult rats, and its protein level was decreased significantly 3 weeks post-birth. Up-regulated Cdc20 expression in the bilateral NAc decreased the dendritic spine density in mature neurons and induced anxiety-like behavior in rats. Cdc20-APC triggered FMRP degradation through K48-linked polyubiquitination in Neuro-2a cells and primary striatal neurons and down-regulated FMRP expression in the NAc of adult rats. These data revealed that up-regulation of Cdc20 in the bilateral NAc reduced dendritic spine density and led to anxiety-like behaviors, possibly by enhancing FMRP degradation via K48-linked polyubiquitination.

Development in multicellular organisms requires the organized generation of differences. A universal mechanism for creating such differences is asymmetric cell division. In plants, as in animals, asymmetric divisions are correlated with the production of cellular diversity and pattern; however, structural constraints imposed by plant cell walls and the absence of homologs of known animal or fungal cell polarity regulators necessitates that plants utilize new molecules and mechanisms to create asymmetries. Here, we identify BASL, a novel regulator of asymmetric divisions in Arabidopsis. In asymmetrically dividing stomatal-lineage cells, BASL accumulates in a polarized crescent at the cell periphery before division, and then localizes differentially to the nucleus and a peripheral crescent in self-renewing cells and their sisters after division. BASL presence at the cell periphery is critical for its function, and we propose that BASL represents a plant-specific solution to the challenge of asymmetric cell division.

The bed nucleus of the stria terminalis is a key part of a ring of cells extending between the centromedial amygdala and bed nucleus of the stria terminalis referred to as the extended amygdala. The present study describes the architecture of the bed nucleus of the stria terminalis and the connections of subnuclei in posterior bed nucleus of the stria terminalis. The hamster bed nucleus of the stria terminalis is readily allotted to anterior and posterior divisions separated by the fibers of the body of the anterior commissure. The anterior division has four subnuclei: anteromedial, anterointermediate, anterolateral, and anteroventral. Within the posterior division, there are three distinct regions: posteromedial, posterointermediate, and posterolateral. In hamsters, the posterior bed nucleus of the stria terminalis contributes to male sexual behavior, particularly chemoinvestigation. Moreover, the posterior bed nucleus of the stria terminalis is part of a neural circuit essential for mating, including the medial amygdaloid nucleus and medial preoptic area. The connections of bed nucleus of the stria terminalis, posteromedial part, bed nucleus of the stria terminalis, posterointermediate part and bed nucleus of the stria terminalis, posterolateral part were visualized by co-injection of anterograde (Phaseolus vulgaris leucoagglutinin) and retrograde (cholera toxin B) tract tracers. The bed nucleus of the stria terminalis, posterointermediate part and bed nucleus of the stria terminalis, posteromedial part have dense bidirectional connections with medial amygdaloid nucleus and cortical amygdala via the stria terminalis and ventral amygdalofugal pathway. These subnuclei also maintain bidirectional connections with steroid-concentrating areas including lateral septum, medial preoptic area, hypothalamus, and periaqueductal gray. The bed nucleus of the stria terminalis, posterointermediate part and bed nucleus of the stria terminalis, posteromedial part receive projections from the subiculum and send projections to deep mesencephalic nuclei. By contrast, the bed nucleus of the stria terminalis, posterolateral part is connected with the central amygdala, lateral hypothalamus, subthalamic nucleus, nucleus accumbens, substantia innominata, substantia nigra and thalamus. Thus, the bed nucleus of the stria terminalis, posterointermediate part and bed nucleus of the stria terminalis, posteromedial part have similar connections with areas involved in social behaviors. The bed nucleus of the stria terminalis, posterolateral part maintains connections with areas involved in motivational circuits. This supports the concept of distinct circuits within the extended amygdala which differentially link the centromedial amygdala and bed nucleus of the stria terminalis.

During mitosis, the genome is transformed from a decondensed, transcriptionally active state to a highly condensed, transcriptionally inactive state. Mitotic chromosome reorganization is marked by the general attenuation of transcription on chromosome arms, yet how the cell regulates nuclear and chromatin-associated RNAs after chromosome condensation and nuclear envelope breakdown is unknown. SAF-A/hnRNPU is an abundant nuclear protein with RNA-to-DNA tethering activity, coordinated by two spatially distinct nucleic acid-binding domains. Here we show that RNA is evicted from prophase chromosomes through Aurora-B-dependent phosphorylation of the SAF-A DNA-binding domain; failure to execute this pathway leads to accumulation of SAF-A-RNA complexes on mitotic chromosomes, defects in metaphase chromosome alignment, and elevated rates of chromosome missegregation in anaphase. This work reveals a role for Aurora-B in removing chromatin-associated RNAs during prophase and demonstrates that Aurora-B-dependent relocalization of SAF-A during cell division contributes to the fidelity of chromosome segregation.

Zika virus (ZIKV) nonstructural protein 5 (NS5) plays a critical role in viral RNA replication and mediates key virus-host cell interactions. As with other flavivirus NS5 proteins, ZIKV NS5 is primarily found in the nucleus. We previously reported that the NS5 protein of dengue virus, another flavivirus, localized to centrosomes during cell division. Here we show that ZIKV NS5 also relocalizes from the nucleus to centrosomes during mitosis. In infected cells with supernumerary centrosomes, NS5 was present at all centrosomes. Transient expression of NS5 in uninfected cells confirmed that centrosomal localization was independent of other viral proteins. Live-cell imaging demonstrated that NS5-GFP accumulated at centrosomes shortly after break down of nuclear membrane and remained there through mitosis. Cells expressing NS5-GFP took longer to complete mitosis than control cells. Finally, an analysis of ZIKV NS5 binding partners revealed several centrosomal proteins, providing potential direct links between NS5 and centrosomes.

In Arabidopsis thaliana, brassinosteroid (BR) signaling and stomatal development are connected through the SHAGGY/GSK3-like kinase BR INSENSITIVE2 (BIN2). BIN2 is a key negative regulator of BR signaling but it plays a dual role in stomatal development. BIN2 promotes or restricts stomatal asymmetric cell division (ACD) depending on its subcellular localization, which is regulated by the stomatal lineage-specific scaffold protein POLAR. BRs inactivate BIN2, but how they govern stomatal development remains unclear. Mapping the single-cell transcriptome of stomatal lineages after triggering BR signaling with either exogenous BRs or the specific BIN2 inhibitor, bikinin, revealed that the two modes of BR signaling activation generate spatiotemporally distinct transcriptional responses. We established that BIN2 is always sensitive to the inhibitor but, when in a complex with POLAR and its closest homolog POLAR-LIKE1, it becomes protected from BR-mediated inactivation. Subsequently, BR signaling in ACD precursors is attenuated, while it remains active in epidermal cells devoid of scaffolds and undergoing differentiation. Our study demonstrates how scaffold proteins contribute to cellular signal specificity of hormonal responses in plants.

Tritrichomonas foetus and Trichomonas vaginalis are extracellular flagellated parasites that inhabit animals and humans, respectively. Cell division is a crucial process in most living organisms that leads to the formation of 2 daughter cells from a single mother cell. It has been assumed that T. vaginalis and T. foetus modes of reproduction are exclusively by binary fission. However, here, we showed that multinuclearity is a phenomenon regularly observed in different T. foetus and T. vaginalis strains in standard culture conditions. Additionally, we revealed that nutritional depletion or nutritional deprivation led to different dormant phenotypes. Although multinucleated T. foetus are mostly observed during nutritional depletion, numerous cells with 1 larger nucleus have been observed under nutritional deprivation conditions. In both cases, when the standard culture media conditions are restored, the cytoplasm of these multinucleated cells separates, and numerous parasites are generated in a short period of time by the fission multiple. We also revealed that DNA endoreplication occurs both in large and multiple nuclei of parasites under nutritional deprivation and depletion conditions, suggesting an important function in stress nutritional situations. These results provide valuable data about the cell division process of these extracellular parasites. IMPORTANCE Nowadays, it's known that T. foetus and T. vaginalis generate daughter cells by binary fission. Here, we report that both parasites are also capable of dividing by multiple fission under stress conditions. We also demonstrated, for the first time, that T. foetus can increase its DNA content per parasite without concluding the cytokinesis process (endoreplication) under stress conditions, which represents an efficient strategy for subsequent fast multiplication when the context becomes favorable. Additionally, we revealed the existence of novel dormant forms of resistance (multinucleated or mononucleated polyploid parasites), different than the previously described pseudocysts, that are formed under stress conditions. Thus, it is necessary to evaluate the role of these structures in the parasites' transmission in the future.

Both cell-cell adhesion and oriented cell division play prominent roles in establishing tissue architecture, but it is unclear how they might be coordinated. Here, we demonstrate that the cell-cell adhesion protein E-cadherin functions as an instructive cue for cell division orientation. This is mediated by the evolutionarily conserved LGN/NuMA complex, which regulates cortical attachments of astral spindle microtubules. We show that LGN, which adopts a three-dimensional structure similar to cadherin-bound catenins, binds directly to the E-cadherin cytosolic tail and thereby localizes at cell-cell adhesions. On mitotic entry, NuMA is released from the nucleus and competes LGN from E-cadherin to locally form the LGN/NuMA complex. This mediates the stabilization of cortical associations of astral microtubules at cell-cell adhesions to orient the mitotic spindle. Our results show how E-cadherin instructs the assembly of the LGN/NuMA complex at cell-cell contacts, and define a mechanism that couples cell division orientation to intercellular adhesion.

Glioblastoma (GBM) is a malignant tumor with a high recurrence rate and has very poor prognosis in humans. The median survival is still <2 years. Therefore, a new treatment strategy should be established. Recently, this cancer has been thought to be heterogeneous, consisting of cancer stem cells (CSCs) that are self-renewable, multipotent, and treatment resistant. So various strategies targeting glioma stem-like cells (GSCs) have been investigated. This study focused on strategies targeting GSCs through the induction of differentiation using bone morphogenetic protein 4 (BMP4). The expression of CD133, a cancer stem cell marker, under BMP4 treatment in GSCs was examined using flow cytometry, western blotting, and quantitative PCR. Immunofluorescent staining of GSCs was also performed to examine the type of cell division: asymmetric cell division (ACD) or symmetric cell division (SCD). We obtained the following results. The BMP4 treatment caused downregulation of CD133 expression. Moreover, it induced ACD in GSCs. While the ACD ratio was 23% without BMP4 treatment, it was 38% with BMP4 treatment (P=0.004). Furthermore, the tumor sphere assay demonstrated that BMP4 suppresses self-renewal ability. In conclusion, these findings may provide a new perspective on how BMP4 treatment reduces the tumorigenicity of GSCs.

Mitogen-activated protein kinases (MAPKs) are a conserved family of protein kinases that regulate signal transduction, proliferation, and development throughout eukaryotes. The apicomplexan parasite Toxoplasma gondii expresses three MAPKs. Two of these, extracellular signal-regulated kinase 7 (ERK7) and MAPKL1, have been implicated in the regulation of conoid biogenesis and centrosome duplication, respectively. The third kinase, MAPK2, is specific to and conserved throughout the Alveolata, although its function is unknown. We used the auxin-inducible degron system to determine phenotypes associated with MAPK2 loss of function in Toxoplasma We observed that parasites lacking MAPK2 failed to duplicate their centrosomes and therefore did not initiate daughter cell budding, which ultimately led to parasite death. MAPK2-deficient parasites initiated but did not complete DNA replication and arrested prior to mitosis. Surprisingly, the parasites continued to grow and replicate their Golgi apparatus, mitochondria, and apicoplasts. We found that the failure in centrosome duplication is distinct from the phenotype caused by the depletion of MAPKL1. As we did not observe MAPK2 localization at the centrosome at any point in the cell cycle, our data suggest that MAPK2 regulates a process at a distal site that is required for the completion of centrosome duplication and the initiation of parasite mitosis.IMPORTANCEToxoplasma gondii is a ubiquitous intracellular protozoan parasite that can cause severe and fatal disease in immunocompromised patients and the developing fetus. Rapid parasite replication is critical for establishing a productive infection. Here, we demonstrate that a Toxoplasma protein kinase called MAPK2 is conserved throughout the Alveolata and essential for parasite replication. We found that parasites lacking MAPK2 protein were defective in the initiation of daughter cell budding and were rendered inviable. Specifically, T. gondii MAPK2 (TgMAPK2) appears to be required for centrosome replication at the basal end of the nucleus, and its loss causes arrest early in parasite division. MAPK2 is unique to the Alveolata and not found in metazoa and likely is a critical component of an essential parasite-specific signaling network.

Plant tissue architecture and organ morphogenesis rely on the proper orientation of cell divisions. Previous attempts to predict division planes from cell geometry in plants mostly focused on 2D symmetric divisions. Using the stereotyped division patterns of Arabidopsis thaliana early embryogenesis, we investigated geometrical principles underlying plane selection in symmetric and in asymmetric divisions within complex 3D cell shapes. Introducing a 3D computational model of cell division, we show that area minimization constrained on passing through the cell centroid predicts observed divisions. Our results suggest that the positioning of division planes ensues from cell geometry and gives rise to spatially organized cell types with stereotyped shapes, thus underlining the role of self-organization in the developing architecture of the embryo. Our data further suggested the rule could be interpreted as surface minimization constrained by the nucleus position, which was validated using live imaging of cell divisions in the stomatal cell lineage.

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.

In the last mitotic division of the epidermal lineage in the ascidian embryo, the cells divide stereotypically along the anterior-posterior axis. During interphase, we found that a unique membrane structure invaginates from the posterior to the centre of the cell, in a microtubule-dependent manner. The invagination projects toward centrioles on the apical side of the nucleus and associates with one of them. Further, a cilium forms on the posterior side of the cell and its basal body remains associated with the invagination. A laser ablation experiment suggests that the invagination is under tensile force and promotes the posterior positioning of the centrosome. Finally, we showed that the orientation of the invaginations is coupled with the polarized dynamics of centrosome movements and the orientation of cell division. Based on these findings, we propose a model whereby this novel membrane structure orchestrates centrosome positioning and thus the orientation of cell division axis.