RESUMO
In the ciliated protozoan Paramecium tetraurelia, Piwi-associated small RNAs are generated upon the elimination of tens of thousands of short transposon-derived DNA segments as part of development. These RNAs then target complementary DNA for elimination in a positive feedback process, contributing to germline defense and genome stability. In this work, we investigate the formation of these RNAs, which we show to be transcribed directly from the short (length mode 27 bp) excised DNA segments. Our data support a mechanism whereby the concatenation and circularization of excised DNA segments provides a template for RNA production. This process allows the generation of a double-stranded RNA for Dicer-like protein cleavage to give rise to a population of small regulatory RNAs that precisely match the excised DNA sequences. VIDEO ABSTRACT.
Assuntos
DNA Concatenado , Paramecium tetraurellia/genética , Núcleo Celular/metabolismo , DNA Ligase Dependente de ATP/metabolismo , Elementos de DNA Transponíveis , Exodesoxirribonucleases/metabolismo , Paramecium tetraurellia/citologia , Paramecium tetraurellia/metabolismo , RNA/genética , Transcrição GênicaRESUMO
Multinucleate cells are found in many eukaryotes, but how multiple nuclei coordinate their functions is still poorly understood. In the cytoplasm of the ciliate Paramecium tetraurelia, two micronuclei (MIC) serving sexual reproduction coexist with a somatic macronucleus (MAC) dedicated to gene expression. During sexual processes, the MAC is progressively destroyed while still ensuring transcription, and new MACs develop from copies of the zygotic MIC. Several gene clusters are successively induced and switched off before vegetative growth resumes. Concomitantly, programmed genome rearrangement (PGR) removes transposons and their relics from the new MACs. Development of the new MACs is controlled by the old MAC, since the latter expresses genes involved in PGR, including the PGM gene encoding the essential PiggyMac endonuclease that cleaves the ends of eliminated sequences. Using RNA deep sequencing and transcriptome analysis, we show that impairing PGR upregulates key known PGR genes, together with â¼600 other genes possibly also involved in PGR. Among these genes, 42% are no longer induced when no new MACs are formed, including 180 genes that are co-expressed with PGM under all tested conditions. We propose that bi-directional crosstalk between the two coexisting generations of MACs links gene expression to the progression of MAC development.
Assuntos
Paramecium tetraurellia , Expressão Gênica , Rearranjo Gênico , Genoma , Paramecium tetraurellia/citologia , Paramecium tetraurellia/genética , MacronúcleoRESUMO
The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline and somatic nuclei, massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs - 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.
Assuntos
Genoma de Protozoário , Paramecium tetraurellia , Meiose , Paramecium tetraurellia/citologia , Paramecium tetraurellia/genética , Paramecium tetraurellia/crescimento & desenvolvimento , RNA não Traduzido/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Ciliary shedding occurs from unicellular organisms to metazoans. Although required during the cell cycle and during neurogenesis, the process remains poorly understood. In all cellular models, this phenomenon occurs distal to the transition zone (TZ), suggesting conserved molecular mechanisms. The TZ module proteins (Meckel Gruber syndrome [MKS]/Nephronophtysis [NPHP]/Centrosomal protein of 290 kDa [CEP290]/Retinitis pigmentosa GTPase regulator-Interacting Protein 1-Like Protein [RPGRIP1L]) are known to cooperate to establish TZ formation and function. To determine whether they control deciliation, we studied the function of 5 of them (Transmembrane protein 107 [TMEM107], Transmembrane protein 216 [TMEM216], CEP290, RPGRIP1L, and NPHP4) in Paramecium. All proteins are recruited to the TZ of growing cilia and localize with 9-fold symmetry at the level of the most distal part of the TZ. We demonstrate that depletion of the MKS2/TMEM216 and TMEM107 proteins induces constant deciliation of some cilia, while depletion of either NPHP4, CEP290, or RPGRIP1L prevents Ca2+/EtOH deciliation. Our results constitute the first evidence for a role of conserved TZ proteins in deciliation and open new directions for understanding motile cilia physiology.
Assuntos
Cílios/metabolismo , Paramecium tetraurellia/citologia , Proteínas de Protozoários/metabolismo , Proliferação de Células , Cílios/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Expressão Gênica , Fusão de Membrana/genética , Paramecium tetraurellia/genética , Domínios Proteicos , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Interferência de RNARESUMO
A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to document previously unreported events, and also perform photobleaching experiments, in conjugating Tetrahymena thermophila.
Assuntos
Técnicas Citológicas/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Animais , Técnicas Citológicas/métodos , Drosophila melanogaster/citologia , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/métodos , Paramecium tetraurellia/citologia , Análise de Célula Única , Tetrahymena thermophila/citologia , Leveduras/citologiaRESUMO
The time-honored assumption about the defensive function of trichocysts in Paramecium against predators was recently verified experimentally against different species of unicellular predators. In the present study, we examined the defensive function of trichocysts against three metazoan predators, Cephalodella sp. (Rotifera), Eucypris sp. (Arthropoda), and Stenostomum sphagnetorum (Platyhelminthes). The results confirmed the defensive function of trichocysts against two of these metazoan predators (Cephalodella sp. and Eucypris sp.), while they seem ineffective against S. sphagnetorum. We also compared the defensive efficiency of the trichocysts of P. tetraurelia with that of toxin-containing extrusomes of two ciliates.
Assuntos
Hidroquinonas/toxicidade , Paramecium tetraurellia/citologia , Paramecium tetraurellia/fisiologia , Comportamento Predatório/efeitos dos fármacos , Resorcinóis/toxicidade , Animais , Artrópodes/efeitos dos fármacos , Estrutura Molecular , Platelmintos/efeitos dos fármacos , Rotíferos/efeitos dos fármacos , Toxinas Biológicas/química , Toxinas Biológicas/toxicidadeRESUMO
We characterized the calcineurin (CaN) gene family, including the subunits CaNA and CaNB, based upon sequence information obtained from the Paramecium genome project. Paramecium tetraurelia has seven subfamilies of the catalytic CaNA subunit and one subfamily of the regulatory CaNB subunit, with each subfamily having two members of considerable identity on the amino acid level (>or=55% between subfamilies, >or=94% within CaNA subfamilies, and full identity in the CaNB subfamily). Within CaNA subfamily members, the catalytic domain and the CaNB binding region are highly conserved and molecular modeling revealed a three-dimensional structure almost identical to a human ortholog. At 14 members, the size of the CaNA family is unprecedented, and we hypothesized that the different CaNA subfamily members were not strictly redundant and that at least some fulfill different roles in the cell. This was tested by selecting two phylogenetically distinct members of this large family for posttranscriptional silencing by RNA interference. The two targets resulted in differing effects in exocytosis, calcium dynamics, and backward swimming behavior that supported our hypothesis that the large, highly conserved CaNA family members are not strictly redundant and that at least two members have evolved diverse but overlapping functions. In sum, the occurrence of CaN in Paramecium spp., although disputed in the past, has been established on a molecular level. Its role in exocytosis and ciliary beat regulation in a protozoan, as well as in more complex organisms, suggests that these roles for CaN were acquired early in the evolution of this protein family.
Assuntos
Calcineurina/metabolismo , Cálcio/metabolismo , Domínio Catalítico , Família Multigênica , Paramecium tetraurellia/enzimologia , Proteínas de Protozoários/metabolismo , Calcineurina/genética , Sinalização do Cálcio/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Conversão Gênica/efeitos dos fármacos , Genes de Protozoários , Íntrons/genética , Modelos Biológicos , Movimento/efeitos dos fármacos , Mutação/genética , Paramecium tetraurellia/citologia , Paramecium tetraurellia/efeitos dos fármacos , Paramecium tetraurellia/genética , Filogenia , Cloreto de Potássio/farmacologia , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Interferência de RNA/efeitos dos fármacos , Homologia de Sequência de Aminoácidos , SoluçõesRESUMO
The heavy metal cadmium is a dangerous environmental toxicant that can be lethal to humans and other organisms. This paper demonstrates that cadmium is lethal to the ciliated protozoan Paramecium tetraurelia and that a circadian clock modulates the sensitivity of the cells to cadmium. Various concentrations of cadmium were shown to increase the number of behavioral responses, decrease the swimming speed of cells, and generate large vacuole formation in cells prior to death. Cells were grown in either 12-h light/12-h dark or constant dark conditions exhibited a toxic response to 500 microM CdCl(2); the sensitivity of the response was found to vary with a 24-h periodicity. Cells were most sensitive to cadmium at circadian time 0 (CT0), while they were least sensitive in the early evening (CT12). This rhythm persisted even when the cells were grown in constant dark. The oscillation in cadmium sensitivity was shown to be temperature-compensated; cells grown at 18 degrees C and 28 degrees C had a similar 24-h oscillation. Finally, phase shifting experiments demonstrated a phase-dependent response to light. These data establish the criteria required for a circadian clock and demonstrate that P. tetraurelia possesses a circadian-influenced regulatory component of the cadmium toxic response. The Paramecium system is shown to be an excellent model system for the study of the effects of biological rhythms on heavy metal toxicity.
Assuntos
Relógios Biológicos/efeitos dos fármacos , Cádmio/toxicidade , Ritmo Circadiano/efeitos dos fármacos , Paramecium tetraurellia/efeitos dos fármacos , Paramecium tetraurellia/fisiologia , Relógios Biológicos/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ritmo Circadiano/efeitos da radiação , Luz , Paramecium tetraurellia/citologia , Paramecium tetraurellia/efeitos da radiação , Natação , Fatores de TempoRESUMO
In the Paramecium tetraurelia genome, 17 genes encoding the 100-kDa-subunit (a-subunit) of the vacuolar-proton-ATPase were identified, representing by far the largest number of a-subunit genes encountered in any organism investigated so far. They group into nine clusters, eight pairs with >82% amino acid identity and one single gene. Green fluorescent protein-tagging of representatives of the nine clusters revealed highly specific targeting to at least seven different compartments, among them dense core secretory vesicles (trichocysts), the contractile vacuole complex, and phagosomes. RNA interference for two pairs confirmed their functional specialization in their target compartments: silencing of the trichocyst-specific form affected this secretory pathway, whereas silencing of the contractile vacuole complex-specific form altered organelle structure and functioning. The construction of chimeras between selected a-subunits surprisingly revealed the targeting signal to be located in the C terminus of the protein, in contrast with the N-terminal targeting signal of the a-subunit in yeast. Interestingly, some chimeras provoked deleterious effects, locally in their target compartment, or remotely, in the compartment whose specific a-subunit N terminus was used in the chimera.
Assuntos
Paramecium tetraurellia/enzimologia , ATPases Vacuolares Próton-Translocadoras/fisiologia , Sequência de Aminoácidos , Animais , Compartimento Celular , Citocalasina B/farmacologia , Inativação Gênica , Proteínas de Fluorescência Verde/análise , Imuno-Histoquímica , Dados de Sequência Molecular , Paramecium tetraurellia/citologia , Paramecium tetraurellia/genética , Fagossomos/efeitos dos fármacos , Filogenia , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiologia , Subunidades Proteicas/análise , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Transporte Proteico , Proteínas Recombinantes de Fusão/análise , Vesículas Secretórias/fisiologia , ATPases Vacuolares Próton-Translocadoras/análise , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
The germ line genome of ciliates is extensively rearranged during development of the somatic macronucleus. Numerous sequences are eliminated, while others are amplified to a high ploidy level. In the Paramecium aurelia group of species, transformation of the maternal macronucleus with transgenes at high copy numbers can induce the deletion of homologous genes in sexual progeny, when a new macronucleus develops from the wild-type germ line. We show that this trans-nuclear effect correlates with homology-dependent silencing of maternal genes before autogamy and with the accumulation of approximately 22- to 23-nucleotide (nt) RNA molecules. The same effects are induced by feeding cells before meiosis with bacteria containing double-stranded RNA, suggesting that small interfering RNA-like molecules can target deletions. Furthermore, experimentally induced macronuclear deletions are spontaneously reproduced in subsequent sexual generations, and reintroduction of the missing gene into the variant macronucleus restores developmental amplification in sexual progeny. We discuss the possible roles of the approximately 22- to 23-nt RNAs in the targeting of deletions and the implications for the RNA-mediated genome-scanning process that is thought to determine developmentally regulated rearrangements in ciliates.
Assuntos
Rearranjo Gênico , Genoma de Protozoário , Paramecium tetraurellia/crescimento & desenvolvimento , Paramecium tetraurellia/genética , RNA de Cadeia Dupla/metabolismo , RNA de Protozoário/metabolismo , Animais , Sequência de Bases , Deleção de Genes , Dosagem de Genes , Inativação Gênica , Vetores Genéticos , Paramecium tetraurellia/citologia , RNA de Cadeia Dupla/genética , RNA de Protozoário/genética , Transformação Genética , TransgenesRESUMO
It has been reported that Paramecium proliferates faster when cultured under microgravity in orbit, and slower when cultured under hypergravity. This shows that the proliferation rate of Paramecium affected by gravity. The effect of gravity on Paramecium proliferation has been argued to be direct in a paper with an axenic culture under hypergravity. To clear up uncertainties with regard to the effect of gravity, Paramecium tetraurelia was cultured axenically under hypergravity (20 x g) and the time course of the proliferation was investigated quantitatively by a new non-invasive method, laser-beam optical slice, for measuring the cell density. This method includes optical slicing a part of the culture and computer-aided counting of cells in the sliced volume. The effects of hypergravity were assessed by comparing the kinetic parameters of proliferation that were obtained through a numerical analysis based on the logistic growth equation. Cells grown under 20 x g conditions had a significantly lower proliferation rate, and had a lower population density at the stationary phase. The lowered proliferation rate continued as long as cells were exposed to hypergravity (> one month). Hypergravity reduced the cell size of Paramecium. The long and short axes of the cell became shorter at 20 x g than those of control cells, which indicates a decrease in volume of the cell grown under hypergravity and is consistent with the reported increase in cell volume under microgravity. The reduced proliferation rate implies changes in biological time defined by fission age. In fact the length of autogamy immaturity decreased by measure of clock time, whereas it remained unchanged by measure of fission age.
Assuntos
Hipergravidade , Paramecium tetraurellia/crescimento & desenvolvimento , Paramecium tetraurellia/fisiologia , Animais , Divisão Celular , Paramecium tetraurellia/citologiaRESUMO
Results of the Cytos M experiment and complementary results of the Cytos I experiment flown aboard the Soviet orbital station Salyut 6 are shown. Space flight of Paramecia cultures resulted in a stimulating effect on cell proliferation, in a larger cell volume, in changes in cell dry weight, cell total protein and the electrolyte content of the culture media in which the organisms were grown. The assumption of a possible effect of weightlessness on membrane permeability is discussed.
Assuntos
Paramecium tetraurellia/citologia , Voo Espacial , Ausência de Peso , Animais , Divisão Celular , Meios de Cultura , Eletrólitos , Paramecium tetraurellia/crescimento & desenvolvimento , Proteínas de ProtozoáriosRESUMO
Space flights resulted in a stimulating effect on kinetics of proliferation in Paramecium tetraurelia. Additional experiments were performed in order to determine the origin of this phenomena. Paramecia were cultivated in balloon flights or in a slow clinostat, or were exposed to different levels of hypergravity. The results suggest that changes in cell proliferation rate are related to cosmic rays and to a direct effect of microgravity.
Assuntos
Radiação Cósmica , Hipergravidade , Paramecium tetraurellia/citologia , Rotação , Voo Espacial , Ausência de Peso , Animais , Divisão Celular , Contagem de Colônia Microbiana , Paramecium tetraurellia/fisiologia , Paramecium tetraurellia/efeitos da radiaçãoRESUMO
Life span is the most interesting and also the most important biologically relevant time to be investigated on the space station. As a model experiment, we proposed an investigation to assess the life span of clone generation of the ciliate Paramecium. In space, clone generation will be artificially started by conjugation or autogamy, and the life span of the cell populations in different gravitational fields (microgravity and onboard 1 x g control) will be precisely assessed in terms of fission age as compared with the clock time. In order to perform the space experiment including long-lasting culture and continuous measurement of cell division, we tested the methods of cell culture and of cell-density measurement, which will be available in closed environments under microgravity. The basic design of experimental hardware and a preliminary result of the cultivation procedure are described.
Assuntos
Longevidade/fisiologia , Paramecium tetraurellia/genética , Paramecium tetraurellia/fisiologia , Voo Espacial/instrumentação , Ausência de Peso , Animais , Técnicas de Cultura de Células/instrumentação , Divisão Celular/fisiologia , Células Clonais , Desenho de Equipamento , Paramecium tetraurellia/citologiaRESUMO
The run of autogamy in temperature sensitive mutant ts401, carrying also the mutation of trichocysts nd3a, at permissive (26 degrees C) and non-permissive temperature (35 degrees C) was studied and compared with this process in wild d4-2 stock of Paramecium tetraurelia. The effect of heat treatment on programmed nuclear and cortical events was investigated using cytological silver impregnation Fernandez-Galiano method and immunofluorescence technique with the application of two anti-alpha-tubulin antibodies. The appearance of cells with some large macronuclear fragments and an excess number of micronuclei suggests that nuclear differentiation is inhibited at restrictive temperature and that macronuclear regeneration takes place in this thermosensitive mutant. In such cells impaired oral reorganization was observed. After slight refeeding at the late autogamy, cells stopped at first postautogamous division were induced by heat treatment. The most striking feature of these abnormal divisions was the lack of a basal body duplication wave, which suggests that production of gamma-tubulin, structural protein being the essential component of the microtubule organizing centres, is disturbed at high temperature.
Assuntos
Paramecium tetraurellia/crescimento & desenvolvimento , Animais , Regulação da Expressão Gênica , Temperatura Alta , Microscopia de Fluorescência , Mutação/genética , Paramecium tetraurellia/citologia , Paramecium tetraurellia/genética , Coloração pela PrataRESUMO
It has been reported that Paramecium proliferates faster under microgravity in space, and slower under hypergravity (Kato et al., 2003). Effects of gravity on cell proliferation could be discussed in terms of energetics of swimming. Because of the characteristics of 'gravikinesis' as well as 'gravitaxis', Paramecium would decrease the energy expenditure under microgravity and increase it under hypergravity. The larger stock of energy would enhance the proliferation under microgravity. In order to simulate the effect of microgravity, we investigated the proliferation under clinorotation. When cells were rotated at 2.5 rpm, the proliferation rate decreased. Similar but less pronounced decrease was also found under low speed clinorotation (0.2 rpm).
Assuntos
Proliferação de Células , Paramecium tetraurellia/crescimento & desenvolvimento , Paramecium tetraurellia/fisiologia , Rotação , Simulação de Ausência de Peso , Animais , Divisão Celular , Paramecium tetraurellia/citologia , NataçãoRESUMO
Previous space CYTOS experiments have shown that space flights resulted in an increase in growth of Paramecia cultures. Microgravity is the major factor responsible of this response: indeed the stimulatory effect disappeared in inflight cultures placed on a 1 g centrifuge aboard the Spacelab. On the other hand, exposure to different levels of hypergravity on Earth resulted in an opposite response, i.e. to a reduced cell growth rate. A possible mechanism of microgravity on paramecia is discussed.
Assuntos
Hipergravidade , Paramecium tetraurellia/crescimento & desenvolvimento , Voo Espacial , Ausência de Peso , Animais , Divisão Celular , Centrifugação , Paramecium tetraurellia/citologiaRESUMO
Paramecium tetraurelia cultured aboard Salyut 6 have shown in increase in cell growth rate, cell volume, water content and changes in electrolyte content. Additional experiments, carried out in balloon flight and on earth, showed that the stimulating effect observed on cell proliferation is related to exposure to cosmic rays. Other changes seem to be due to a direct effect of microgravity on cell. Mechanism of gravity action on cell is discussed.
Assuntos
Radiação Cósmica , Paramecium tetraurellia/crescimento & desenvolvimento , Paramecium tetraurellia/efeitos da radiação , Voo Espacial , Ausência de Peso , Animais , Biomassa , Cálcio/metabolismo , Contagem de Colônia Microbiana , Magnésio/metabolismo , Paramecium tetraurellia/citologia , Paramecium tetraurellia/metabolismo , Proteínas de Protozoários/metabolismoRESUMO
The paper shows the results of investigations carried out in a single cell organism. Paramecium tetraurelia exposed to different gravitational levels. Hypergravity resulted in a decrease in cell growth rate. The responses depend on g level and angular speed of the centrifuge; furthermore they depend also on small short fluctuations in g levels, delta g, due to the swimming of the cells inside the culture tubes. Delta g depends on angular speed and size of the holding device. The inhibitory effect of hypergravity, for the same angular speed, increases with respect of the diameter of the culture tubes.
Assuntos
Hipergravidade , Paramecium tetraurellia/crescimento & desenvolvimento , Animais , Divisão Celular , Centrifugação , Força Coriolis , Paramecium tetraurellia/citologia , RotaçãoRESUMO
The contractile vacuole complex (CVC) of freshwater protists serves the extrusion of water and ions, including Ca(2+). No vesicle trafficking based on SNAREs has been detected so far in any CVC. SNAREs (soluble NSF [N-ethylmaleimide sensitive factor] attachment protein receptors) are required for membrane-to-membrane interaction, i.e. docking and fusion also in Paramecium. We have identified three v-/R- and three t/Q-SNAREs selectively in the CVC. Posttranscriptional silencing of Syb2, Syb6 or Syx2 slows down the pumping cycle; silencing of the latter two also causes vacuole swelling. Increase in extracellular Ca(2+) after Syb2, Syb6 or Syx2 silencing causes further swelling of the contractile vacuole and deceleration of its pulsation. Silencing of Syx14 or Syx15 entails lethality in the Ca(2+) stress test. Thus, the effects of silencing strictly depend on the type of the silenced SNARE and on the concentration of Ca(2+) in the medium. This shows the importance of organelle-resident SNARE functions (which may encompass the vesicular delivery of other organelle-resident proteins) for Ca(2+) tolerance. A similar principle may be applicable also to the CVC in widely different unicellular organisms. In addition, in Paramecium, silencing particularly of Syx6 causes aberrant positioning of the CVC during de novo biogenesis before cytokinesis.