Midwifery and assisted reproduction in Dictyostelium and Entamoeba

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Leishmanial proteins can correct human mitochondrial mutations

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Delinking of S phase and cytokinesis in the protozoan parasite

The alternation of DNA replication in S phase and chromosome segregation in M phase is a hallmark in the cell cycle of most well-studied eukaryotes and ensures that the progeny do not have more than the normal complement of genes and chromosomes. An exception to this rule has been described in cancer cells that occasionally become polyploid as a result of failure to restrain S phase despite the failure to undergo complete mitosis. Here, we describe the cell division cycle of the human pathogen, Entamoeba histolytica, which routinely accumulates polyploid cells. We have studied DNA synthesis in freshly subcultured cells and show that, unlike most eukaryotes, Entamoeba cells reduplicate their genome several times before cell division occurs. Furthermore, polyploidy may occur without nuclear division so that single nuclei may contain 1-10 times or more genome contents. Multinucleated cells may also accumulate several genome contents in each nuclei of one cell. Thus, checkpoints that normally prevent DNA reduplication until after cytokinesis in most eukaryotes are not observed in E. histolytica

Bipolar spindle frequency and genome content are inversely regulated by the activity of two N-type kinesins in Entamoeba histolytica

Bipolar microtubular spindles are seen infrequently in Entamoeba histolytica trophozoites while monopolar or radial microtubular assemblies are common. Additionally, heterogeneity in nuclear DNA content and multi-nucleation is found in amoeba cells growing in axenic culture. Taken together these observations indicate that genome segregation is irregular in these cells. In order to identify proteins involved in regulating genome segregation, we have focused on studying E. histolytica homologues of kinesin motor proteins that are known to affect stability of bipolar mitotic spindles. We have demonstrated earlier that increased levels of the kinesin - Eh Klp5 - led to increased frequency of bipolar spindles accompanied with a reduction in the heterogeneity of genome content, showing that bipolar spindle frequency was inversely linked to genome content in E. histolytica. In this study, we have investigated the role of E. histolytica kinesins (Eh KlpA1, 2-4) in regulating bipolar spindle frequency and genome content. While downregulation of Eh Klp3, 4 and A1 showed no effect, downregulation of Eh Klp2 led to increased frequency of bipolar spindles and homogenization of genome content, similar to the effect of increased expression of Eh Klp5. In addition to microtubules, Eh Klp2-4 associated with F-actin in the cytoplasm, suggesting that these kinesins are multi-functional

Eukaryotic checkpoints are absent in the cell division cycle of Entamoeba histolytica

Fidelity in transmission of genetic characters is ensured by the faithful duplication of the genome, followed by equal segregation of the genetic material in the progeny. Thus, alternation of DNA duplication (S-phase) and chromosome segregation during the M-phase are hallmarks of most well studied eukaryotes. Several rounds of genome reduplication before chromosome segregation upsets this cycle and leads to polyploidy. Polyploidy is often witnessed in cells prior to differentiation, in embryonic cells or in diseases such as cancer. Studies on the protozoan parasite, Entamoeba histolytica suggest that in its proliferative phase, this organism may accumulate polyploid cells. It has also been shown that although this organism contains sequence homologs of genes which are known to control the cell cycle of most eukaryotes, these genes may be structurally altered and their equivalent function yet to be demonstrated in amoeba. The available information suggests that surveillance mechanisms or 'checkpoints' which are known to regulate the eukaryotic cell cycle may be absent or altered in E. histolytica

Eh Klp5 is a divergent member of the kinesin 5 family that regulates genome content and microtubular assembly in Entamoeba histolytica

Earlier studies have established two unusual features in the cell division cycle of Entamoeba histolytica. First, microtubules form a radial assembly instead of a bipolar mitotic spindle, and second, the genome content of E. histolytica cells varied from 1X to 6X or more. In this study, Eh Klp5 was identified as a divergent member of the BimC kinesin family that is known to regulate formation and stabilization of the mitotic spindle in other eukaryotes. In contrast to earlier studies, we show here that bipolar microtubular spindles were formed in E. histolytica but were visible only in 8-12% of the cells after treatment with taxol. The number of bipolar spindles was significantly increased in Eh Klp5 stable transformants (20-25%) whereas Eh Klp5 double-stranded RNA (dsRNA) transformants did not show any spindles (< 1%). The genome content of Eh Klp5 stable transformants was regulated between 1X and 2X unlike control cells. Binucleated cells accumulated in Eh Klp5 dsRNA transformants and after inhibition of Eh Klp5 with small molecule inhibitors in control cells, suggesting that cytokinesis was delayed in the absence of Eh Klp5. Taken together, our results indicate that Eh Klp5 regulates microtubular assembly, genome content and cell division in E. histolytica. Additionally, Eh Klp5 showed alterations in its drug-binding site compared with its human homologue, Hs Eg5 and this was reflected in its reduced sensitivity to Eg5 inhibitors - monastrol and HR22C16 analogues

Lysis of vibrio cholerae cells: direct isolation of the outer membrane from whole cells by treatment with urea

Cells of Vibrio cholerae underwent rapid autolysis when suspended in media of low osmolarity under non-growing conditions. Chaotropes like urea and guanidine. HCl which are potent protein denaturants caused complete and immediate lysis of whole cells. This unique sensitivity of V. cholerae to protein denaturants led to the development of a rapid method for the selective isolation of the outer membrane upon treatment of whole cells with urea. The composition of the outer membrane isolated from both whole cells and crude envelopes by treatment with urea was comparable with that of the outer membrane isolated by other conventional methods

The mcm17 mutation of yeast shows a size-dependent segregational defect of a mini-chromosome

Mini-chromosome-maintenance (mcm) mutants were described earlier as yeast mutants which could not stably maintain mini-chromosomes. Out of these, the ARS-specific class has been more extensively studied and is found to lose chromosomes and mini-chromosomes due to a defect in the initiation of DNA replication at yeast ARSs. In the present study we have identified a number of mcm mutants which show size-dependent loss of mini-chromosomes. When the size of the mini-chromosome was increased, from about 15 kb to about 60 kb, there was a dramatic increase in its mitotic stability in these mutants, but not in the ARS-specific class of mutants. One mutant, mcm17, belonging to the size-dependent class was further characterized. In this mutant, cells carried mini-chromosomes in significantly elevated copy numbers, suggesting a defect in segregation. This defect was largely suppressed in the 60-kb mini-chromosome. A non-centromeric plasmid, the TRP1ARS1 circle, was not affected in its maintenance. This mutant also displayed enhanced chromosome-III loss during mitosis over the wild-type strain, without elevating mitotic recombination. Cloning and sequencing of MCM17 has shown it to be the same as CHL4, a gene required for chromosome stability. This gene is non-essential for growth, as its disruption or deletion from the chromosome did not affect the growth-rate of cells at 23 °C or 37 °C. This work suggests that centromere-directed segregation of a chromosome in yeast is strongly influenced by its length

Constitutive association of Mcm2-3-5 proteins with chromatin in Entamoeba histolytica

Eukaryotic cells duplicate their genome once and only once per cell cycle. Our earlier studies with the protozoan parasite, Entamoeba histolytica, have shown that genome reduplication may occur several times ?without ?nuclear ?or ?cellular ?division. ?The Mcm2-7 protein complex is required for licensing of DNA replication. In an effort to understand whether genome reduplication occurs due to absence or failure of the DNA replication licensing system, we analysed the function of Mcm2-3-5 proteins in E. histolytica. In this study, we have cloned E. histolytica (Eh) MCM2 and Eh MCM5 genes, while Eh MCM3 was cloned earlier. The sequence of Eh MCM2-3-5 genes is well conserved with other eukaryotic homologues. We have shown that Eh Mcm2,3 proteins are functional in Saccharomyces cerevisiae. Our studies in E. histolytica showed that Eh Mcm2-3-5 proteins are associated with chromatin constitutively in cycling cells and during arrest of DNA synthesis induced by serum starvation. Alternation of genome duplication with mitosis is regulated by association-dissociation of Mcm2-7 proteins with chromatin in other eukaryotes. Our results suggest that constitutive association of Mcm proteins with chromatin could be one of the reasons why genome reduplication occurs in E. histolytica

Genome re-duplication and irregular segregation occur during the cell cycle of Entamoeba histolytica

Heterogeneity of genome content is commonly observed in axenic cultures of Entamoeba histolytica. Cells with multiple nuclei and nuclei with heterogenous genome contents suggest that regulatory mechanisms that ensure alternation of DNA synthesis and mitosis are absent in this organism. Therefore, several endo-reduplicative cycles may occur without mitosis. The data also shows that unlike other endo-reduplicating organisms, E.histolytica does not undergo a precise number of endo-reduplicative cycles. We propose that irregular endo-reduplication and genome partitioning lead to heterogeneity in the genome content of E.histolytica trophozoites in their proliferative phase. The goal of future studies should be aimed at understanding the mechanisms that are involved in (a) accumulation of multiple genome contents in a single nucleus; (b) genome segregation in nuclei that contain multiple genome contents and (c) maintenance of genome fidelity in E. histolytica