Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.     

Imaging Centrosomes in Fly Testes

Centrosomes are conserved microtubule-based organelles whose structure and function change dramatically throughout the cell cycle and cell differentiation. Centrosomes are essential to determine the cell division axis during mitosis and to nucleate cilia during interphase. The identity of the proteins that mediate these dynamic changes remains only partially known, and the function of many of the proteins that have been implicated in these processes is still rudimentary. Recent work has shown that Drosophila spermatogenesis provides a powerful system to identify new proteins critical for centrosome function and formation as well as to gain insight into the particular function of known players in centrosome-related processes. Drosophila is an established genetic model organism where mutants in centrosomal genes can be readily obtained and easily analyzed. Furthermore, recent advances in the sensitivity and resolution of light microscopy and the development of robust genetically tagged centrosomal markers have transformed the ability to use Drosophila testes as a simple and accessible model system to study centrosomes. This paper describes the use of genetically-tagged centrosomal markers to perform genetic screens for new centrosomal mutants and to gain insight into the specific function of newly identified genes.     

Prokaryotic ParA-ParB-parS system links bacterial chromosome segregation with the cell cycle

While the essential role of episomal par loci in plasmid DNA partitioning has long been appreciated, the function of chromosomally encoded par loci is less clear. The chromosomal parA-parB genes are conserved throughout the bacterial kingdom and encode proteins homologous to those of the plasmidic Type I active partitioning systems. The third conserved element, the centromere-like sequence called parS, occurs in several copies in the chromosome. Recent studies show that the ParA-ParB-parS system is a key player of a mitosis-like process ensuring proper intracellular localization of certain chromosomal regions such as oriC domain and their active and directed segregation. Moreover, the chromosomal par systems link chromosome segregation with initiation of DNA replication and the cell cycle.     

Upstream open reading frames regulate the cell cycle-dependent expression of the RNA helicase Rok1 in Saccharomyces cerevisiae

The RNA helicase Rok1 plays a role in rRNA processing and in control of cell cycle progression in Saccharomyces cerevisiae. We identified two upstream open reading frames (uORFs) within the ROK1 5′ untranslated region, which inhibited Rok1 translation. Mutating uATG to uAAG or generation of a premature stop codon in the uORFs resulted in increased Rok1p levels. Rok1 protein levels oscillated during the cell cycle, declining at G1/S and increasing at G2. The uAAG1 mutation caused a constitutive level of Rok1 proteins throughout the cell cycle, resulting in significant delays in mitotic bud emergence and recovery from pheromone arrest. Our study reveals that the Rok1 protein level is regulated by uORFs, which is critical in cell cycle progression.     

The small ubiquitin-like modifier (SUMO) is essential in cell cycle regulation in Trypanosoma brucei

SUMO, a reversible post-translational protein modifier, plays important roles in many processes of higher eukaryotic cell life. Although SUMO has been identified in many eukaryotes, SUMO and SUMO system are still unknown in some eukaryotic unicellular organisms, such as Trypanosoma brucei (T. brucei). In this study, only one SUMO homologue (TbSUMO) was identified in T. brucei. Expression of TbSUMO was knocked down by using RNA interference technique in procyclic-form T. brucei. The growth of TbSUMO-deficient cells was significantly inhibited. TbSUMO-deficient cells were arrested in G₂/M phase accompanied with an obvious increase of 0N1K cells (zoids), and failed in chromosome segregation. These results indicate that TbSUMO is essential in cell cycle regulation, with one important role in mitosis. Meanwhile, the enrichment of zoids suggests the inhibition of mitosis does not prevent the cell division in procyclic-form T. brucei. HA-tagged TbSUMO was overexpressed in T. brucei and was shown to be localized to the nucleus through the whole cell cycle, further revealing its distinguished functions in nucleus. All these accumulated data imply that a SUMO system essential for regulating cell cycle progression might exist in the procyclic-form T. brucei.     

RNA interference screen reveals a high proportion of mitochondrial proteins essential for correct cell cycle progress in Trypanosoma brucei

Background

Trypanosomatid parasites possess a single mitochondrion which is classically involved in the energetic metabolism of the cell, but also, in a much more original way, through its single and complex DNA (termed kinetoplast), in the correct progress of cell division. In order to identify proteins potentially involved in the cell cycle, we performed RNAi knockdowns of 101 genes encoding mitochondrial proteins using procyclic cells of Trypanosoma brucei.

Results

A major cell growth reduction was observed in 10 cases and a moderate reduction in 29 other cases. These data are overall in agreement with those previously obtained by a case-by-case approach performed on chromosome 1 genes, and quantitatively with those obtained by “high-throughput phenotyping using parallel sequencing of RNA interference targets” (RIT-seq). Nevertheless, a detailed analysis revealed many qualitative discrepancies with the RIT-seq-based approach. Moreover, for 37 out of 39 mutants for which a moderate or severe growth defect was observed here, we noted abnormalities in the cell cycle progress, leading to increased proportions of abnormal cell cycle stages, such as cells containing more than 2 kinetoplasts (K) and/or more than 2 nuclei (N), and modified proportions of the normal phenotypes (1N1K, 1N2K and 2N2K).

Conclusions

These data, together with the observation of other abnormal phenotypes, show that all the corresponding mitochondrial proteins are involved, directly or indirectly, in the correct progress or, less likely, in the regulation, of the cell cycle in T. brucei. They also show how post-genomics analyses performed on a case-by-case basis may yield discrepancies with global approaches.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1505-5) contains supplementary material, which is available to authorized users.     

ELONGATA3 is required for shoot meristem cell cycle progression in Arabidopsis thaliana seedlings

A key feature of the development of a higher plant is the continuous formation of new organs from the meristems. Originally patterned during embryogenesis, the meristems must activate cell division de novo at the time of germination, in order to initiate post-embryonic development. In a mutagenesis screen aimed at finding new players in early seedling cell division control, we identified ELONGATA3 (ELO3) as a key regulator of meristem cell cycle activation in Arabidopsis. Our results show that plants carrying a hypomorphic allele of ELO3 fail to activate cell division in the meristems following germination, which leads to seedling growth arrest and lethality. Further analyses suggest that this is due to a failure in DNA replication, followed by cell cycle arrest, in the meristematic tissue. Interestingly, the meristem cell cycle arrest in elo3 mutants, but not the later leaf developmental defects that have been linked to the loss of ELO3 activities, can be relieved by the addition of metabolic sugars in the growth medium. This finding points to a new role by which carbohydrate availability promotes meristem growth. Furthermore, growth arrested elo3 mutants suffer a partial loss of shoot meristem identity, which provides further evidence that cell cycle activities can influence the control of tissue identity.     

Molecular Docking of Known Carcinogen 4- (Methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) with Cyclin Dependent Kinases towards Its Potential Role in Cell Cycle Perturbation

Cell cycle is maintained almost all the times and is controlled by various regulatory proteins and their complexes (Cdk+Cyclin) in different phases of interphase (G₁, S and G₂) and mitosis of cell cycle. A number of mechanisms have been proposed for the initiation and progression of carcinogenesis by abruption in cell cycle process. One of the important features of cancer/carcinogenesis is functional loss of these cell cycle regulatory proteins particularly in CDKs and cyclins. We hypothesize that there is a direct involvement of these cell cycle regulatory proteins not only at the genetic level but also proteins level, during the initiation of carcinogenesis. Therefore, it becomes significant to determine inconsistency in the functioning of regulatory proteins due to interaction with carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Hence, we investigated the interaction efficiency of NNK, against cell cycle regulatory proteins. We found a different value of ΔG (free energy of binding) among the studied proteins ranging between -3.29 to -7.25 kcal/mol was observed. To validate the results, we considered Human Oxy-Hemoglobin at 1.25 Å Resolution, [PDB_ID:1HHO] as a +ve control, (binding energy -6.06 kcal/mol). Finally, the CDK8 (PDB_ID:3RGF) and CDK2 (PDB_ID:3DDP) regulatory proteins showing significantly strong molecular interaction with NNK -7.25 kcal/mol, -6.19 kcal/mol respectively were analyzed in details. In this study we predicted that CDK8 protein fails to form functional complex with its complementary partner cyclin C in presence of NNK. Consequently, inconsistency of functioning in regulatory proteins might lead to the abruption in cell cycle progression; contribute to the loss of cell cycle control and subsequently increasing the possibility of carcinogenesis.     

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 µm of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.     

A Leishmania donovani gene that confers accelerated recovery from stationary phase growth arrest

We have isolated a gene, LdGF1, from the protozoan parasite Leishmania donovani. Overexpression of this gene confers a strong selective advantage in liquid culture after stationary phase growth arrest. We could show that recombinant L. donovani or Leishmania major, when overexpressing LdGF1, recover faster from a stationary phase growth arrest than control parasite strains. While no advantage of LdGF1 overexpression could be observed in log phase cultures or after a hydroxyurea-induced S-phase growth arrest, recovery from a cell cycle arrest due to serum deprivation was faster in LdGF1-overexpressing strains. This was found to be due to an accelerated release from a G₁ cell cycle arrest. By contrast, in a BALB/c mouse infection system, overexpression of LdGF1 in L. major resulted in reduced virulence. We conclude that increased levels of LdGF1 are beneficiary during recovery from G₁ cell cycle arrest, but pose a disadvantage inside a mammalian host. These results are discussed in the context of the observed loss of virulence during in vitro passage of Leishmania parasites.     

2-Methoxy-4-vinylphenol can induce cell cycle arrest by blocking the hyper-phosphorylation of retinoblastoma protein in benzo[a]pyrene-treated NIH3T3 cells

Benzo[a]pyrene (BaP) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. In this study, the effects of 2M4VP on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in BaP-treated NIH 3T3 cells to establish the molecular mechanisms of 2M4VP as anti-proliferative agents. 2M4VP exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed 2M4VP increased expression of CDK inhibitor, p21Waf1/Cip1 and p15 INK4b, decreased expression of cyclin D1 and cyclin E, and inhibited kinase activities of CDK4 and CDK2. However, 2M4VP did not affect the expression of CDK4 and CDK2. Also, 2M4VP inhibited the hyper-phosphorylation of Rb induced by BaP. Our results suggest that 2M4VP induce growth arrest of BaP-treated NIH 3T3 cells by blocking the hyper-phosphorylation of Rb via regulating the expression of cell cycle-related proteins.     

Revealing oxidative damage to enzymes of carbohydrate metabolism in yeast: An integration of 2D DIGE,quantitative proteomics,and bioinformatics

Clinical usage of lidocaine, a pro‐oxidant has been linked with severe, mostly neurological complications. The mechanism(s) causing these complications is independent of the blockade of voltage‐gated sodium channels. The budding yeast Saccharomyces cerevisiae lacks voltage‐gated sodium channels, thus provides an ideal system to investigate lidocaine‐induced protein and pathway alterations. Whole‐proteome alterations leading to these complications have not been identified. To address this, S. cerevisiae was grown to stationary phase and exposed to an LC₅₀ dose of lidocaine. The differential proteomes of lidocaine treatment and control were resolved 6 h post exposure using 2D DIGE. Amine reactive dyes and carbonyl reactive dyes were used to assess protein abundance and protein oxidation, respectively. Quantitative analysis of these dyes (? 1.5‐fold alteration, p ? 0.05) revealed a total of 33 proteoforms identified by MS differing in abundance and/or oxidation upon lidocaine exposure. Network analysis showed enrichment of apoptotic proteins and cell wall maintenance proteins, while the abundance of proteins central to carbohydrate metabolism, such as triosephosphate isomerase and glyceraldehyde‐3‐phosphate dehydrogenase, and redox proteins superoxide dismutase and peroxiredoxin were significantly decreased. Enzymes of carbohydrate metabolism, such as phosphoglycerate kinase and enolase, the TCA cycle enzyme aconitase, and multiple ATP synthase subunits were found to be oxidatively modified. Also, the activity of aconitase was found to be decreased. Overall, these data suggest that toxic doses of lidocaine induce significant disruption of glycolytic pathways, energy production, and redox balance, potentially leading to cell malfunction and death.     

Cell cycle independent role of Cyclin E during neural cell fate specification in Drosophila is mediated by its regulation of Prospero function

During development, neural progenitor cells or neuroblasts generate a great intra- and inter-segmental diversity of neuronal and glial cell types in the nervous system. In thoracic segments of the embryonic central nervous system of Drosophila, the neuroblast NB6-4t undergoes an asymmetric first division to generate a neuronal and a glial sublineage, while abdominal NB6-4a divides once symmetrically to generate only 2 glial cells. We had earlier reported a critical function for the G1 cyclin, CyclinE (CycE) in regulating asymmetric cell division in NB6-4t. Here we show that (i) this function of CycE is independent of its role in cell cycle regulation and (ii) the two functions are mediated by distinct domains at the protein level. Results presented here also suggest that CycE inhibits the function of Prospero and facilitates its cortical localization, which is critical for inducing stem cell behaviour, i.e. asymmetric cell division of NB6-4t. Furthermore our data imply that CycE is required for the maintenance of stem cell identity of most other neuroblasts.     

A cAMP receptor-like G protein-coupled receptor with roles in growth regulation and development

Dictyostelium discoideum uses G protein-mediated signal transduction for many vegetative and developmental functions, suggesting the existence of G protein-coupled receptors (GPCRs) other than the four known cyclic adenosine monophosphate (cAMP) receptors (cAR1-4). Sequences of the cAMP receptors were used to identify Dictyostelium genes encoding cAMP receptor-like proteins, CrlA-C. Limited sequence identity between these putative GPCRs and the cAMP receptors suggests the Crl receptors are unlikely to be receptors for cAMP. The crl genes are expressed at various times during growth and the developmental life cycle. Disruption of individual crl genes did not impair chemotactic responses to folic acid or cAMP or alter cAMP-dependent aggregation. However, crlA⁻ mutants grew to a higher cell density than did wild-type cells and high-copy-number crlA expression vectors were detrimental to cell viability, suggesting that CrlA is a negative regulator of cell growth. In addition, crlA⁻ mutants produce large aggregates with delayed anterior tip formation indicating a role for the CrlA receptor in the development of the anterior prestalk cell region. The scarcity of GFP-expressing crlA⁻ mutants in the anterior prestalk cell region of chimeric organisms supports a cell-autonomous role for the CrlA receptor in prestalk cell differentiation.