Cdc2 Kinase Research Articles

A noncanonical GTPase signaling mechanism controls exit from mitosis in budding yeast

In the budding yeast Saccharomyces cerevisiae, exit from mitosis is coupled to spindle position to ensure successful genome partitioning between mother and daughter cells. This coupling occurs through a GTPase signaling cascade known as the mitotic exit network (MEN). The MEN senses spindle position via a Ras-like GTPase Tem1 which localizes to the spindle pole bodies (SPBs, yeast equivalent of centrosomes) during anaphase and signals to its effector protein kinase Cdc15. How Tem1 couples the status of spindle position to MEN activation is not fully understood. Here, we show that Cdc15 has a relatively weak preference for Tem1GTP and Tem1's nucleotide state does not change upon MEN activation. Instead, we find that Tem1's nucleotide cycle establishes a localization-based concentration difference in the cell where only Tem1GTP is recruited to the SPB, and spindle position regulates the MEN by controlling Tem1 localization to the SPB. SPB localization of Tem1 primarily functions to promote Tem1-Cdc15 interaction for MEN activation by increasing the effective concentration of Tem1. Consistent with this model, we demonstrate that artificially tethering Tem1 to the SPB or concentrating Tem1 in the cytoplasm with genetically encoded multimeric nanoparticles could bypass the requirement of Tem1GTP and correct spindle position for MEN activation. This localization/concentration-based GTPase signaling mechanism for Tem1 differs from the canonical Ras-like GTPase signaling paradigm and is likely relevant to other localization-based signaling scenarios.

Antiproliferative Activity of Cephalotaxus Esters: Overcoming Chemoresistance.

Omacetaxine, a semisynthetic form of Homoharringtonine (HHT), was approved for the treatment of Chronic Myeloid Leukemia (CML). Previously, we have published the synthesis of this natural alkaloid and three of its derivatives: Deoxyharringtonine (DHT), Deoxyhomoharringtonine (DHHT), and Bis(demethyl)-deoxyharringtonine (BDHT), and reported its refractory activity against the HL-60/RV+ cells over-expressing P-glycoprotein 1 (MDR1). In this study, we have explored the extent of this resistance by first expanding the panel of established cell lines and using a panel of 21 leukemia patient-derived primary cells. Herein, we have reported consistent resistance to HTT of K562-derived cells and to mitoxantrone of MES-SA/MX2-derived cells; all of them have been found to overexpress MDR1, while we have found U87MG-ABCG2 and H69AR cells to be very sensitive to HTT. In contrast, DHT, DHHT, and BDHT seemingly overcame this resistance due to the changes made to the acyl chain of HTT, rendering the derivatives less susceptible to efflux. Surprisingly, the leukemia primary cells were very sensitive to HHT and its derivatives with low nanomolar potencies, followed by a new class of CDC7 kinase inhibitors, the anthracycline class of topoisomerase inhibitors, the DNA intercalator actinomycin-D, and the vinca alkaloid class of microtubule inhibitors. The mechanism of cell death induced by HTT and DHHT was found to be mediated via caspase 3 cleavage, leading to apoptosis. Taken together, our results confirm that HHT is a substrate for MDR1. It opens the door to a new opportunity to clinically evaluate HHT and its derivatives for the treatment of AML and other cancers.

DBF4, not DRF1, is the crucial regulator of CDC7 kinase at replication forks.

CDC7 kinase is crucial for DNA replication initiation and is involved in fork processing and replication stress response. Human CDC7 requires the binding of either DBF4 or DRF1 for its activity. However, it is unclear whether the two regulatory subunits target CDC7 to a specific set of substrates, thus having different biological functions, or if they act redundantly. Using genome editing technology, we generated isogenic cell lines deficient in either DBF4 or DRF1: these cells are viable but present signs of genomic instability, indicating that both can independently support CDC7 for bulk DNA replication. Nonetheless, DBF4-deficient cells show altered replication efficiency, partial deficiency in MCM helicase phosphorylation, and alterations in the replication timing of discrete genomic regions. Notably, we find that CDC7 function at replication forks is entirely dependent on DBF4 and not on DRF1. Thus, DBF4 is the primary regulator of CDC7 activity, mediating most of its functions in unperturbed DNA replication and upon replication interference.

Gene Expression Analysis of Yeast Strains with a Nonsense Mutation in the eRF3-Coding Gene Highlights Possible Mechanisms of Adaptation.

In yeast Saccharomyces cerevisiae, there are two translation termination factors, eRF1 (Sup45) and eRF3 (Sup35), which are essential for viability. Previous studies have revealed that presence of nonsense mutations in these genes leads to amplification of mutant alleles (sup35-n and sup45-n), which appears to be necessary for the viability of such cells. However, the mechanism of this phenomenon remained unclear. In this study, we used RNA-Seq and proteome analysis to reveal the complete set of gene expression changes that occur during cellular adaptation to the introduction of the sup35-218 nonsense allele. Our analysis demonstrated significant changes in the transcription of genes that control the cell cycle: decreases in the expression of genes of the anaphase promoting complex APC/C (APC9, CDC23) and their activator CDC20, and increases in the expression of the transcription factor FKH1, the main cell cycle kinase CDC28, and cyclins that induce DNA biosynthesis. We propose a model according to which yeast adaptation to nonsense mutations in the translation termination factor genes occurs as a result of a delayed cell cycle progression beyond the G2-M stage, which leads to an extension of the S and G2 phases and an increase in the number of copies of the mutant sup35-n allele.

Proteostatic tuning underpins the evolution of novel multicellular traits.

The evolution of multicellularity paved the way for the origin of complex life on Earth, but little is known about the mechanistic basis of early multicellular evolution. Here, we examine the molecular basis of multicellular adaptation in the multicellularity long-term evolution experiment (MuLTEE). We demonstrate that cellular elongation, a key adaptation underpinning increased biophysical toughness and organismal size, is convergently driven by down-regulation of the chaperone Hsp90. Mechanistically, Hsp90-mediated morphogenesis operates by destabilizing the cyclin-dependent kinase Cdc28, resulting in delayed mitosis and prolonged polarized growth. Reinstatement of Hsp90 or Cdc28 expression resulted in shortened cells that formed smaller groups with reduced multicellular fitness. Together, our results show how ancient protein folding systems can be tuned to drive rapid evolution at a new level of biological individuality by revealing novel developmental phenotypes.

A First-in-Human, Phase 1, Dose Escalation Study of Sgr-2921 As Monotherapy in Subjects with Relapsed/Refractory Acute Myeloid Leukemia or Myelodysplastic Syndrome

Background Cell division cycle 7-related protein kinase (CDC7), or DBF4-dependent CDC7 kinase (DDK), is a cell cycle kinase that maintains DNA replication by phosphorylation and activation of the minichromosome maintenance protein 2 and 4 (MCMs), components of the replicative DNA helicase. Due to the central role of CDC7 in maintenance of the replication fork integrity, chemical inhibition of CDC7 kinase can ultimately lead to cancer cell death. SGR-2921 is an oral, small molecule inhibitor of CDC7. Preclinical studies demonstrate that, among all cell lines tested, SGR-2921 has the most potent anti-proliferative activity in AML. Potent antitumor activity has also been demonstrated in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) AML models. This antitumor response is observed in animal models representative of difficult to treat patient populations and appears to be agnostic of serious mutations (including those with p53 mutations and FLT3 mutations), BTK resistance, and multiple prior lines of treatment. Patients with relapsed or refractory (R/R) acute myeloid leukemia (AML) or high-risk (HR) or very high-risk (VHR) myelodysplastic syndrome (MDS) have low response rates and poor overall survival. Considering the potent preclinical activity of SGR-2921 in AML and MDS models, coupled with the high unmet medical need in this population, SGR-2921 is being evaluated in patients with R/R AML and HR/VHR MDS. Study Design and Methods This is a phase 1, FIH, open-label, single-agent, two-arm, dose escalation study (NCT#05961839) designed to evaluate safety and tolerability and identify the recommended phase 2 dose (RP2D) of SGR-2921 as monotherapy in subjects with R/R AML, HR MDS, or VHR MDS. The study utilizes a hybrid accelerated titration design with single patient cohorts that transitions to a 3+3 design once a single Grade 2 event or DLT is observed. This is a multicenter global study (US, Spain and France) with an estimated study recruitment start date in October, 2023. SGR-2921 will be administered orally, once daily, utilizing a 5-day on and 9-day off dosing schedule over a 28-day cycle. Up to a maximum of 144 patients will be enrolled in the dose escalation and exploratory cohort phase of the study. To evaluate the effect of CYP3A4 inhibition on SGR-2921 exposure, subjects will be enrolled into one of two staggered, parallel study treatment arms, according to concomitant administration with (Arm B) or without (Arm A) azole antifungals that are strong CYP3A4 inhibitors at time of first dose. Safety and tolerability must be demonstrated in treatment Arm A, at the first two dose levels before initiating treatment Arm B. A single dose PK run-in will be required for subjects enrolled into the first 3 cohorts of each treatment arm. Subjects will be treated at increasing doses of study drug until all dose levels have been investigated or any dose level is found to exceed the maximum tolerated dose (MTD). A RP2D will be selected from one of the tolerable dose levels which will not exceed the MTD (see Figure 1 for study design). Key study inclusion criteria include: Age ≥ 18 years of age; Life expectancy ≥ 8 weeks; Confirmed diagnosis of R/R AML or HR and VHR MDS; Eastern Cooperative Oncology Group (ECOG) performance status ≤ 2. Key study exclusion criteria include: Active malignancies not related to AML or MDS within two years prior to the first dose or requiring ongoing treatment; Clinical evidence of central nervous system (CNS) or pulmonary leukostasis; ≥ Grade 3 disseminated intravascular coagulation, or active CNS leukemia; QT interval corrected for heart rate per Fridericia's formula ≥470 msec during screening ECG. The study primary objectives are to evaluate the safety and tolerability of SGR-2921 as monotherapy and identify RP2D including MTD. Secondary objectives include evaluating the pharmacokinetics (PK) of SGR-2921 and investigating preliminary antitumor activity (composite complete remission rate, objective response rate, duration of response, etc.).

Protein kinase Cdc7 supports viral replication by phosphorylating Avibirnavirus VP3 protein.

The Avibirnavirus infectious bursal disease virus is still an important agent which largely threatens global poultry farming industry economics. VP3 is a multifunctional scaffold structural protein that is involved in virus morphogenesis and the regulation of diverse cellular signaling pathways. However, little is known about the roles of VP3 phosphorylation during the IBDV life cycle. In this study, we determined that IBDV infection induced the upregulation of Cdc7 expression and phosphorylated the VP3 Ser13 site to promote viral replication. Moreover, we confirmed that the negative charge addition of phosphoserine on VP3 at the S13 site was essential for IBDV proliferation. This study provides novel insight into the molecular mechanisms of VP3 phosphorylation-mediated regulation of IBDV replication.

Glucose to lactate shift reprograms CDK-dependent mitotic decisions and its communication with MAPK Sty1 in Schizosaccharomyces pombe.

Cell cycle regulation in response to biochemical cues is a fundamental event associated with many diseases. The regulation of such responses in complex metabolic environments is poorly understood. This study reveals unknown aspects of the metabolic regulation of cell division in Schizosaccharomyces pombe. We show that changing the carbon source from glucose to lactic acid alters the functions of the cyclin-dependent kinase (CDK) Cdc2 and mitogen-activated protein kinase (MAPK) Sty1, leading to unanticipated outcomes in the behavior and fate of such cells. Functional communication of Cdc2 with Sty1 is known to be an integral part of the cellular response to aberrant Cdc2 activity in S. pombe. Our results show that cross-talk between Cdc2 and Sty1, and the consequent Sty1-dependent regulation of Cdc2 activity, appears to be compromised and the relationship between Cdc2 activity and mitotic timing is also reversed in the presence of lactate. We also show that the biochemical status of cells under these conditions is an important determinant of the altered molecular functions mentioned above as well as the altered behavior of these cells.

Cdc7 kinase is required for postnatal brain development.

The evolutionally conserved Cdc7 kinase plays crucial roles in initiation of DNA replication as well as in other chromosomal events. To examine the roles of Cdc7 in brain development, we have generated mice carrying Cdc7 knockout in neural stem cells by using Nestin-Cre. The Cdc7Fl/Fl NestinCre mice were born, but exhibited severe growth retardation and impaired postnatal brain development. These mice exhibited motor dysfunction within 9 days after birth and did not survive for more than 19 days. The cerebral cortical layer formation was impaired, although the cortical cell numbers were not altered in the mutant. In the cerebellum undergoing hypoplasia, granule cells (CGC) decreased in number in Cdc7Fl/F l NestinCre mice compared to the control at E15-18, suggesting that Cdc7 is required for DNA replication and cell proliferation of CGC at mid embryonic stage (before embryonic day 15). On the other hand, the Purkinje cell numbers were not altered but its layer formation was impaired in the mutant. These results indicate differential roles of Cdc7 in DNA replication/cell proliferation in brain. Furthermore, the defects of layer formation suggest a possibility that Cdc7 may play an additional role in cell migration during neural development.

Strain variation in gene expression impact of hyphal cyclin Hgc1 in Candida albicans.

Formation of hyphae is a key virulence trait of the fungal pathogen Candida albicans. Hypha morphogenesis depends upon the cyclin Hgc1, which acts together with cyclin-dependent protein kinase Cdc28 to phosphorylate effectors that drive polarized growth. Hgc1 has also been implicated in gene regulation through its effects on 2 transcription factors, Efg1 and Ume6. Here, we report RNA-sequencing (RNA-seq) analysis of 2 pairs of hgc1Δ/Δ mutants and their respective wild-type strains, which lie in 2 different genetic backgrounds. We find that hgc1Δ/Δ mutations alter expression of 271 genes in both genetic backgrounds and 266 of those genes respond consistently with regard to up- or down-regulation. Consistency is similar to what has been observed with efg1Δ/Δ mutations and greater than observed with nrg1Δ/Δ mutations in these 2 backgrounds. The gene expression response includes genes under Efg1 control, as expected from prior studies. Hgc1-responsive genes also include ergosterol biosynthetic genes and bud neck-related genes, which may reflect interactions between Hgc1 and additional transcription factors as well as effects of Hgc1 on cellular length-to-width ratios.

ATR kinase supports normal proliferation in the early S phase by preventing replication resource exhaustion

The ATR kinase, which coordinates cellular responses to DNA replication stress, is also essential for the proliferation of normal unstressed cells. Although its role in the replication stress response is well defined, the mechanisms by which ATR supports normal cell proliferation remain elusive. Here, we show that ATR is dispensable for the viability of G0-arrested naïve B cells. However, upon cytokine-induced proliferation, Atr-deficient B cells initiate DNA replication efficiently, but by mid-S phase they display dNTP depletion, fork stalling, and replication failure. Nonetheless, productive DNA replication and dNTP levels can be restored in Atr-deficient cells by suppressing origin firing, such as partial inhibition of CDC7 and CDK1 kinase activities. Together, these findings indicate that ATR supports the proliferation of normal unstressed cells by tempering the pace of origin firing during the early S phase to avoid exhaustion of dNTPs and importantly also other replication factors.

PTBP1 enforces ATR-CHK1 signaling determining the potency of CDC7 inhibitors

CDC7 kinase is crucial for DNA replication initiation and fork processing. CDC7 inhibition mildly activates the ATR pathway, which further limits origin firing; however, to date the relationship between CDC7 and ATR remains controversial. We show that CDC7 and ATR inhibitors are either synergistic or antagonistic depending on the degree of inhibition of each individual kinase. We find that Polypyrimidine Tract Binding Protein 1 (PTBP1) is important for ATR activity in response to CDC7 inhibition and genotoxic agents. Compromised PTBP1 expression makes cells defective in RPA recruitment, genomically unstable, and resistant to CDC7 inhibitors. PTBP1 deficiency affects the expression and splicing of many genes indicating a multifactorial impact on drug response. We find that an exon skipping event in RAD51AP1 contributes to checkpoint deficiency in PTBP1-deficient cells. These results identify PTBP1 as a key factor in replication stress response and define how ATR activity modulates the activity of CDC7 inhibitors.

Homozygous DBF4 mutation as a cause of severe congenital neutropenia

Severe congenital neutropenia presents with recurrent infections early in life as a result of arrested granulopoiesis. Multiple genetic defects are known to block granulocyte differentiation; however, a genetic cause remains unknown in approximately 40% of cases. We aimed to characterize a patient with severe congenital neutropenia and syndromic features without a genetic diagnosis. Whole exome sequencing results were validated using flow cytometry, Western blotting, coimmunoprecipitation, quantitative PCR, cell cycle and proliferation analysis of lymphocytes and fibroblasts and granulocytic differentiation of primary CD34+ and HL-60 cells. We identified a homozygous missense mutation in DBF4 in a patient with mild extra-uterine growth retardation, facial dysmorphism and severe congenital neutropenia. DBF4 is the regulatory subunit of the CDC7 kinase, together known as DBF4-dependent kinase (DDK), the complex essential for DNA replication initiation. The DBF4 variant demonstrated impaired ability to bind CDC7, resulting in decreased DDK-mediated phosphorylation, defective S-phase entry and progression and impaired differentiation of granulocytes associated with activation of the p53-p21 pathway. The introduction of wild-type DBF4 into patient CD34+ cells rescued the promyelocyte differentiation arrest. Hypomorphic DBF4 mutation causes autosomal-recessive severe congenital neutropenia with syndromic features.

CCNB2 as a potential biomarker of bladder cancer via the high throughput technology.

Bladder cancer and oral squamous cell carcinoma (OSCC) seriously affect people's health. However, the relationship between bladder cancer and OSCC remains unclear. Got GSE138206, GSE146483, GSE184616, and bladder cancer datasets GSE65635, GSE100926 from Gene Expression Omnibus database. Weighted gene co-expression network analysis was used to identify the significant module. Functional enrichment analysis was performed via the Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes. Furthermore, the Gene Set Enrichment Analysis was also used to complete the enrichment analysis. Comparative Toxicogenomics Database found most relevant diseases to core genes. TargetScan is used to forecast analysis of microRNA and target genes. In Gene Ontology analysis, differentially expressed genes were mostly concentrated in cell differentiation, extrallular region, structural molecule activity, and actin binding. In Kyoto Encyclopedia of Genes and Genomes analysis, the differentially expressed genes were mainly enriched in PI3K-Akt signaling pathway, pathway in cancer, and extracellular matrix-receptor interaction. Seven hub genes (cyclin B2 [CCNB2], TK1, CDC20, PCNA, CKS1B, CDCA5, MCM4) were obtained. Hub genes (CCNB2, CDC20) are highly expressed in OSCC and bladder cancer samples. CCNB2 was one common oncogene of bladder cancer and OSCC.

Analysis of the expression and prognostic significance of DDK complex in Hepatocarcinoma

BackgroundHepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide. Although DBF4-dependent kinase (DDK) complex composed of CDC7 kinase and its regulatory subunit DBF4 has been shown to be overexpressed in primary tumors and promotes tumor development, while its role and prognostic value in HCC remain largely unknown. In the present study, the expression of DBF4 and CDC7 and their relationship with clinical characteristics were comprehensively analyzed.MethodsThe mRNA expression profiles of HCC and the corresponding clinical data of HCC patients were downloaded from TCGA and GEO databases, respectively. The differences in DBF4 and CDC7 expression in tumor tissues and adjacent normal tissues were analyzed. HCC-derived tissue microarray (TMA) was used to evaluate and score the expression of CDC7 by immunohistochemistry (IHC) staining. The Kaplan–Meier method and the Cox regression method were used to analyze the relationship between overall survival and clinical characteristics of the patients. Gene set enrichment analysis (GSEA) was used to analyze the pathway enrichment of DBF4 and CDC7.ResultsDBF4 and CDC7 had similar expression patterns in HCC patients. Detailly, compared with adjacent tissues, both mRNA and protein of DBF4 and CDC7 were significantly higher in HCC, and their expression was positively correlated with AJCC_T stage, clinical stage and G stage (grade) of liver cancer patients, and higher DBF4 or CDC7 expression predicted a worse prognosis in HCC patients with shorter overall survival (OS), recurrence-free survival (RFS), disease-specific survival (DSS) and progress-free survival (PFS). Cox regression analysis suggested that both DBF4 and CDC7 were independent risk factors for the prognosis of HCC patients in TCGA dataset. GSEA suggested that both DBF4 and CDC7 were positively correlated with cell cycle and DNA replication. Finally, the prognostic value of CDC7 was furtherly confirmed by TMA-based IHC staining results.ConclusionsOur study showed that DDK complex was significantly increased in HCC. Both DBF4 and CDC7 may be potential diagnostic and prognostic markers for HCC, and high expression of DDK members predicts a worse prognosis in patients with HCC, which may be associated with high tumor cell proliferation rate.

Claspin is Required for Growth Recovery from Serum Starvation through Regulating the PI3K-PDK1-mTOR Pathway in Mammalian Cells.

Claspin plays multiple important roles in regulation of DNA replication as a mediator for the cellular response to replication stress, an integral replication fork factor that facilitates replication fork progression and a factor that promotes initiation by recruiting Cdc7 kinase. Here, we report a novel role of Claspin in growth recovery from serum starvation, which requires the activation of PI3 kinase (PI3K)-PDK1-Akt-mTOR pathways. In the absence of Claspin, cells do not proceed into S phase and eventually die partially in a ROS- and p53-dependent manner. Claspin directly interacts with PI3K and mTOR, and is required for activation of PI3K-PDK1-mTOR and for that of mTOR downstream factors, p70S6K and 4EBP1, but not for p38 MAPK cascade during the recovery from serum starvation. PDK1 physically interacts with Claspin, notably with CKBD, in a manner dependent on phosphorylation of the latter protein, and is required for interaction of mTOR with Claspin. Thus, Claspin plays a novel role as a key regulator for nutrition-induced proliferation/survival signaling by activating the mTOR pathway. The results also suggest a possibility that Claspin may serve as a common mediator that receives signals from different PI3K-related kinases and transmit them to specific downstream kinases.

Absence of Wee1 alters global transcriptional response to oxidative stress in Schizosaccharomyces pombe.

Stress response and checkpoint activation are the main determinants of cellular survival in adverse conditions. In Schizosaccharomyces pombe, these are controlled by the Mitogen Activated Protein Kinase Spc1 and the Cyclin dependent Kinase Cdc2 respectively. Cdc2 is regulated positively by Cdc25 and negatively by Wee1. Changes in Cdc2 activity can be sensed by Spc1 resulting in the modulation of mitotic timing by Spc1. Functional cross talks between cell cycle regulation and MAPK machinery during regulation of mitotic timing are well characterised but the presence of similar communication during stress response remains unexplored. In this study we report how the checkpoint activator kinase Wee1 can also influence the transcriptional response to oxidative stress. We show that deletion of Wee1 results in changes in gene expression of the cells, especially with respect to genes whose expression is known to be regulated by Spc1. These differences are seen in unperturbed cells as well as during oxidative stress. Moreover, such variations extend beyond what could be expected to occur due to the known enhanced Spc1 activity of these cells. This is the first depiction of the influence of Wee1 and consequently Cdc2 activity on transcriptional response to oxidative stress.

Bacillus subtilis KLBMPGC81 suppresses appressorium-mediated plant infection by altering the cell wall integrity signaling pathway and multiple cell biological processes in Magnaporthe oryzae.

Magnaporthe oryzae is one of the most destructive crop pathogens in the world, causing huge losses in rice harvest every year. Bacillus subtilis is a potential biocontrol agent that has been explored in many crop systems because it is a potent producer of bioactive compounds. However, the mechanisms by which these agents control rice blasts are not fully understood. We show that B. subtilis KLBMPGC81 (KC81) and its supernatant (SUP) have high antimicrobial activity against M. oryzae strain Guy11. To better exploit KC81 as a biocontrol agent, the mechanism by which KC81 suppresses rice blast pathogens was investigated. This study shows that KC81 SUP is effective in controlling rice blast disease. The SUP has a significant effect on suppressing the growth of M. oryzae and appressorium-mediated plant infection. KC81 SUP compromises cell wall integrity, microtubules and actin cytoskeleton, mitosis, and autophagy, all of which are required for M. oryzae growth, appressorium development, and host infection. We further show that the SUP reduces the activity of the cyclin-dependent kinase Cdc2 by enhancing the phosphorylation of Cdc2 Tyr 15, thereby impairing mitosis in M. oryzae cells. SUP induces the cell wall sensor MoWsc1 to activate the cell wall integrity pathway and Mps1 and Pmk1 mitogen-activated protein kinases. Taken together, our findings reveal that KC81 is an effective fungicide that suppresses M. oryzae growth, appressorium formation, and host infection by abnormally activating the cell wall integrity pathway, disrupting the cytoskeleton, mitosis, and autophagy.

A quantitative and spatial analysis of cell cycle regulators during the fission yeast cycle

We have carried out a systems-level analysis of the spatial and temporal dynamics of cell cycle regulators in the fission yeast Schizosaccharomyces pombe. In a comprehensive single-cell analysis, we have precisely quantified the levels of 38 proteins previously identified as regulators of the G2 to mitosis transition and of 7 proteins acting at the G1- to S-phase transition. Only 2 of the 38 mitotic regulators exhibit changes in concentration at the whole-cell level: the mitotic B-type cyclin Cdc13, which accumulates continually throughout the cell cycle, and the regulatory phosphatase Cdc25, which exhibits a complex cell cycle pattern. Both proteins show similar patterns of change within the nucleus as in the whole cell but at higher concentrations. In addition, the concentrations of the major fission yeast cyclin-dependent kinase (CDK) Cdc2, the CDK regulator Suc1, and the inhibitory kinase Wee1 also increase in the nucleus, peaking at mitotic onset, but are constant in the whole cell. The significant increase in concentration with size for Cdc13 supports the view that mitotic B-type cyclin accumulation could act as a cell size sensor. We propose a two-step process for the control of mitosis. First, Cdc13 accumulates in a size-dependent manner, which drives increasing CDK activity. Second, from mid-G2, the increasing nuclear accumulation of Cdc25 and the counteracting Wee1 introduce a bistability switch that results in a rapid rise of CDK activity at the end of G2 and thus, brings about an orderly progression into mitosis.

The interaction between the Dbf4 ortholog Chiffon and Gcn5 is conserved in Dipteran insect species.

Chiffon is the sole Drosophila ortholog of Dbf4, the regulatory subunit for the cell-cycle kinase Cdc7 that initiates DNA replication. In Drosophila, the chiffon gene encodes two polypeptides with independent activities. Chiffon-A contains the conserved Dbf4 motifs and interacts with Cdc7 to form the Dbf4-dependent Kinase (DDK) complex, which is essential for a specialized form of DNA replication. In contrast, Chiffon-B binds the histone acetyltransferase Gcn5 to form the Chiffon histone acetyltransferase (CHAT) complex, which is necessary for histone H3 acetylation and viability. Previous studies have shown that the Chiffon-B region is only present within insects. However, it was unclear how widely the interaction between Chiffon-B and Gcn5 was conserved among insect species. To examine this, we performed yeast two-hybrid assays using Chiffon-B and Gcn5 from a variety of insect species and found that Chiffon-B and Gcn5 interact in Diptera species such as Australian sheep blowfly and yellow fever mosquito. Protein domain analysis identified that Chiffon-B has features of acidic transcriptional activators such as Gal4 or VP16. We propose that the CHAT complex plays a critical role in a biological process that is unique to Dipterans and could therefore be a potential target for pest control strategies.