Cyclin B3 is a dominant fast-acting cyclin that drives rapid early embryonic mitoses.

Mitosis in early embryos often proceeds at a rapid pace, but how this pace is achieved is not understood. Here, we show that cyclin B3 is the dominant driver of rapid embryonic mitoses in the C. elegans embryo. Cyclins B1 and B2 support slow mitosis (NEBD to anaphase ∼600 s), but the presence of cyclin B3 dominantly drives the approximately threefold faster mitosis observed in wildtype. Multiple mitotic events are slowed down in cyclin B1 and B2-driven mitosis, and cyclin B3-associated Cdk1 H1 kinase activity is ∼25-fold more active than cyclin B1-associated Cdk1. Addition of cyclin B1 to fast cyclin B3-only mitosis introduces an ∼60-s delay between completion of chromosome alignment and anaphase onset; this delay, which is important for segregation fidelity, is dependent on inhibitory phosphorylation of the anaphase activator Cdc20. Thus, cyclin B3 dominance, coupled to a cyclin B1-dependent delay that acts via Cdc20 phosphorylation, sets the rapid pace and ensures mitotic fidelity in the early C. elegans embryo.

The Expression of Cell Cycle Cyclins in a Human Megakaryoblast Cell Line Exposed to Simulated Microgravity.

The study of the physiological and pathophysiological processes under extreme conditions facilitates a better understanding of the state of a healthy organism and can also shed light on the pathogenesis of diseases. In recent years, it has become evident that gravitational stress affects both the whole organism and individual cells. We have previously demonstrated that simulated microgravity inhibits proliferation, induces apoptosis, changes morphology, and alters the surface marker expression of megakaryoblast cell line MEG-01. In the present work, we investigate the expression of cell cycle cyclins in MEG-01 cells. We performed several experiments for 24 h, 72 h, 96 h and 168 h. Flow cytometry and Western blot analysis demonstrated that the main change in the levels of cyclins expression occurs under conditions of simulated microgravity after 96 h. Thus, the level of cyclin A expression showed an increase in the RPM group during the first 4 days, followed by a decrease, which, together with the peak of cyclin D, may indicate inhibition of the cell cycle in the G2 phase, before mitosis. In addition, based on the data obtained by PCR analysis, we were also able to see that both cyclin A and cyclin B expression showed a peak at 72 h, followed by a gradual decrease at 96 h. STED microscopy data also confirmed that the main change in cyclin expression of MEG-01 cells occurs at 96 h, under simulated microgravity conditions, compared to static control. These results suggested that the cell cycle disruption induced by RPM-simulated microgravity in MEG-01 cells may be associated with the altered expression of the main regulators of the cell cycle. Thus, these data implicate the development of cellular stress in MEG-01 cells, which may be important for proliferating human cells exposed to microgravity in real space.

Analysis of nondegradable cyclins reveals distinct roles of the mitotic cyclins in Drosophila meiosis.

Meiosis is a complex variant of the mitotic cell cycle, and as such relies on many of the same proteins involved in mitosis, but utilizes these in novel ways. As in mitosis, Cdk1 and its cyclin partners, Cyclin A, B, and B3 are required at multiple steps in meiosis. Here, we study the effect of stabilized forms of the three mitotic cyclins to study the consequences of failure to degrade the cyclins in meiosis. We find that stabilized Cyclin B3 promotes ectopic microtubule polymerization throughout the egg, dependent on APC/C activity and apparently due to the consequent destruction of Cyclin A and Cyclin B. We present data that suggests CycB, and possibly CycA, can also promote APC/C activity at specific stages of meiosis. We also present evidence that in meiosis APC/CCort and APC/CFzy are able to target Cyclin B via a novel degron. Overall, our findings highlight the distinct functions of the three mitotic Cdk-cyclin complexes in meiosis.

Recombinant cyclin B-Cdk1-Suc1 capable of multi-site mitotic phosphorylation in vitro.

Cyclin-dependent kinase 1 (Cdk1) complexed with cyclin B phosphorylates multiple sites on hundreds of proteins during mitosis. However, it is not fully understood how multi-site mitotic phosphorylation by cyclin B-Cdk1 controls the structures and functions of individual substrates. Here we develop an easy-to-use protocol to express recombinant vertebrate cyclin B and Cdk1 in insect cells from a single baculovirus vector and to purify their complexes with excellent homogeneity. A series of in-vitro assays demonstrate that the recombinant cyclin B-Cdk1 can efficiently and specifically phosphorylate the SP and TP motifs in substrates. The addition of Suc1 (a Cks1 homolog in fission yeast) accelerates multi-site phosphorylation of an artificial substrate containing TP motifs. Importantly, we show that mitosis-specific multi-subunit and multi-site phosphorylation of the condensin I complex can be recapitulated in vitro using recombinant cyclin B-Cdk1-Suc1. The materials and protocols described here will pave the way for dissecting the biochemical basis of critical mitotic processes that accompany Cdk1-mediated large-scale phosphorylation.

Cell-type-specific interacting proteins collaborate to regulate the timing of Cyclin B protein expression in male meiotic prophase.

During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5 days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. Here, we show that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8 h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase.

Mutations in CCNB3 affect its location thus causing a multiplicity of phenotypes in human oocytes maturation by aberrant CDK1 activity and APC/C activity at different stages.

BACKGROUND: Oocyte maturation arrest results in female infertility and the genetic etiology of this phenotype remains largely unknown. Previous studies have proven that cyclins play a significant role in the cell cycle both in meiosis and mitosis. Cyclin B3 (CCNB3) is one of the members of the cyclin family and its function in human oocyte maturation is poorly understood. METHODS: 118 infertile patients were recruited and WES was performed for 68 independent females that experienced oocyte maturation arrest. Four mutations in CCNB3 were found and effects of these mutations were validated by Sanger sequencing and in vitro functional analyses. RESULTS: We found these mutations altered the location of cyclin B3 which affected the function of cyclin dependent kinase 1 (CDK1) and led to mouse oocyte arrested at germinal vesicle (GV) stage. And then, low CDK1 activity influenced the degradation of cadherin 1 (CDH1) and the accumulation of cell division cycle 20 (CDC20) which are two types of anaphase-promoting complex/cyclosome (APC/C) activators and act in different stages of the cell cycle. Finally, APC/C activity was downregulated due to insufficient CDC20 level and resulted in oocyte metaphase I (MI) arrest. Moreover, we also found that the addition of PP1 inhibitor Okadic acid and CDK1 inhibitor Roscovitine at corresponding stages during oocyte in vitro maturation (IVM) significantly improved the maturation rates in CCNB3 mutant cRNAs injected oocytes. The above experiments were performed in mouse oocytes. CONCLUSION: Here, we report five independent patients in which mutations in CCNB3 may be the cause of oocyte maturation arrest. Our findings shed lights on the critical role of CCNB3 in human oocyte maturation.

Spectrum of Histopathological, Immunohistochemical, Molecular and Radiological Features in 12 Cases of BCOR::CCNB3-positive Sarcomas With Literature Review.

Introduction BCOR::CCNB3-positive undifferentiated sarcomas are rare. Herein, we present clinicopathological features including immunohistochemical and molecular data, along with the radiological profile of 12 such tumors. Methods Tumors were tested for BCOR::CCNB3 fusion by reverse transcription polymerase chain reaction (RT-PCR) technique. Eight tumors were tested for EWSR1 and three for SS18 gene rearrangements by fluorescence in situ hybridization, and two for SS18::SSX fusion by fragment analysis. Results Ten of 12 patients were male with ages ranging between 4 and 17 years (median = 13, average = 14.4). Nine tumors occurred in bones and three in soft tissues (median size = 8 cm). Four of five tumors within the appendicular bones were metadiaphyseal and appeared as permeative lesions, invariably associated with cortical thickening. Three tumors displayed mineralization. Histopathologically, the tumors comprised round to epithelioid cells with round to oval to spindle-shaped nuclei, mostly diffusely arranged in a myxoid stroma with intervening thin-walled vessels. Immunohistochemically, tumor cells were positive for BCOR (10/11), SATB2 (8/9), TLE1 (5/6), cyclinD1 (4/4), and EMA (3/8). All tumors revealed BCOR::CCNB3 fusion transcript. Nine patients underwent neoadjuvant chemotherapy, including five who underwent surgical resection, with two patients, who received adjuvant radiation therapy. A single patient, each, underwent palliative chemotherapy and palliative radiotherapy, respectively. Four patients developed pulmonary metastasis and three developed local recurrences. Four patients were alive-with-disease and two were free-of-disease. Conclusions It is crucial to identify BCOR::CCNB3 fusion-positive sarcomas, given significant treatment-associated implications. Certain clinicoradiological, histopathological features, absent EWSR1 rearrangement and BCOR, SATB2, and TLE1 immunoexpression are useful for triaging these tumors for molecular testing. A review of the literature on these ultra-rare tumors, including their diagnostic mimics is presented.

[The diagnostic value of CCNB3 and BCOR expression in BCOR-CCNB3 sarcoma].

Objective: To investigate the diagnostic value of expression of CCNB3 and BCOR in BCOR-CCNB3 sarcoma (BCS). Methods: Fifteen cases of BCS confirmed by fluorescence in situ hybridization (FISH) and/or reverse transcription-polymerase chain reaction (RT-PCR) from January 2014 to October 2021 at Beijing Jishuitan Hospital were collected. Immunohistochemical EnVision method was used to detect the expression of CCNB3 and BCOR in 15 cases of BCS and in 65 non-BCS tumors (54 cases of Ewing's sarcoma, 5 cases of CIC rearranged sarcoma, 4 cases of synovial sarcoma, 1 case of mesenchymal chondrosarcoma and 1 case of soft tissue clear cell sarcoma). Results: Immunohistochemical staining for CCNB3 revealed strongly diffuse nuclear staining in 14 of 15 (14/15) BCS cases, whereas none of the 65 non-BCS tumors showed any staining. Immunohistochemical staining for BCOR showed strongly diffuse nuclear staining in 11 (11/14) BCS cases; seven of the 65 (7/65, 10.8%) non-BCS tumors showed variable staining (five cases of Ewing sarcoma, one cases of synovial sarcoma, and one case of mesenchymal chondrosarcoma). The sensitivity and specificity of CCNB3 in diagnosing BCS were 93.3% and 100% and these of BCOR were 78.6% and 89.2%, respectively. Conclusions: CCNB3 is a highly sensitive and specific marker for BCS.The antibody may help screening BCS.

RNA Interference Analysis of the Functions of Cyclin B in Male Reproductive Development of the Oriental River Prawn (Macrobrachium nipponense).

Cyclin B (CycB) plays essential roles in cell proliferation and promotes gonad development in many crustaceans. The goal of this study was to investigate the regulatory roles of this gene in the reproductive development of male oriental river prawns (Macrobrachium nipponense). A phylo-genetic tree analysis revealed that the protein sequence of Mn-CycB was most closely related to those of freshwater prawns, whereas the evolutionary distance from crabs was much longer. A quantitative PCR analysis showed that the expression of Mn-CycB was highest in the gonad of both male and female prawns compared to that in other tissues (p < 0.05), indicating that this gene may play essential roles in the regulation of both testis and ovary development in M. nipponense. In males, Mn-CycB expression in the testis and androgenic gland was higher during the reproductive season than during the non-reproductive season (p < 0.05), implying that CycB plays essential roles in the reproductive development of male M. nipponense. An RNA interference analysis revealed that the Mn-insulin-like androgenic gland hormone expression decreased as the Mn-CycB expression decreased, and that few sperm were detected 14 days after the dsCycB treatment, indicating that CycB positively affects testis development in M. nipponense. The results of this study highlight the functions of CycB in M. nipponense, and they can be applied to studies of male reproductive development in other crustacean species.

Control of division in Chlamydomonas by cyclin B/CDKB1 and the anaphase-promoting complex.

In yeast and animals, cyclin B binds and activates the cyclin-dependent kinase ('CDK') CDK1 to drive entry into mitosis. We show that CYCB1, the sole cyclin B in Chlamydomonas, activates the plant-specific CDKB1 rather than the CDK1 ortholog CDKA1, confirming and extending previous results. Time-lapse microscopy shows that CYCB1 is synthesized before each division in the multiple fission cycle, then is rapidly degraded 3-5 minutes before division occurs. CYCB1 degradation is dependent on the anaphase-promoting complex (APC). Like CYCB1, CDKB1 is not synthesized until late G1; however, CDKB1 is not degraded with each division within the multiple fission cycle, but is degraded after all divisions have ceased. The microtubule plus-end-binding protein EB1 labeled with mNeonGreen allowed detection of mitotic events in live cells. The earliest detectable step in mitosis, splitting of polar EB1 signal into two foci, likely associated with future spindle poles, was dependent on CYCB1. CYCB1-GFP localized close to these foci immediately before spindle formation. Spindle breakdown, cleavage furrow formation and accumulation of EB1 in the furrow were dependent on the APC. In interphase, rapidly growing microtubules are marked by 'comets' of EB1; comets are absent in the absence of APC function. Thus CYCB1/CDKB1 and the APC modulate microtubule function and assembly while regulating mitotic progression. Genetic results suggest an independent additional role for the APC in regulating sister chromatid cohesion; this role is likely conserved across eukaryotes.

BCOR-CCNB3 sarcoma with concurrent RNF213-SLC26A11 gene fusion: a rare sarcoma with altered histopathological features after chemotherapy.

BACKGROUND: Chemotherapy is a common approach for cancer treatment, but intrinsic genetic mutations in different individuals may cause different responses to chemotherapy, resulting in unique histopathological changes. The genetic mutation along with the distinct histopathological features may indicate new tumor entities. BCOR-CCNB3 sarcomas is a kind of Ewing-like sarcomas (ELS) occurring mostly in bone and soft tissues. No gene fusion other than BCOR-CCNB3 has been found in this type of tumor. CASE PRESENTATION: We herein report a case of 17-year-old male patient, presented with a mass on his left shoulder that was diagnosed as undifferentiated small round cell sarcoma according to core biopsy. The patient received 5 courses of preoperational chemotherapy, and the tumor was resected and analyzed. Primitive small round cells and larger myoid cells in the resected tumor tissue but not in biopsy were observed, and arterioles stenosis and occlusion were also detected, indicating a dramatic change of histopathological features of this tumor. In addition, the immunohistochemical results showed the altered staining patterns of BCOR, bcl2, CyclinD1, TLE1, AR, SMA, CD117, STAB2, CD56, and CD99 in tumor tissues after chemotherapy. Notably, RNA sequencing revealed a RNF213-SLC26A11 fusion in the tumor sample. CONCLUSIONS: The BCOR-CCNB3 sarcoma with RNF213-SLC26A11 fusion may indicate a subset of tumors that undergo histopathological changes in response to chemotherapy. More similar cases in the future may help to clarify the clinical meanings of RNF213-SLC26A11 fusion in BCOR-CCNB3 sarcomas and the underlying mechanisms.

Cyclin B/CDK1 and Cyclin A/CDK2 phosphorylate DENR to promote mitotic protein translation and faithful cell division.

DENR and MCTS1 have been identified as oncogenes in several different tumor entities. The heterodimeric DENR·MCTS1 protein complex promotes translation of mRNAs containing upstream Open Reading Frames (uORFs). We show here that DENR is phosphorylated on Serine 73 by Cyclin B/CDK1 and Cyclin A/CDK2 at the onset of mitosis, and then dephosphorylated as cells exit mitosis. Phosphorylation of Ser73 promotes mitotic stability of DENR protein and prevents its cleavage at Asp26. This leads to enhanced translation of mRNAs involved in mitosis. Indeed, we find that roughly 40% of all mRNAs with elevated translation in mitosis are DENR targets. In the absence of DENR or of Ser73 phosphorylation, cells display elevated levels of aberrant mitoses and cell death. This provides a mechanism how the cell cycle regulates translation of a subset of mitotically relevant mRNAs during mitosis.

Multisite phosphorylation by Cdk1 initiates delayed negative feedback to control mitotic transcription.

Cell-cycle progression is driven by the phosphorylation of cyclin-dependent kinase (Cdk) substrates.1-3 The order of substrate phosphorylation depends in part on the general rise in Cdk activity during the cell cycle,4-7 together with variations in substrate docking to sites on associated cyclin and Cks subunits.3,6,8-10 Many substrates are modified at multiple sites to provide more complex regulation.10-14 Here, we describe an elegant regulatory circuit based on multisite phosphorylation of Ndd1, a transcriptional co-activator of budding yeast genes required for mitotic progression.11,12 As cells enter mitosis, Ndd1 phosphorylation by Cdk1 is known to promote mitotic cyclin (CLB2) gene transcription, resulting in positive feedback.13-16 Consistent with these findings, we show that low Cdk1 activity promotes CLB2 expression at mitotic entry. We also find, however, that when high Cdk1 activity accumulates in a mitotic arrest, CLB2 expression is inhibited. Inhibition is accompanied by Ndd1 degradation, and we present evidence that degradation is triggered by multisite Ndd1 phosphorylation by high mitotic Cdk1-Clb2 activity. Complete Ndd1 phosphorylation by Clb2-Cdk1-Cks1 requires the phosphothreonine-binding site of Cks1, as well as a recently identified phosphate-binding pocket on the cyclin Clb2.17 We therefore propose that initial phosphorylation by Cdk1 primes Ndd1 for delayed secondary phosphorylation at suboptimal sites that promote degradation. Together, our results suggest that rising levels of mitotic Cdk1 activity act at multiple phosphorylation sites on Ndd1, first triggering rapid positive feedback and then promoting delayed negative feedback, resulting in a pulse of mitotic gene expression.

The Aurora B gradient sustains kinetochore stability in anaphase.

Kinetochores assemble on chromosomes in mitosis to allow microtubules to attach and bring about accurate chromosome segregation. The kinases Cyclin B-Cdk1 and Aurora B are crucial for the formation of stable kinetochores. However, the activity of these two kinases appears to decline dramatically at centromeres during anaphase onset, precisely when microtubule attachments are required to move chromosomes toward opposite poles of the dividing cell. We find that, although Aurora B leaves centromeres at anaphase, a gradient of Aurora B activity centered on the central spindle is still able to phosphorylate kinetochore substrates such as Dsn1 to modulate kinetochore stability in anaphase and to regulate kinetochore disassembly as cells enter telophase. We provide a model to explain how Aurora B co-operates with Cyclin B-Cdk1 to maintain kinetochore function in anaphase.

Quantitative differences between cyclin-dependent kinases underlie the unique functions of CDK1 in human cells.

One of the most intriguing features of cell-cycle control is that, although there are multiple cyclin-dependent kinases (CDKs) in higher eukaryotes, a single CDK is responsible for both G1-S and G2-M in yeasts. By leveraging a rapid conditional silencing system in human cell lines, we confirm that CDK1 assumes the role of G1-S CDK in the absence of CDK2. Unexpectedly, CDK1 deficiency does not prevent mitotic entry. Nonetheless, inadequate phosphorylation of mitotic substrates by noncanonical cyclin B-CDK2 complexes does not allow progression beyond metaphase and underscores deleterious late mitotic events, including the uncoupling of anaphase A and B and cytokinesis. Elevation of CDK2 to a level similar to CDK1 overcomes the mitotic defects caused by CDK1 deficiency, indicating that the relatively low concentration of CDK2 accounts for the defective anaphase. Collectively, these results reveal that the difference between G2-M and G1-S CDKs in human cells is essentially quantitative.

Cis-acting elements involved in the G2/M-phase-specific transcription of the cyclin B gene in the unicellular alga Cyanidioschyzon merolae.

M-specific activator (MSA) cis-acting elements have been determined to be involved in the regulation of G2/M-phase-specific transcription in spermatophytes. In this study, the involvement of MSA-core elements in G2/M-phase-specific transcription was examined in the unicellular red alga Cyanidioschyzon merolae. In the C. merolae genome, MSA-core elements do not accumulate specifically in the upstream of mitosis-specific transcriptional genes. Mutations of the four MSA-core elements of the cyclin B gene, which encodes a central factor of the G2-to-M-phase transition, have resulted in the abolishment of transcription or permission of transcription even in the G1 phase. These results suggest that all four MSA-core elements located in the upstream region of cyclin B are involved in G2/M-phase-specific transcription in C. merolae; however, the nature of the involvement of MSA-core elements in G2/M-phase-specific transcription differed among the four elements. Thus, MSA-core-element-mediated G2/M-phase-specific transcription in C. merolae seems to be regulated by a complex mechanism.

Modulating the bicoid gradient in space and time.

BACKGROUND: The formation of the Bicoid (Bcd) gradient in the early Drosophila is one of the most fascinating observations in biology and serves as a paradigm for gradient formation, yet its mechanism is still not fully understood. Two distinct models were proposed in the past, the SDD and the ARTS model. RESULTS: We define novel cis- and trans-acting factors that are indispensable for gradient formation. The first one is the poly A tail length of the bcd mRNA where we demonstrate that it changes not only in time, but also in space. We show that posterior bcd mRNAs possess a longer poly tail than anterior ones and this elongation is likely mediated by wispy (wisp), a poly A polymerase. Consequently, modulating the activity of Wisp results in changes of the Bcd gradient, in controlling downstream targets such as the gap and pair-rule genes, and also in influencing the cuticular pattern. Attempts to modulate the Bcd gradient by subjecting the egg to an extra nuclear cycle, i.e. a 15th nuclear cycle by means of the maternal haploid (mh) mutation showed no effect, neither on the appearance of the gradient nor on the control of downstream target. This suggests that the segmental anlagen are determined during the first 14 nuclear cycles. Finally, we identify the Cyclin B (CycB) gene as a trans-acting factor that modulates the movement of Bcd such that Bcd movement is allowed to move through the interior of the egg. CONCLUSIONS: Our analysis demonstrates that Bcd gradient formation is far more complex than previously thought requiring a revision of the models of how the gradient is formed.