Cosuppression of eukaryotic release factor 1-1 in Arabidopsis affects cell elongation and radial cell division

The role of the eukaryotic release factor 1 (eRF1) in translation termination has previously been established in yeast; however, only limited characterization has been performed on any plant homologs. Here, we demonstrate that cosuppression of eRF1-1 in Arabidopsis (Arabidopsis thaliana) has a profound effect on plant morphology, resulting in what we term the broomhead phenotype. These plants primarily exhibit a reduction in internode elongation causing the formation of a broomhead-like cluster of malformed siliques at the top of the inflorescence stem. Histological analysis of broomhead stems revealed that cells are reduced in height and display ectopic lignification of the phloem cap cells, some phloem sieve cells, and regions of the fascicular cambium, as well as enhanced lignification of the interfascicular fibers. We also show that cell division in the fascicular cambial regions is altered, with the majority of vascular bundles containing cambial cells that are disorganized and possess enlarged nuclei. This is the first attempt at functional characterization of a release factor in vivo in plants and demonstrates the importance of eRF1-1 function in Arabidopsis.     

Viral infections and cell cycle G2／M regulation

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyrl5) on Cdc2, which is phosphorylated by Weel kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two wellcharacterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-Ⅰ) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.     

Evolutionarily conserved pressure for the existence of distinct G2/M cell cycle arrest and A3H inactivation functions in HIV-1 Vif

HIV-1 Vif assembles the Cul5-EloB/C E3 ubiquitin ligase to induce proteasomal degradation of the cellular antiviral APOBEC3 proteins. Detailed structural studies have confirmed critical functional domains in Vif that we have previously identified as important for the interaction of EloB/C, Cul5, and CBFβ. However, the mechanism by which Vif recognizes substrates remains poorly understood. Specific regions of Vif have been identified as being responsible for binding and depleting APOBEC3G and APOBEC3F. Interestingly, we have now identified distinct yet overlapping domains that are required for HIV-1 Vif-mediated G2/M-phase cell cycle arrest and APOBEC3H degradation, but not for the inactivation of APOBEC3G or APOBEC3F. Surprisingly, Vif molecules from primary HIV-1 variants that caused G2/M arrest were unable to inactivate APOBEC3H; on the other hand, HIV-1 Vif variants that could inactivate APOBEC3H were unable to induce G2/M arrest. All of these Vif variants still maintained the ability to inactivate APOBEC3G/F. Thus, primary HIV-1 variants have evolved to possess distinct functional activities that allow them to suppress APOBEC3H or cause G2 cell cycle arrest, using mutually exclusive interface domains. APOBEC3H depletion and G2 arrest are apparently evolutionary selected features that cannot co-exist on a single Vif molecule. The existence and persistence of both types of HIV-1 Vif variant suggests the importance of APOBEC3H suppression and cell cycle regulation for HIV-1''s survival in vivo.     

Over-expression of Arabidopsis CAP causes decreased cell expansion leading to organ size reduction in transgenic tobacco plants

Cyclase-associated proteins (CAP) are multifunctional proteins involved in Ras-cAMP signalling and regulation of the actin cytoskeleton. It has recently been demonstrated that over-expression of AtCAP1 in transgenic arabidopsis plants causes severe morphological defects owing to loss of actin filaments. To test the generality of the function of AtCAP1 in plants, transgenic tobacco plants over-expressing an arabidopsis CAP (AtCAP1) under the regulation of a glucocorticoid-inducible promoter were produced. Over-expression of AtCAP1 in transgenic tobacco plants led to growth abnormalities, in particular a reduction in the size of leaves. Morphological alterations in leaves were the result of reduced elongation of epidermal and mesophyll cells.     

The E2F functional analogue SBF recruits the Rpd3(L) HDAC,via Whi5 and Stb1, and the FACT chromatin reorganizer,to yeast G1 cyclin promoters

Regulation of the CLN1 and CLN2 G1 cyclin genes controls cell cycle progression. The SBF activator binds to these promoters but is kept inactive by the Whi5 and Stb1 inhibitors. The Cdc28 cyclin‐dependent kinase phosphorylates Whi5, ending the inhibition. Our chromatin immunoprecipitation (ChIP) experiments show that SBF, Whi5 and Stb1 recruit both Cdc28 and the Rpd3(L) histone deacetylase to CLN promoters, extending the analogy with mammalian G1 cyclin promoters in which Rb recruits histone deacetylases. Finally, we show that the SBF subunit Swi6 recruits the FACT chromatin reorganizer to SBF‐ and MBF‐regulated genes. Mutations affecting FACT reduce the transient nucleosome eviction seen at these promoters during a normal cell cycle and also reduce expression. Temperature‐sensitive mutations affecting FACT and Cdc28 can be suppressed by disruption of STB1 and WHI5, suggesting that one critical function of FACT and Cdc28 is overcoming chromatin repression at G1 cyclin promoters. Thus, SBF recruits complexes to promoters that either enhance (FACT) or repress (Rpd3L) accessibility to chromatin, and also recruits the kinase that activates START.     

Engagement of DYRK2 in proper control for cell division

Dysregulation of cell cycle machinery causes abnormal cell division, leading to cancer development. To drive cell cycle properly, expression levels of cell cycle regulators are tightly regulated through the cell cycle. Dual specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is a Ser/Thr kinase, and its intracellular functions had not been elucidated for decades. Recent studies have shown that DYRK2 down-regulates key molecules on cell cycle control. This review mainly highlights the DYRK2 function during cell division. In addition, we summarize tumor suppressive role of DYRK2 in cancer cells and discuss future research directions for DYRK2 toward the novel cancer therapies.     

The WEE1 regulators CPEB1 and miR-15b switch from inhibitor to activators at G2/M

MicroRNAs (miRNAs) in the AGO-containing RISC complex control messenger RNA (mRNA) translation by binding to mRNA 3′ untranslated region (3′UTR). The relationship between miRNAs and other regulatory factors that also bind to mRNA 3′UTR, such as CPEB1 (cytoplasmic polyadenylation element-binding protein), remains elusive. We found that both CPEB1 and miR-15b control the expression of WEE1, a key mammalian cell cycle regulator. Together, they repress WEE1 protein expression during G1 and S-phase. Interestingly, the 2 factors lose their inhibitory activity at the G2/M transition, at the time of the cell cycle when WEE1 expression is maximal, and, moreover, rather activate WEE1 translation in a synergistic manner. Our data show that translational regulation by RISC and CPEB1 is essential in cell cycle control and, most importantly, is coordinated, and can be switched from inhibition to activation during the cell cycle.     

Ectopic expression of the NtSET1 histone methyltransferase inhibits cell expansion, and affects cell division and differentiation in tobacco plants

The tobacco NtSET1 gene encodes a member of the SUV39H family of histone methyltransferases. Ectopic expression of NtSET1 causes an increase in methylated histone H3 lysine 9 and abnormal chromosome segregation in tobacco suspension cells, and inhibits tobacco plant growth. Here we show that the inhibition of plant growth was caused by reduced cell expansion as well as by abnormal cell division and differentiation. We found that deletion of the C-terminally located catalytic domain of the protein abolished the ectopic effects of NtSET1 on plant growth. Our results indicate that histone H3 lysine 9 methylation is a critical mark of epigenetic control for plant development.     

Cell cycle-dependent proteolysis and ectopic overexpression of cyclin B1 in tobacco BY2 cells

Activation of cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Although cyclin gene expression has been extensively studied in plants, not much is known at the level of the protein stability and function. Here, we demonstrated by using the highly synchronizable tobacco BY2 cell culture, that endogenous cyclin B1 protein undergoes cell cycle-dependent proteolysis and is stabilized when the spindle checkpoint has been activated. Furthermore, we established transgenic tobacco BY2 cell cultures expressing under the control of an inducible promoter, cyclin B1 protein as well as its non-degradable form as fusion proteins with GFP and found that the ectopic expression of these proteins did not dramatically disturb the cell cycle progression. These results indicate that, to a certain extent, cell cycle exit is possible without cyclin B1 proteolysis.     

Human cytochrome c enters murine J774 cells and causes G1 and G2/M cell cycle arrest and induction of apoptosis

Cytochrome c is well known as a carrier of electrons during respiration. Current evidence indicates that cytochrome c also functions as a major component of apoptosomes to induce apoptosis in eukaryotic cells as well as an antioxidant. More recently, a prokaryotic cytochrome c, cytochrome c(551) from Pseudomonas aeruginosa, has been shown to enter in mammalian cells such as the murine macrophage-like J774 cells and causes inhibition of cell cycle progression. Much less is known about such functions by mammalian cytochromes c, particularly the human cytochrome c. We now report that similar to P. aeruginosa cytochrome c(551), the purified human cytochrome c protein can enter J774 cells and induce cell cycle arrest at the G(1) to S phase, as well as at the G(2)/M phase at higher concentrations. Unlike P. aeruginosa cytochrome c(551) which had no effect on the induction of apoptosis, human cytochrome c induces significant apoptosis and cell death in J774 cells, presumably through inhibition of the cell cycle at the G(2)/M phase. When incubated with human breast cancer MCF-7 and normal mammary epithelial cell line MCF-10A1 cells, human cytochrome c entered in both types of cells but induced cell death only in the normal MCF-10A1 cells. The ability of human cytochrome c to enter J774 cells was greatly reduced at 4 degrees C, suggesting energy requirement in the entry process.     

Intracellular acidification delays hormonal G2/M transition and inhibits G2/M transition triggered by thiophosphorylated MAPK in Xenopus oocytes

Xenopus oocyte maturation is analogous to G2/M transition and characterized by germinal vesicle breakdown (GVBD), spindle formation, activation of MPF and Mos-Xp42(Mpk1) pathways. It is accompanied prior to GVBD by a transient increase in intracellular pH. We determined that a well known acidifying compound, NH(4)Cl, delayed progesterone-induced GVBD in a dose-dependent manner. GVBD(50) was delayed up to 2.3-fold by 10 mM NH(4)Cl. Cyclin B2 phosphorylation, Cdk1 Tyr15 dephosphorylation as well as p39(Mos) accumulation, Xp42(Mpk1) and p90(Rsk) phosphorylation induced by progesterone were also delayed by incubation of oocyte in NH(4)Cl. The delay induced by NH(4)Cl was prevented by injection of MOPS buffer pH 7.7. In contrast to acidifying medium, alkalyzing treatment such as Tris buffer pH 9 injections, accelerated GVBD, MPF and Xp42(Mpk1) activation, indicating that pHi changes control early steps of G2/M dynamics. When injected in an immature recipient oocyte, egg cytoplasm triggers GVBD through MPF auto-amplification, independently of protein synthesis. In these conditions, GVBD and Xp42(Mpk1) activation were delayed by high concentration of NH(4)Cl, which never prevented or delayed MPF activation. Strickingly, NH(4)Cl strongly inhibited thiophosphorylated active MAPK-induced GVBD and MPF activation. Nevertheless, Tris pH 9 did not have any effects on egg cytoplasm- or active MAPK-induced GVBD. Taken together, our results suggest that dynamic of early events driving Xp42(Mpk1) and MPF activation induced by progesterone may be negatively or positively regulated by pH(i) changes. However Xp42(Mpk1) pathway was inhibited by acidification alone. Finally, MPF auto-amplification loop was not sensitive to pH(i) changes.     

Inhibition of topoisomerase II overrides the G2/M check points of the cell cycle in EBV-lymphocytes

Epstein-Barr virus (EBV) is associated with a number of human malignancies. In vitro EBV infection transforms human lymphocytes into proliferating cell lines (EBV-lymphocytes). Etoposide, topoisomerase II inhibitor, induced apoptosis in EBV-lymphocytes as shown by expression of phosphatidylserine, loss of DNA and mitochondrial membrane potential, and cell shrinkage. In contrast, those cells, which had yet to display signs of apoptosis, grew to exceed their normal size. These EBV-lymphocytes had unusual cellular and nuclear morphology, higher mitochondrial membrane potential, increased expression of proteins and an amount of DNA that exceeded the maximum DNA content in normal EBV-lymphocytes by more than two-fold. Application of the caspase inhibitor Z-VAD-FMK in the presence of etoposide increased the numbers of such large cells. This data suggests that inhibition of topoisomerase II by etoposide does not inhibit DNA synthesis but rather overrides the G₂/M check points of the cell cycle, resulting in cells growth over their genetically determined size. This may trigger apoptosis to eliminate cells, which failed to complete mitosis.