Mps1Mph1 Kinase Phosphorylates Mad3 to Inhibit Cdc20Slp1-APC/C and Maintain Spindle Checkpoint Arrests.

The spindle checkpoint is a mitotic surveillance system which ensures equal segregation of sister chromatids. It delays anaphase onset by inhibiting the action of the E3 ubiquitin ligase known as the anaphase promoting complex or cyclosome (APC/C). Mad3/BubR1 is a key component of the mitotic checkpoint complex (MCC) which binds and inhibits the APC/C early in mitosis. Mps1(Mph1) kinase is critical for checkpoint signalling and MCC-APC/C inhibition, yet few substrates have been identified. Here we identify Mad3 as a substrate of fission yeast Mps1(Mph1) kinase. We map and mutate phosphorylation sites in Mad3, producing mutants that are targeted to kinetochores and assembled into MCC, yet display reduced APC/C binding and are unable to maintain checkpoint arrests. We show biochemically that Mad3 phospho-mimics are potent APC/C inhibitors in vitro, demonstrating that Mad3p modification can directly influence Cdc20(Slp1)-APC/C activity. This genetic dissection of APC/C inhibition demonstrates that Mps1(Mph1) kinase-dependent modifications of Mad3 and Mad2 act in a concerted manner to maintain spindle checkpoint arrests.

CPF-associated phosphatase activity opposes condensin-mediated chromosome condensation.

Functional links connecting gene transcription and condensin-mediated chromosome condensation have been established in species ranging from prokaryotes to vertebrates. However, the exact nature of these links remains misunderstood. Here we show in fission yeast that the 3' end RNA processing factor Swd2.2, a component of the Cleavage and Polyadenylation Factor (CPF), is a negative regulator of condensin-mediated chromosome condensation. Lack of Swd2.2 does not affect the assembly of the CPF but reduces its association with chromatin. This causes only limited, context-dependent effects on gene expression and transcription termination. However, CPF-associated Swd2.2 is required for the association of Protein Phosphatase 1 PP1(Dis2) with chromatin, through an interaction with Ppn1, a protein that we identify as the fission yeast homologue of vertebrate PNUTS. We demonstrate that Swd2.2, Ppn1 and PP1Dis2 form an independent module within the CPF, which provides an essential function in the absence of the CPF-associated Ssu72 phosphatase. We show that Ppn1 and Ssu72, like Swd2.2, are also negative regulators of condensin-mediated chromosome condensation. We conclude that Swd2.2 opposes condensin-mediated chromosome condensation by facilitating the function of the two CPF-associated phosphatases PP1 and Ssu72.

Bub3p facilitates spindle checkpoint silencing in fission yeast.

Although critical for spindle checkpoint signaling, the role kinetochores play in anaphase promoting complex (APC) inhibition remains unclear. Here we show that spindle checkpoint proteins are severely depleted from unattached kinetochores in fission yeast cells lacking Bub3p. Surprisingly, a robust mitotic arrest is maintained in the majority of bub3 Delta cells, yet they die, suggesting that Bub3p is essential for successful checkpoint recovery. During recovery, two defects are observed: (1) cells mis-segregate chromosomes and (2) anaphase onset is significantly delayed. We show that Bub3p is required to activate the APC upon inhibition of Aurora kinase activity in checkpoint-arrested cells, suggesting that Bub3p is required for efficient checkpoint silencing downstream of Aurora kinase. Together, these results suggest that spindle checkpoint signals can be amplified in the nucleoplasm, yet kinetochore localization of spindle checkpoint components is required for proper recovery from a spindle checkpoint-dependent arrest.

Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint.

Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/C(Cdc20) is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C( Cdc20) inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble 'degron' signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation.

Embryonic stem cells as a source of differentiated neural cells for pharmacological screens.

The process of bringing a new pharmacologically active drug to market is laborious, time consuming, and costly. From drug discovery to safety assessment, new methods are constantly sought to develop faster and more efficient procedures to eliminate drugs from further investigation because of their limited effectiveness or high toxicity. Because in vitro cell assays are an important arm of this discovery process, it is therefore somewhat unsurprising that there is an emerging contribution of embryonic stem (ES) cell technology to this area. This technology utilizes the in vitro differentiation of ES cells into somatic cell target populations that, when coupled to the use of "lineage selection" protocols, allows for the production of infinite numbers of pure populations of the desired cells for both bioactivity and toxicological screens. Unlike the use of transformed cell lines, ES-derived cells remain karyotypically normal and therefore better reflect the potential responses of cells in vivo, and when selected are more homogeneous than those obtained using primary cultures. In this chapter we discuss the use of ES cell-derived somatic cells in pharmacological screens, with particular emphasis on neural cells, and describe the methods and protocols associated with the development of ES cell-derived neural cell assays.

Embryonic stem cells: understanding their history, cell biology and signalling.

Embryonic stem cells offer enormous potential as a source of a variety of differentiated cells for cell therapy, drug discovery and toxicology screening. With the creation of human embryonic stem cell lines we now have a resource with the potential to differentiate into every tissue of the body. To fully harness this resource it is necessary to understand their biology. Here we give a background to their history, describe interesting elements of their cell biology and introduce the underlying signalling mechanisms that control their ability to self-renew and differentiate.

Epigenetic regulation of mammalian pericentric heterochromatin in vivo by HP1.

We developed a model system whereby HP1 can be targeted to pericentric heterochromatin in ES cells lacking Suv(3)9h1/2 histone methyltransferase (HMTase) activities. HP1 so targeted can reconstitute tri-methylated lysine 9 of histone H3 (Me(3)K9H3) and tri-methylated lysine 20 of histone H4 (Me(3)K20H4) at pericentric heterochromatin, indicating that HP1 can regulate the distribution of these histone modifications in vivo. Both homo- and hetero-typic interactions between the HP1 isotypes were demonstrated in vivo as were HP1 interactions with the ESET/SETDB1 HMTase and the ATRX chromatin remodelling enzyme. We conclude that HP1 not only "deciphers" the histone code but can also "encode it".

Highly efficient mRNA-based gene transfer in feeder-free cultured H9 human embryonic stem cells.

Several protocols have been described for virus-based gene transfer in human embryonic stem (hES) cells, while efficient non-viral methods are currently non-existing. In this study, we investigated the efficiency of mRNA-based gene transfer in feeder-free cultured H9 hES cells, based on electroporation of in vitro transcribed mRNA encoding the enhanced green fluorescent protein (EGFP). Optimisation of culture and electroporation conditions for feeder-free cultured H9 hES cells resulted a highly pure, transgene-expressing (90% positive cells) H9 hES cell population.

Loss of heterochromatin protein 1 (HP1) chromodomain function in mammalian cells by intracellular antibodies causes cell death.

The chromodomain (CD) is a highly conserved motif present in a variety of animal and plant proteins, and its probable role is to assemble a variety of macromolecular complexes in chromatin. The importance of the CD to the survival of mammalian cells has been tested. Accordingly, we have ablated CD function using two single-chain intracellular Fv (scFv) fragments directed against non-overlapping epitopes within the HP1 CD motif. The scFv fragments can recognize both CD motifs of HP1 and Polycomb (Pc) in vitro and, when expressed intracellularly, interact with and dislodge the HP1 protein(s) from their heterochromatin localization in vivo. Mouse and human fibroblasts expressing anti-chromodomain scFv fragments show a cell-lethal phenotype and an apoptotic morphology becomes apparent soon after transfection. The mechanism of cell death appears to be p53 independent, and the cells are only partly rescued by incubation with the wide spectrum caspase inhibitor Z-VAD fmk. We conclude that expression of anti-chromodomain intracellular antibodies is sufficient to trigger a p53-independent apoptotic pathway that is only partly dependent on the known Z-VAD-inhibitable caspases, suggesting that CD function is essential for cell survival.

The Ki-67 protein interacts with members of the heterochromatin protein 1 (HP1) family: a potential role in the regulation of higher-order chromatin structure.

The expression of the nuclear protein Ki-67 (pKi-67) is strictly correlated with cell proliferation. Because of this, anti-Ki-67 antibodies can be used as operational markers to estimate the growth fraction of human neoplasia in situ. For a variety of tumours, the assessment of pKi-67 expression has repeatedly been proven to be of prognostic value for survival and tumour recurrence, but no cellular function has yet been ascribed to the Ki-67 protein. This study shows that a C-terminal domain of pKi-67 (Kon21) is able to bind to all three members of the mammalian heterochromatin protein 1 (HP1) family in vitro and in vivo. This interaction can be manipulated in living cells, as evidenced by ectopic expression of GFP-tagged HP1 proteins in HeLa cells, which results in a dramatic relocalization of endogenous pKi-67. Taken together, the data presented in this study suggest a role for pKi-67 in the control of higher-order chromatin structure.