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Breaking the Cycle a Sperm Cytoskeletal Protein That Signals Oocyte a Broken Leg Is a Minor Inconve- the Authors Used a Site-Specific Meiotic Maturation and Ovulation

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Breaking the Cycle a Sperm Cytoskeletal Protein That Signals Oocyte a Broken Leg Is a Minor Inconve- the Authors Used a Site-Specific Meiotic Maturation and Ovulation HIGHLIGHTS IN BRIEF DNA REPAIR REPRODUCTION Breaking the cycle A sperm cytoskeletal protein that signals oocyte A broken leg is a minor inconve- The authors used a site-specific meiotic maturation and ovulation. nience — it may keep you in bed for a endonuclease to introduce one or two Miller, M. A. et al. Science 291, 2144–2147 (2001) few days, but eventually you’ll need to DSBs that could not be repaired. get up and carry on as normal. So Although cells adapt to one DSB, they In many animals, oocytes arrest during meiotic prophase. They are too, it seems, for yeast with broken remain arrested with two. triggered to resume meiosis by sperm, which also cause the gonadal DNA. A double-stranded DNA break Interestingly, however, it has been sheath cells (needed for ovulation) to contract. Miller and (DSB) brings the cell cycle to a halt, previously shown that yeast cells do colleagues show here that the major sperm cytoskeletal protein allowing time for the break to be not decide to adapt by measuring the (MSP) is a signal for both of these events. They go on to show that repaired. After 8–12 hours, however, number of DSBs per se. Rather, they MSP has a function in sperm locomotion, analogous to that of the cells may escape this arrest — seem to monitor the amount of sin- actin, and conclude that MSP has acquired both extracellular even if the DSB persists — in a gle-stranded DNA that is generated signalling and intracellular cytoskeletal functions during evolution. process known as adaptation. as exonucleases nibble back at the How can cells do this? James broken ends. So deletion of the DNA- PROTEIN METHYLATION Haber, Marco Foiani and colleagues end-binding protein yKu70 prevents have investigated this problem and, adaptation with one DSB, as exonu- α β Arginine methylation of STAT1 modulates IFN / - reporting in Molecular Cell, they show cleases are more free to attack the induced transcription. that adaptation is accompanied by a exposed DNA ends. Mowen, K. A. et al. Cell 104, 731–741 (2001) loss of the Rad53 checkpoint kinase Haber, Foiani and colleagues Previous analysis of STAT transcription factors has focused on activity. found that, in cells that adapt, there their regulation by phosphorylation, shadowing the importance The DNA-damage checkpoint in is a concomitant loss of Rad53 of another modification — arginine methylation. Here, Mowen Saccharomyces cerevisiae involves a kinase activity. Conversely, in those and colleagues show that STAT1 is methylated on a conserved cascade of protein kinases, including cells that fail to adapt — because arginine residue, a modification mediated by the arginine Rad53, Chk1, Mec1 and Cdc5 (which they contain two DSBs or lack func- methyltransferase PRMT1 and required for interferon α/β- is phosphorylated by Rad53 and is tional Cdc5 or yKu70 — the levels of induced transcription. Moreover, they find that the known to be involved in adaptation). Rad53 activity remain high. When methyltransferase inhibitor MTA inhibits this methylation, One way for cells to adapt would be cells adapt, phosphorylation of Chk1 leading to increased association of STAT with its inhibitor PIAS1 to turn off this kinase cascade, even in also decreases, indicating that both and so provide a model for the lack of interferon responsiveness the presence of a DSB, so the authors the Rad53 and Chk1 pathways are observed in certain cancers. asked what happens to Rad53 and involved in adaptation. Moreover, Chk1 in cells that adapt versus those maintenance of the arrested state CELL DIVISION that remain permanently arrested. depends on continued activity of the They chose Rad53 and Chk1 because Mec1 kinase. This means, say the Requirement of a centrosomal activity for cell cycle both are phosphorylated and activat- authors, that adaptation is due to progression through G1 into S phase ed by the upstream kinase Mec1 in inactivation of the kinase cascade response to DNA damage. itself, rather than, for example, mod- Hinchcliffe, E. H. et al. Science 291, 1547–1550 (2001) ification of a downstream receptor Centrosome-dependent exit of cytokinesis in such that it is no longer sensitive to animal cells the cascade. Piel, M. et al. Science 291, 1550–1553 (2001) But why do yeast cells adapt at all? The authors speculate that it may The centrosome — the main microtubule organizing centre in allow the DSB to be repaired in a sub- animal cells — has long been thought to change its microtubule sequent cell cycle, and there is evi- organizing properties in response to cell-cycle progression. These dence that some repair mechanisms two papers now show that the centrosome, in turn, is required at may be more efficient during S phase two distinct stages of the cell cycle — the G1 to S transition and the than at other times in the cell cycle. completion of cytokinesis. Piel and colleagues show that, in animal For the moment, though, a next step cells, the centrosome is repositioned after anaphase, and is required will be to find out how the kinase cas- for microtubule release from the midbody and the completion of cade is inactivated — by proteolytic cell division. Hinchcliffe et al. removed the centrosome from destruction of phosphorylated kinas- primate somatic cells, and found that they arrested before S phase, es, perhaps, or by dephosphorylation indicating that a centrosomal-associated factor mediates entry into of Rad53 and Chk1? S phase. They also find that, once in mitosis, the centrosome is not Alison Mitchell required for the G2 to M transition. Together these papers indicate References and links cells may have checkpoints that monitor centrosome duplication ORIGINAL RESEARCH PAPER Pellicioli, A. et al. or, say the authors,“core centrosomal structures could bind cell Regulation of Saccharomyces Rad53 checkpoint kinase during adaptation from DNA damage- cycle regulatory molecules in a way that activates their function”. induced G2/M arrest. Mol. Cell 7, 293–300 (2001) 230 | APRIL 2001 | VOLUME 2 www.nature.com/reviews/molcellbio © 2001 Macmillan Magazines Ltd HIGHLIGHTS WEB WATCH … and the virtual cell project The Virtual Cell is described by its creators as “a general computational framework for modelling cell biological processes”. Access to this software is available over the internet through a JAVA- based interface, and although there is no charge for academic use of the Virtual Cell, users are required to register. So what is the Virtual Cell? It has been developed at the National Resource for Cell Analysis and Modeling (NRCAM), a US-based resource centre supported by the National Center for Research Resources. Those behind it claim that the technology links biochemical and electrophysiological data Organelles in the Golgi region. C1 (cis-Golgi cisterna), light blue; C5, dark blue; C7, red; ER, yellow; microtubules and with experimentally derived mitochondria, green; dense core vesicles, blue; clathrin-negative vesicles, white; clathrin-positive vesicles and compartments, microscopic images showing red; clathrin-negative compartments, purple; free ribosomes, orange. Reproduced with permission from National Academy of the subcellular localizations of Sciences © (2001). the molecules involved. In this way, they say, “physiological TECHNIQUE results can be simulated within the empirically derived geometries, thus facilitating the direct comparison of The visible cell project . model predictions with experiment”. Pancreatic β-cells (HIT-T15) were put to death light on the relationship between the ER and the Golgi, There are two interfaces to through high-pressure freezing and freeze- which has been controversial over the past years. the Virtual Cell — one substitution, cut into ribbons of serial 400-nm-thick Microtubules seem to follow closely the membranes biologically orientated, the sections and studied by electron tomography. Marsh of the C1 cisterna of the Golgi and of endosomal other mathematical. The biological interface has been and colleagues collected 480 images of three serial compartments. However, the ER seems to be anchored designed to allow users to sections by tilting the grid that holds the specimen by along microtubules at a few points only. Here again, define cellular geometry, 1.5° over a range of 120° about two orthogonal axes. this observation could provide a lead for further create models and They then combined all these images to produce a investigation into the interaction of organelles with simulations, and to analyse single, high resolution three-dimensional microtubules. the results of such reconstruction of a 3.1 × 3.2 × 1.2 µm3 area around the The technique is not only qualitative but also simulations. Golgi apparatus of a cell.You would think this is a lot of provides a means to quantify organelles in situ in For those unfamiliar with the technology or software work for a single image, but just look at the result! three dimensions, and to measure accurately their there’s a ‘User’s Guide’, The freezing procedure immobilized all cellular physical associations with other organelles. For backed up by a tutorial activity within milliseconds, so the final image example, at first sight, you might get the impression designed to work in probably reflects the situation found in a live cell. In that the Golgi region is very crowded, but Marsh and conjunction with it, and a addition to being incredibly pretty, this snapshot of the colleagues calculated that only about 34% of the ‘User Discussion’ page for cell also gives us some insights into the organization of volume is taken up by organelles, the rest being made troubleshooting. There are also examples of what the organelles around the Golgi. up of cytoplasmic matrix. site can do — the There were not many surprises about the structure Although many of the findings in this study are not applications shown include of the Golgi itself, which is here made up of seven truly novel or revolutionary, being able to see use of the Virtual Cell to study cisternae (C1 to C7, going from cis to trans Golgi).
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