- Email: [email protected]
Orchestrating cell division
In the April to May transition of this year, a conference in Roscoff, France* paid tribute to Yoshio Masui one of the founders of the cell-cycle field and was a forum for discussion of some of the central features that cause cells to transit from one cell-cycle stage to another. Although this meeting was billed as ‘The Cell Division Cycle’, the choice of speakers clearly pointed to an intense interest in the architecture and regulation of M phase. Are centrosomes an essential structure? Centrosomes are thought to be required for the assembly of the mitotic spindle and thus for correct chromosome segregation. This view has been challenged lately by a number of studies that highlight alternative mechanisms for the formation of the spindle and the maintenance of its bipolarity. Whether or not there is an absolute need for a centrosome is controversial, and work presented by K. Sluder (Worcester, USA) fuelled the debate. He described a delicate microsurgical procedure developed to cut cells in two just between the nucleus and centrosome. The resulting acentrosomal karyoplasts are able to form a bipolar spindle and can go through a round of chromosomal division but cannot complete the final stages of cytokinesis. These binucleate acentrosomal karyoplasts are unable to enter the next cell cycle and consequently arrest in G1 phase.
57 Zhang, X.P. et al. (1999) Interaction of mitochondrial presequences with DnaK and mitochondrial hsp70. J. Mol. Biol. 288, 177–190 58 Bolter, B. et al. (1998) Origin of a chloroplast protein importer. Proc. Natl. Acad. Sci. U. S. A. 95, 15831–15836 59 Reumann, S. and Keegstra, K. (1999) The endosymbiotic origin of the protein import machinery of chloroplastic envelope membranes. Trends Plant Sci. 4, 302–307 60 Reumann, S. et al. (1999) The evolutionary origin of the proteintranslocating channel of chloroplastic envelope membranes: identification of a cyanobacterial homolog. Proc. Natl. Acad. Sci. U. S. A. 96, 784–789 61 McFadden, G.I. (1999) Endosymbiosis and evolution of the plant cell. Curr. Opin. Plant Biol. 2, 513–519 62 de Souza, S.J. et al. (1996) Intron positions correlate with module boundaries in ancient proteins. Proc. Natl. Acad. Sci. U. S. A. 93, 14632–14636 63 Quigley, F. et al. (1988) Intron conservation across the prokaryoteeukaryote boundary: structure of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize. Proc. Natl. Acad. Sci. U. S. A. 85, 2672–2676 64 Gregerson, R.G. et al. (1994) Genomic structure, expression and evolution of the alfalfa aspartate aminotransferase genes. Plant Mol. Biol. 25, 387–399 65 Long, M. et al. (1996) Exon shuffling and the origin of the mitochondrial targeting function in plant cytochrome c1 precursor. Proc. Natl. Acad. Sci. U. S. A. 93, 7727–7731 66 Liaud, M.F. et al. (1990) Differential intron loss and endosymbiotic transfer of chloroplast glyceraldehyde-3-phosphate dehydrogenase genes to the nucleus. Proc. Natl. Acad. Sci. U. S. A. 87, 8918–8922
MEETING REPORT
45 Lawrence, S.D. and Kindle, K.L. (1997) Alterations in the Chlamydomonas plastocyanin transit peptide have distinct effects on in vitro import and in vivo protein accumulation. J. Biol. Chem. 272, 20357–20363 46 Schatz, G. and Dobberstein, B. (1996) Common principles of protein translocation across membranes. Science 271, 1519–1526 47 May, T. and Soll, J. (2000) 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants. Plant Cell 12, 53–64 48 Komiya, T. et al. (1997) Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones. EMBO J. 16, 4267–4275 49 Waegemann, K. and Soll, J. (1996) Phosphorylation of the transit sequence of chloroplast precursor proteins. J. Biol. Chem. 271, 6545–6554 50 Muslin, A.J. et al. (1996) Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84, 889–897 51 Pilon, M. et al. (1992) Kinetic analysis of translocation into isolated chloroplasts of the purified ferredoxin precursor. FEBS Lett. 302, 65–68 52 Pilon, M. and Schekman, R. (1999) Protein translocation: how Hsp70 pulls it off. Cell 97, 679–682 53 Marshall, J.S. et al. (1990) Identification of heat shock protein hsp70 homologues in chloroplasts. Proc. Natl. Acad. Sci. U. S. A. 87, 374–378 54 Ivey, R.A. and Bruce, B.D. (2000) In vivo and in vitro interaction of DnaK and a chloroplast transit peptide. Cell Stress Chaperones 5, 62–71 55 Ivey, R.A., 3rd et al. (2000) Identification of a hsp70 recognition domain within the rubisco small subunit transit peptide. Plant Physiol. 122, 1289–1300 56 Stahl, T. et al. (1999) Tic40, a new ‘old’ subunit of the chloroplast protein import translocon. J. Biol. Chem. 274, 37467–37472
Orchestrating cell division Kirsten C. Sadler and Maria do Carmo Avides Other observations do point to a central role of the centrosome in the establishment of a functional bipolar mitotic spindle. As an example, Drosophila mutants in key centrosomal components display gross spindle morphological and functional defects. One such mutant, dd4, a mutant in the spc98 Drosophila homologue dGRIP91, has abnormal mitotic spindles with a reduced number of microtubules (D. Glover, Cambridge, UK; Fig. 1). It is often said that a book is better than a movie, but, in cell biology, it is just the opposite. Many presentations contained spectacular movies of gyrating spindles, dancing centrosomes and dividing chromosomes. For example, the movies shown by M. Bornens (Paris, France) that accompanied a recent publication were certainly more entertaining and informative than the corresponding print version1. GFP-labelled centrin labels the mother centriole more brightly than the daughter, providing a means to discriminate the two as they perform their separation dance during anaphase. The daughter centriole leaps and twitches around the unmoving mother and, if she
trends in CELL BIOLOGY (Vol. 10) October 2000
dances near the midbody, the connection between two recently formed daughter cells is severed and the cells are free to move apart. Until recently, the process of centrosome duplication has been poorly understood, and the first insights came from work on the budding yeast spindle pole body (SPB). It soon became apparent that the proteins involved in SPB duplication had functional homologues in higher eukaryotes. One such example is the S. cerevisiae kinase MPS1, also required for the spindle integrity checkpoint2. Two informative MPS1 mutant alleles, mps1-7 and mps1-8, were described by M. Winey (Boulder, USA). Mps1-7 mutants are defective in the spindle checkpoint and show abnormalities in meiotic chromosome segregation, implying a new function for MPS1 during meiosis. Mps1-8 defects are exclusively on the duplication of the SPB and relate to the structure of the half-bridge. A link between regulatory and structural cell-cycle events was provided by E. Nigg (Martinsreid, Germany). The nuclear cell cycle and the centrosome cycle appear to be linked through
0962-8924/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(00)01820-1
Kirsten Sadler is in the Dept of Molecular Biology and Genetics, Bosphorus University, Istanbul, Turkey; and Maria do Carmo Avides is in the Dept of Genetics, University of Cambridge, Cambridge, UK CB23EH. E-mail: kirsten_sadler_ phd98@post. harvard.edu *The Jacques Monod Conference on the Cell Division Cycle; Roscoff, France; 30 April – 3 May, 2000. Organized by Eric Nigg and Bernard Maro.
447
meeting report (a)
(b)
(c)
FIGURE 1 The Drosophila mutant dd4 shows an abnormal localization of Asp in larval neuroblasts. (a) A wild-type cell in anaphase. (b) Asp contacts the tips of microtubules (arrows) in the abnormally broad spindle poles of dd42 cells. (c) A dd4S cell in which one spindle pole is no longer focussed. Punctate Asp staining can be seen at the tips of microtubules (arrows). DNA is shown in blue, Asp in red and b-tubulin in green. Bars, 5 mm. Image courtesy of David M. Glover.
the central S-phase regulators Rb and E2F. When entry into S-phase is prevented by blocking either Rb inactivation, activation of E2F or activation of cdk2, the centrosome duplication cycle is also blocked. Nigg suggested that the uncoupling between the centrosomal cycle and the cell cycle, which often occurs in cancer, may result from the disruption of molecules that are common to both cycles.
448
Stabilizing and destabilizing the mitotic spindle Although much progress has been made in our understanding of spindle microtubule dynamics, it is not yet fully understood how the dynamics change from one cell-cycle stage to another. For instance, during Mphase, microtubules polymerize and depolymerize at an astounding rate, much faster than what is observed in interphase. In a Herculean effort to identify Caenorhabditis elegans genes involved in spindle behaviour, A. Hyman (Heidelberg, Germany) reported the use of RNA interference (RNAi) to knock out all of the 2291 genes on chromosome III followed by video taping the first cleavage of each of the resulting embryos. He reported 128 genes required for the first cleavage, seven of which are directly related to the function of the spindle. One of the seven was identified as ZYG8, a novel microtubule-binding protein containing both a kinase and a doublecortin domain. RNAi deletion of ZYG8 causes spindle collapse in mid-M phase. Although this is the first gene from the Hyman screen to be described, the remaining 127 have been classified into categories such as spindle or cytokinesis defects and are open to investigation. Studying C. elegans mutants defective in cytokinesis was also fruitful in uncovering CYK4 and ZEN4 as central to the mysterious relationship between the spindle and the cleavage furrow (M. Glotzer, Vienna, Austria). Cyk4 is a GTPase-activating protein crucial for microtubule bundling and for cleavage furrow formation. Cyk4 is probably tethered to the plus ends of microtubules at the spindle midzone through its attachment to Zen4. Another link between cytokinesis and the spindle was demonstrated by studies of mammalian INCENP, the inner centromere protein that is left behind at the midbody during anaphase to define the site of the future cleavage furrow (W. Earnshaw, Edinburgh, UK). Interestingly, when the INCENP domain responsible for targeting it to the inner centromere is swapped with the centromere-targeting domain of CENP-B, the cells carrying this hybrid do not complete cytokinesis. Xenopus egg extracts were used by I. Vernos (Heidelberg, Germany) to investigate the role of kinesin-related proteins in spindle formation. The kinesin-related protein Xklp2 and its partner, Tpx2, interact with microtubules and localize to the centro-
some throughout mitosis. Depletion of Xklp2 disrupts spindle formation, and its targeting to microtubules is dependent upon Tpx2 (Ref. 3). Another kinesin required for chromosome segregation is Xkid1 (Ref. 4) (A. Murray, San Francisco, USA), a protein involved in the spindle assembly checkpoint that was identified in a screen for proteins that adhere to the chromosomes during M phase but not during interphase. The MAP kinase dogma changes A molecular pathway that has been ingrained in the minds of most cellcycle researchers is the c-Mos-MEKMAP kinase cascade leading to the activation of MPF and entry into meiotic M-phase in frog oocytes. Scores of experiments over the past 10 years have shown that the members of this pathway are both necessary and sufficient for activation of MPF. However, studies from other organisms have led to the uncomfortable realization that c-Mos and MAP kinase are not needed for meiosis re-entry in any other animal, and perhaps this discomfort has inspired a re-examination of the role of the Mos–MAP kinase pathway in Xenopus. Data presented from many labs both in the recent literature and in Roscoff support the notion that MAP kinase is in fact not centre stage in the pathway leading to MPF activation in Xenopus (Fig. 2). For instance, a poster from the Haccard lab (Paris, France) showed that injecting oocytes with Xenopus Ras results in MPF activation even in the absence of MAP kinase activity, and M. Dorée (Montpellier, France) has shown that MAP kinase is phosphorylated very soon after progesterone stimulation, well in advance of any c-Mos translation. Although this doctrine shift has left some feeling more comfortable with the notion that Xenopus oocytes do after all match the rest of the animal kingdom, it has left many wondering what is the trigger kinase for MPF activation and what MAP kinase is doing during meiosis. A recent study from the Nebreda lab (Heidelberg, Germany) addressed the latter issue, showing that MAP kinase interacts with and phosphorylates pp90rsk, which, in turn, phosphorylates and inactivates Myt1 kinase, the sole component that keeps Cdc2 inactive in G2-arrested Xenopus oocytes5. The Maller lab (Denver, USA) has identified the polo-like kinase homologue (Plx1) as the trigger kinase that phosphorylates and activates the MPF
trends in CELL BIOLOGY (Vol. 10) October 2000
meeting report Hormone receptor ?
mos mRNA c-Mos protein (unstable)
APC/C PKA
? ?
XAIRK-1
c-Mos—hsp90 (stable) CPEB
?
?
?
MEK
XAIRK-1 (Eg2) ?
Plkk1
MAPK
?
Xenopus & starfish
Ras Xplk-1
pp90rsk ? cdc25
Myt-1 Interphase/ DNA replication MPF wee1
Mitosis Meiosis
trends in Cell Biology
FIGURE 2 Signalling pathway controlling meiosis I in animal oocytes. It is not known whether all of the components depicted are functional in all animals. However, recent data point to a more conserved pathway than originally thought. Data presented at the Roscoff meeting are shown in colours corresponding to the lab from which they came: Maller (purple), Nebreda (green), Dorée (brown), Jessus (orange), Kishimoto (pink), Sagata (cyan), Haccard (grey) and Ruderman (blue; published data8,12 but not presented). Abbreviations: CPEB, cytoplasmic polyadenylation element binding factor; hsp90, heat shock protein 90; PKA, protein kinase A.
activator cdc25. Injection of constitutively active Plx1 into oocytes causes them to enter the cell cycle in the absence of progesterone, confirming its central role in MPF activation. Plx1 can also phosphorylate and activate its activator, Plx1-kinase (Xplkk1), although it is clear that there are other factors required for Plx1 activation too6. What does Eg2 do? Oocytes have traditionally been used for studying the G2–M transition because many oocytes are naturally arrested in G2 of meiosis I until an external stimulus catapults their entry into M phase. Although there is a significant amount of data on the regulation MPF in these cells, much less is know about the steps that lead to MPF activation (see Fig. 2 for summary). Eg2 kinase has emerged as an important molecule in the pathway leading to MPF activation in Xenopus oocytes and was the focus of no fewer than three talks. Eg2 is a member of the Aurora/Ipl family of kinases that serve diverse yet crucial functions in several stages of M phase. C. Prigent
(Rennes, France) first isolated Eg2 (Ref. 7) and has since been investigating this family, all of which have confusing names. He began his talk with a sensible appeal to adopt a common nomenclature for this fast-growing family. The proposed AIRK family members will each have a prefix to denote their species, followed by a numerical suffix that will be assigned to the functional homologues in different species. He showed experiments in Xenopus egg extracts in which XAIRK-1 (formerly known as Eg2) had been depleted or inactivated. This resulted in both the failure to form bipolar spindles as well as causing preformed spindles to collapse into monopolar spindles that retain their attachments to chromosomes. This effect is thought to be due to XAIRK-1 association with the kinesin-like molecule Eg5. Is XAIRK-1 involved in MPF activation? A previous study showed that overexpression of XAIRK-1 accelerates progesterone-induced c-Mos translation, MAP kinase activation and cellcycle re-entry8. This was confirmed by the Dorée lab, but with the surprising
trends in CELL BIOLOGY (Vol. 10) October 2000
caveat that MAP kinase activation occurs even in the absence of Mos or detectable MEK activity. This one result proposes a hitherto unknown mechanism for activation of MAP kinase in Xenopus and deserves greater attention. A study from the Jessus lab (Paris, France) showed that that XAIRK-1 is a target of MPF, and phosphorylation by MPF is a requisite for XAIRK-1 to be degraded by the anaphase-promoting complex/cyclosome (APC/C), the instrument responsible for the destruction of many cell-cycle regulatory molecules controlling key cellcycle transitions. M, no S For the M-phase aficionados in attendance, the meiotic cell cycle is a haven, providing a double-dose of M without any intervening S or G phases to confuse matters. There are two hypotheses to explain the absence of S-phase during meiosis. One is that meiotic cells lack a factor that is required for entry into interphase following meiosis I, whereas the other proposes the existence of a factor that actively suppresses S during
449
meeting report interkinesis (the period between meiosis I and II). N. Sagata (Fukuoka, Japan) presented evidence supporting the first hypothesis by showing that introduction of the interphasepromoting molecule and MPF inactivator, wee1, during meiosis I causes the oocytes to exit M phase and to replicate their DNA during interkinesis. In support of the suppressor model, several years ago Sagata showed that c-Mos was required for suppression of S-phase during Xenopus meiosis9, but, until this conference, it was presumed that it was not a universal mechanism because c-Mos had only been identified in vertebrates. In one of the most exciting reports of the meeting, Kishimoto (Yokohama, Japan) described the function of starfish c-Mos. Overexpression of c-Mos in starfish oocytes results in MAP kinase activation, but not cell-cycle activation. Prevention of Mos translation during meiosis I, however, converts the meiotic divisions to a mitosis-like pattern in which the cells undergo S phase after meiosis I, then re-activate MPF to undergo a periodic S- and Mphase cycle, just like the mitotic divisions of embryonic cleavage (albeit without cytokinesis; Fig. 2). Separating sisters A commonly held view is that the chromosomal glue is distributed along the chromosomal arms and in the kinetochore region and this global cohesion is maintained until it is degraded in anaphase A. An elegant experiment in budding yeast challenged this idea. A technique to monitor sister-chromatid separation in yeast involves placing a GFP tag on one chromosome so that, when the chromatids are together, there is one dot and when the sisters separate, two dots are observed10. G. Goshima (Yanagida lab, Kyoto, Japan) described an experiment in which the GFP tag was placed either 1.8 Kb or 23 Kb from the centromere. Surprisingly, when the tag was near the centromere, two dots were seen in Sphase, meaning that the kinetochore region had separated as soon as the spindle is formed. By contrast, the more distal tag (which is the site most commonly used) remains a single dot until anaphase. This suggests that, following DNA replication, sisters are held together along their arms, but not in their centromere region. C. Lehner (Bayreuth, Germany) described the Drosophila mutant pimples as a dramatic example of what happens if sisters do not separate11.
450
In most systems, a defect in sister separation results in M-phase arrest, with stable cyclin B. By contrast, sister chromatids in pimples never separate, even though the cells go through multiple rounds of S and M phases, resulting in multivalent chromosomes. Pimples contains a destruction box (D-box), the sequence that targets proteins for degradation by the APC/C. Lehner showed that Pimples is degraded during late metaphase, just like cyclin B, and mutating the D-box to create stable Pimples causes cells to arrest in what appears to be an M-phase, although cyclin B is degraded. This result contradicts the accepted view that stabilization of cyclin B is a defining characteristic of M-phase-arrested cells and suggests a possible need for rethinking the criteria by which cell-cycle stages are defined. For Roscoff The setting of this meeting at the Station Biologique in Roscoff befitted the field’s inextricable link to the use of marine invertebrates to uncover some of the most interesting and crucial components of the cell-cycle machinery. Species native to Roscoff are still being used to probe some important cell-cycle issues, such as how the polarized cell division observed in the zygotes of the common sea weed, Fucus, is achieved (F. Bouget, Roscoff, France) and how can we interrupt the unwanted cell divisions in cancer. Answers to the latter question are being sought by L. Meijer (Roscoff, France) who has established a network of chemists, biologists and cell-cycle specialists to design or extract compounds capable of inhibiting starfish MPF in vitro, with the hopes that one will also inhibit cancer cell division in vivo. Finally, in the straightforward and stately style befitting one of the field’s founders, Masui (Toronto, Canada) described his ongoing observations of the cell-division cycle in Xenopus embryos. During the mid-blastula transition, these cells switch from their synchronous cycles to seemingly irregular ones. His closer look at the asynchronously dividing cells revealed a rhythm to their cell-cycle times, with the duration of each division lasting for a multiple of 30 minutes. These sorts of simple, yet revealing, experiments in embryonic systems formed the heart of the field some 30 years ago and today provide inspiration to take a walk on a beach and bring some of the findings back to the lab for a look.
References 1 Piel, M. et al. (2000) The respective contributions of the mother and daughter centrioles to centrosome activity and behaviour in vertebrate cells. J. Cell Biol. 149, 317–330 2 Weiss, E. and Winey, M. (1996) The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint. J. Cell Biol. 132, 111–123 3 Wittman, T. et al. (1998) Localization of the kinesin-like protein Xklp2 to spindle poles requires a leucine zipper, a microtubuleassociated protein, and dynein. J. Cell Biol. 143, 673–685 4 Tokay, N. et al. (1996) Kid, a novel kinesinlike DNA binding protein, is localized to chromosomes and the mitotic spindle. EMBO J. 15, 457–467 5 Gavin, A.C. et al. (1999) A p90(rsk) mutant constitutively interacting with MAP kinase uncouples MAP kinase from p34(cdc2)/cyclin B activation in Xenopus oocytes. Mol. Biol. Cell 10, 2971–2986 6 Qian, Y.W. et al. (1998) Purification and cloning of a protein kinase that phosphorylates and activates polo-like kinase Plx1. Science 282, 1701–1704 7 Roghi C. et al. (1998) The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly. J. Cell Sci. 111, 557–572 8 Andresson, T. and Ruderman, J.V. (1998) The kinase Eg2 is a component of the Xenopus oocyte progesterone-activated signalling pathway. EMBO J. 17, 5627–5637 9 Furuno, N. et al. (1994) Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes. EMBO J. 13, 2399–2410 10 Straight, A.F. et al. (1997) Mitosis in living budding yeast: anaphase A but no metaphase plate. Science 277, 574–578 11 Stratmann, R. and Lehner, C.F. (1996) Separation of sister chromatids in mitosis requires the Drosophila pimples product, a protein degraded after the metaphase/anaphase transition. Cell 84, 25–35 12 Mendez, R. et al. (2000) Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. Nature 404, 302–307
Students Did you know that you are entitled to a 50% discount on a subscription to TCB? See the bound-in subscription order card for details
trends in CELL BIOLOGY (Vol. 10) October 2000
57 Zhang, X.P. et al. (1999) Interaction of mitochondrial presequences with DnaK and mitochondrial hsp70. J. Mol. Biol. 288, 177–190 58 Bolter, B. et al. (1998) Origin of a chloroplast protein importer. Proc. Natl. Acad. Sci. U. S. A. 95, 15831–15836 59 Reumann, S. and Keegstra, K. (1999) The endosymbiotic origin of the protein import machinery of chloroplastic envelope membranes. Trends Plant Sci. 4, 302–307 60 Reumann, S. et al. (1999) The evolutionary origin of the proteintranslocating channel of chloroplastic envelope membranes: identification of a cyanobacterial homolog. Proc. Natl. Acad. Sci. U. S. A. 96, 784–789 61 McFadden, G.I. (1999) Endosymbiosis and evolution of the plant cell. Curr. Opin. Plant Biol. 2, 513–519 62 de Souza, S.J. et al. (1996) Intron positions correlate with module boundaries in ancient proteins. Proc. Natl. Acad. Sci. U. S. A. 93, 14632–14636 63 Quigley, F. et al. (1988) Intron conservation across the prokaryoteeukaryote boundary: structure of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize. Proc. Natl. Acad. Sci. U. S. A. 85, 2672–2676 64 Gregerson, R.G. et al. (1994) Genomic structure, expression and evolution of the alfalfa aspartate aminotransferase genes. Plant Mol. Biol. 25, 387–399 65 Long, M. et al. (1996) Exon shuffling and the origin of the mitochondrial targeting function in plant cytochrome c1 precursor. Proc. Natl. Acad. Sci. U. S. A. 93, 7727–7731 66 Liaud, M.F. et al. (1990) Differential intron loss and endosymbiotic transfer of chloroplast glyceraldehyde-3-phosphate dehydrogenase genes to the nucleus. Proc. Natl. Acad. Sci. U. S. A. 87, 8918–8922
MEETING REPORT
45 Lawrence, S.D. and Kindle, K.L. (1997) Alterations in the Chlamydomonas plastocyanin transit peptide have distinct effects on in vitro import and in vivo protein accumulation. J. Biol. Chem. 272, 20357–20363 46 Schatz, G. and Dobberstein, B. (1996) Common principles of protein translocation across membranes. Science 271, 1519–1526 47 May, T. and Soll, J. (2000) 14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants. Plant Cell 12, 53–64 48 Komiya, T. et al. (1997) Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones. EMBO J. 16, 4267–4275 49 Waegemann, K. and Soll, J. (1996) Phosphorylation of the transit sequence of chloroplast precursor proteins. J. Biol. Chem. 271, 6545–6554 50 Muslin, A.J. et al. (1996) Interaction of 14-3-3 with signaling proteins is mediated by the recognition of phosphoserine. Cell 84, 889–897 51 Pilon, M. et al. (1992) Kinetic analysis of translocation into isolated chloroplasts of the purified ferredoxin precursor. FEBS Lett. 302, 65–68 52 Pilon, M. and Schekman, R. (1999) Protein translocation: how Hsp70 pulls it off. Cell 97, 679–682 53 Marshall, J.S. et al. (1990) Identification of heat shock protein hsp70 homologues in chloroplasts. Proc. Natl. Acad. Sci. U. S. A. 87, 374–378 54 Ivey, R.A. and Bruce, B.D. (2000) In vivo and in vitro interaction of DnaK and a chloroplast transit peptide. Cell Stress Chaperones 5, 62–71 55 Ivey, R.A., 3rd et al. (2000) Identification of a hsp70 recognition domain within the rubisco small subunit transit peptide. Plant Physiol. 122, 1289–1300 56 Stahl, T. et al. (1999) Tic40, a new ‘old’ subunit of the chloroplast protein import translocon. J. Biol. Chem. 274, 37467–37472
Orchestrating cell division Kirsten C. Sadler and Maria do Carmo Avides Other observations do point to a central role of the centrosome in the establishment of a functional bipolar mitotic spindle. As an example, Drosophila mutants in key centrosomal components display gross spindle morphological and functional defects. One such mutant, dd4, a mutant in the spc98 Drosophila homologue dGRIP91, has abnormal mitotic spindles with a reduced number of microtubules (D. Glover, Cambridge, UK; Fig. 1). It is often said that a book is better than a movie, but, in cell biology, it is just the opposite. Many presentations contained spectacular movies of gyrating spindles, dancing centrosomes and dividing chromosomes. For example, the movies shown by M. Bornens (Paris, France) that accompanied a recent publication were certainly more entertaining and informative than the corresponding print version1. GFP-labelled centrin labels the mother centriole more brightly than the daughter, providing a means to discriminate the two as they perform their separation dance during anaphase. The daughter centriole leaps and twitches around the unmoving mother and, if she
trends in CELL BIOLOGY (Vol. 10) October 2000
dances near the midbody, the connection between two recently formed daughter cells is severed and the cells are free to move apart. Until recently, the process of centrosome duplication has been poorly understood, and the first insights came from work on the budding yeast spindle pole body (SPB). It soon became apparent that the proteins involved in SPB duplication had functional homologues in higher eukaryotes. One such example is the S. cerevisiae kinase MPS1, also required for the spindle integrity checkpoint2. Two informative MPS1 mutant alleles, mps1-7 and mps1-8, were described by M. Winey (Boulder, USA). Mps1-7 mutants are defective in the spindle checkpoint and show abnormalities in meiotic chromosome segregation, implying a new function for MPS1 during meiosis. Mps1-8 defects are exclusively on the duplication of the SPB and relate to the structure of the half-bridge. A link between regulatory and structural cell-cycle events was provided by E. Nigg (Martinsreid, Germany). The nuclear cell cycle and the centrosome cycle appear to be linked through
0962-8924/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0962-8924(00)01820-1
Kirsten Sadler is in the Dept of Molecular Biology and Genetics, Bosphorus University, Istanbul, Turkey; and Maria do Carmo Avides is in the Dept of Genetics, University of Cambridge, Cambridge, UK CB23EH. E-mail: kirsten_sadler_ phd98@post. harvard.edu *The Jacques Monod Conference on the Cell Division Cycle; Roscoff, France; 30 April – 3 May, 2000. Organized by Eric Nigg and Bernard Maro.
447
meeting report (a)
(b)
(c)
FIGURE 1 The Drosophila mutant dd4 shows an abnormal localization of Asp in larval neuroblasts. (a) A wild-type cell in anaphase. (b) Asp contacts the tips of microtubules (arrows) in the abnormally broad spindle poles of dd42 cells. (c) A dd4S cell in which one spindle pole is no longer focussed. Punctate Asp staining can be seen at the tips of microtubules (arrows). DNA is shown in blue, Asp in red and b-tubulin in green. Bars, 5 mm. Image courtesy of David M. Glover.
the central S-phase regulators Rb and E2F. When entry into S-phase is prevented by blocking either Rb inactivation, activation of E2F or activation of cdk2, the centrosome duplication cycle is also blocked. Nigg suggested that the uncoupling between the centrosomal cycle and the cell cycle, which often occurs in cancer, may result from the disruption of molecules that are common to both cycles.
448
Stabilizing and destabilizing the mitotic spindle Although much progress has been made in our understanding of spindle microtubule dynamics, it is not yet fully understood how the dynamics change from one cell-cycle stage to another. For instance, during Mphase, microtubules polymerize and depolymerize at an astounding rate, much faster than what is observed in interphase. In a Herculean effort to identify Caenorhabditis elegans genes involved in spindle behaviour, A. Hyman (Heidelberg, Germany) reported the use of RNA interference (RNAi) to knock out all of the 2291 genes on chromosome III followed by video taping the first cleavage of each of the resulting embryos. He reported 128 genes required for the first cleavage, seven of which are directly related to the function of the spindle. One of the seven was identified as ZYG8, a novel microtubule-binding protein containing both a kinase and a doublecortin domain. RNAi deletion of ZYG8 causes spindle collapse in mid-M phase. Although this is the first gene from the Hyman screen to be described, the remaining 127 have been classified into categories such as spindle or cytokinesis defects and are open to investigation. Studying C. elegans mutants defective in cytokinesis was also fruitful in uncovering CYK4 and ZEN4 as central to the mysterious relationship between the spindle and the cleavage furrow (M. Glotzer, Vienna, Austria). Cyk4 is a GTPase-activating protein crucial for microtubule bundling and for cleavage furrow formation. Cyk4 is probably tethered to the plus ends of microtubules at the spindle midzone through its attachment to Zen4. Another link between cytokinesis and the spindle was demonstrated by studies of mammalian INCENP, the inner centromere protein that is left behind at the midbody during anaphase to define the site of the future cleavage furrow (W. Earnshaw, Edinburgh, UK). Interestingly, when the INCENP domain responsible for targeting it to the inner centromere is swapped with the centromere-targeting domain of CENP-B, the cells carrying this hybrid do not complete cytokinesis. Xenopus egg extracts were used by I. Vernos (Heidelberg, Germany) to investigate the role of kinesin-related proteins in spindle formation. The kinesin-related protein Xklp2 and its partner, Tpx2, interact with microtubules and localize to the centro-
some throughout mitosis. Depletion of Xklp2 disrupts spindle formation, and its targeting to microtubules is dependent upon Tpx2 (Ref. 3). Another kinesin required for chromosome segregation is Xkid1 (Ref. 4) (A. Murray, San Francisco, USA), a protein involved in the spindle assembly checkpoint that was identified in a screen for proteins that adhere to the chromosomes during M phase but not during interphase. The MAP kinase dogma changes A molecular pathway that has been ingrained in the minds of most cellcycle researchers is the c-Mos-MEKMAP kinase cascade leading to the activation of MPF and entry into meiotic M-phase in frog oocytes. Scores of experiments over the past 10 years have shown that the members of this pathway are both necessary and sufficient for activation of MPF. However, studies from other organisms have led to the uncomfortable realization that c-Mos and MAP kinase are not needed for meiosis re-entry in any other animal, and perhaps this discomfort has inspired a re-examination of the role of the Mos–MAP kinase pathway in Xenopus. Data presented from many labs both in the recent literature and in Roscoff support the notion that MAP kinase is in fact not centre stage in the pathway leading to MPF activation in Xenopus (Fig. 2). For instance, a poster from the Haccard lab (Paris, France) showed that injecting oocytes with Xenopus Ras results in MPF activation even in the absence of MAP kinase activity, and M. Dorée (Montpellier, France) has shown that MAP kinase is phosphorylated very soon after progesterone stimulation, well in advance of any c-Mos translation. Although this doctrine shift has left some feeling more comfortable with the notion that Xenopus oocytes do after all match the rest of the animal kingdom, it has left many wondering what is the trigger kinase for MPF activation and what MAP kinase is doing during meiosis. A recent study from the Nebreda lab (Heidelberg, Germany) addressed the latter issue, showing that MAP kinase interacts with and phosphorylates pp90rsk, which, in turn, phosphorylates and inactivates Myt1 kinase, the sole component that keeps Cdc2 inactive in G2-arrested Xenopus oocytes5. The Maller lab (Denver, USA) has identified the polo-like kinase homologue (Plx1) as the trigger kinase that phosphorylates and activates the MPF
trends in CELL BIOLOGY (Vol. 10) October 2000
meeting report Hormone receptor ?
mos mRNA c-Mos protein (unstable)
APC/C PKA
? ?
XAIRK-1
c-Mos—hsp90 (stable) CPEB
?
?
?
MEK
XAIRK-1 (Eg2) ?
Plkk1
MAPK
?
Xenopus & starfish
Ras Xplk-1
pp90rsk ? cdc25
Myt-1 Interphase/ DNA replication MPF wee1
Mitosis Meiosis
trends in Cell Biology
FIGURE 2 Signalling pathway controlling meiosis I in animal oocytes. It is not known whether all of the components depicted are functional in all animals. However, recent data point to a more conserved pathway than originally thought. Data presented at the Roscoff meeting are shown in colours corresponding to the lab from which they came: Maller (purple), Nebreda (green), Dorée (brown), Jessus (orange), Kishimoto (pink), Sagata (cyan), Haccard (grey) and Ruderman (blue; published data8,12 but not presented). Abbreviations: CPEB, cytoplasmic polyadenylation element binding factor; hsp90, heat shock protein 90; PKA, protein kinase A.
activator cdc25. Injection of constitutively active Plx1 into oocytes causes them to enter the cell cycle in the absence of progesterone, confirming its central role in MPF activation. Plx1 can also phosphorylate and activate its activator, Plx1-kinase (Xplkk1), although it is clear that there are other factors required for Plx1 activation too6. What does Eg2 do? Oocytes have traditionally been used for studying the G2–M transition because many oocytes are naturally arrested in G2 of meiosis I until an external stimulus catapults their entry into M phase. Although there is a significant amount of data on the regulation MPF in these cells, much less is know about the steps that lead to MPF activation (see Fig. 2 for summary). Eg2 kinase has emerged as an important molecule in the pathway leading to MPF activation in Xenopus oocytes and was the focus of no fewer than three talks. Eg2 is a member of the Aurora/Ipl family of kinases that serve diverse yet crucial functions in several stages of M phase. C. Prigent
(Rennes, France) first isolated Eg2 (Ref. 7) and has since been investigating this family, all of which have confusing names. He began his talk with a sensible appeal to adopt a common nomenclature for this fast-growing family. The proposed AIRK family members will each have a prefix to denote their species, followed by a numerical suffix that will be assigned to the functional homologues in different species. He showed experiments in Xenopus egg extracts in which XAIRK-1 (formerly known as Eg2) had been depleted or inactivated. This resulted in both the failure to form bipolar spindles as well as causing preformed spindles to collapse into monopolar spindles that retain their attachments to chromosomes. This effect is thought to be due to XAIRK-1 association with the kinesin-like molecule Eg5. Is XAIRK-1 involved in MPF activation? A previous study showed that overexpression of XAIRK-1 accelerates progesterone-induced c-Mos translation, MAP kinase activation and cellcycle re-entry8. This was confirmed by the Dorée lab, but with the surprising
trends in CELL BIOLOGY (Vol. 10) October 2000
caveat that MAP kinase activation occurs even in the absence of Mos or detectable MEK activity. This one result proposes a hitherto unknown mechanism for activation of MAP kinase in Xenopus and deserves greater attention. A study from the Jessus lab (Paris, France) showed that that XAIRK-1 is a target of MPF, and phosphorylation by MPF is a requisite for XAIRK-1 to be degraded by the anaphase-promoting complex/cyclosome (APC/C), the instrument responsible for the destruction of many cell-cycle regulatory molecules controlling key cellcycle transitions. M, no S For the M-phase aficionados in attendance, the meiotic cell cycle is a haven, providing a double-dose of M without any intervening S or G phases to confuse matters. There are two hypotheses to explain the absence of S-phase during meiosis. One is that meiotic cells lack a factor that is required for entry into interphase following meiosis I, whereas the other proposes the existence of a factor that actively suppresses S during
449
meeting report interkinesis (the period between meiosis I and II). N. Sagata (Fukuoka, Japan) presented evidence supporting the first hypothesis by showing that introduction of the interphasepromoting molecule and MPF inactivator, wee1, during meiosis I causes the oocytes to exit M phase and to replicate their DNA during interkinesis. In support of the suppressor model, several years ago Sagata showed that c-Mos was required for suppression of S-phase during Xenopus meiosis9, but, until this conference, it was presumed that it was not a universal mechanism because c-Mos had only been identified in vertebrates. In one of the most exciting reports of the meeting, Kishimoto (Yokohama, Japan) described the function of starfish c-Mos. Overexpression of c-Mos in starfish oocytes results in MAP kinase activation, but not cell-cycle activation. Prevention of Mos translation during meiosis I, however, converts the meiotic divisions to a mitosis-like pattern in which the cells undergo S phase after meiosis I, then re-activate MPF to undergo a periodic S- and Mphase cycle, just like the mitotic divisions of embryonic cleavage (albeit without cytokinesis; Fig. 2). Separating sisters A commonly held view is that the chromosomal glue is distributed along the chromosomal arms and in the kinetochore region and this global cohesion is maintained until it is degraded in anaphase A. An elegant experiment in budding yeast challenged this idea. A technique to monitor sister-chromatid separation in yeast involves placing a GFP tag on one chromosome so that, when the chromatids are together, there is one dot and when the sisters separate, two dots are observed10. G. Goshima (Yanagida lab, Kyoto, Japan) described an experiment in which the GFP tag was placed either 1.8 Kb or 23 Kb from the centromere. Surprisingly, when the tag was near the centromere, two dots were seen in Sphase, meaning that the kinetochore region had separated as soon as the spindle is formed. By contrast, the more distal tag (which is the site most commonly used) remains a single dot until anaphase. This suggests that, following DNA replication, sisters are held together along their arms, but not in their centromere region. C. Lehner (Bayreuth, Germany) described the Drosophila mutant pimples as a dramatic example of what happens if sisters do not separate11.
450
In most systems, a defect in sister separation results in M-phase arrest, with stable cyclin B. By contrast, sister chromatids in pimples never separate, even though the cells go through multiple rounds of S and M phases, resulting in multivalent chromosomes. Pimples contains a destruction box (D-box), the sequence that targets proteins for degradation by the APC/C. Lehner showed that Pimples is degraded during late metaphase, just like cyclin B, and mutating the D-box to create stable Pimples causes cells to arrest in what appears to be an M-phase, although cyclin B is degraded. This result contradicts the accepted view that stabilization of cyclin B is a defining characteristic of M-phase-arrested cells and suggests a possible need for rethinking the criteria by which cell-cycle stages are defined. For Roscoff The setting of this meeting at the Station Biologique in Roscoff befitted the field’s inextricable link to the use of marine invertebrates to uncover some of the most interesting and crucial components of the cell-cycle machinery. Species native to Roscoff are still being used to probe some important cell-cycle issues, such as how the polarized cell division observed in the zygotes of the common sea weed, Fucus, is achieved (F. Bouget, Roscoff, France) and how can we interrupt the unwanted cell divisions in cancer. Answers to the latter question are being sought by L. Meijer (Roscoff, France) who has established a network of chemists, biologists and cell-cycle specialists to design or extract compounds capable of inhibiting starfish MPF in vitro, with the hopes that one will also inhibit cancer cell division in vivo. Finally, in the straightforward and stately style befitting one of the field’s founders, Masui (Toronto, Canada) described his ongoing observations of the cell-division cycle in Xenopus embryos. During the mid-blastula transition, these cells switch from their synchronous cycles to seemingly irregular ones. His closer look at the asynchronously dividing cells revealed a rhythm to their cell-cycle times, with the duration of each division lasting for a multiple of 30 minutes. These sorts of simple, yet revealing, experiments in embryonic systems formed the heart of the field some 30 years ago and today provide inspiration to take a walk on a beach and bring some of the findings back to the lab for a look.
References 1 Piel, M. et al. (2000) The respective contributions of the mother and daughter centrioles to centrosome activity and behaviour in vertebrate cells. J. Cell Biol. 149, 317–330 2 Weiss, E. and Winey, M. (1996) The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint. J. Cell Biol. 132, 111–123 3 Wittman, T. et al. (1998) Localization of the kinesin-like protein Xklp2 to spindle poles requires a leucine zipper, a microtubuleassociated protein, and dynein. J. Cell Biol. 143, 673–685 4 Tokay, N. et al. (1996) Kid, a novel kinesinlike DNA binding protein, is localized to chromosomes and the mitotic spindle. EMBO J. 15, 457–467 5 Gavin, A.C. et al. (1999) A p90(rsk) mutant constitutively interacting with MAP kinase uncouples MAP kinase from p34(cdc2)/cyclin B activation in Xenopus oocytes. Mol. Biol. Cell 10, 2971–2986 6 Qian, Y.W. et al. (1998) Purification and cloning of a protein kinase that phosphorylates and activates polo-like kinase Plx1. Science 282, 1701–1704 7 Roghi C. et al. (1998) The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly. J. Cell Sci. 111, 557–572 8 Andresson, T. and Ruderman, J.V. (1998) The kinase Eg2 is a component of the Xenopus oocyte progesterone-activated signalling pathway. EMBO J. 17, 5627–5637 9 Furuno, N. et al. (1994) Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes. EMBO J. 13, 2399–2410 10 Straight, A.F. et al. (1997) Mitosis in living budding yeast: anaphase A but no metaphase plate. Science 277, 574–578 11 Stratmann, R. and Lehner, C.F. (1996) Separation of sister chromatids in mitosis requires the Drosophila pimples product, a protein degraded after the metaphase/anaphase transition. Cell 84, 25–35 12 Mendez, R. et al. (2000) Phosphorylation of CPE binding factor by Eg2 regulates translation of c-mos mRNA. Nature 404, 302–307
Students Did you know that you are entitled to a 50% discount on a subscription to TCB? See the bound-in subscription order card for details
trends in CELL BIOLOGY (Vol. 10) October 2000