meeting report

Journey to the centre of the cellAndrew W. Murray

Over the last 20 years, studies of the biochemical oscillator that drives cell reproduction have revolutionized our understanding of the cell cycle. A recent Jaques Monod Conference, at the Station Biologique in Roscoff (30 April – 3 May 2000), concentrated on dissecting the elaborate structural rearrangements that the oscillator induces in order to push cells from interphase to mitosis and then to divide them in two.

ell biologists first observed the centro-some, the mysterious organelle thatdefines the geographic centre of the

cell, more than 150 years ago. The centro-some’s core is a pair of centrioles — short,highly organized cylinders of specializedmicrotubules, which direct the assembly ofa sphere of pericentriolar material. Thispoorly defined collection of proteinsanchors the �-tubulin-containing com-plexes that nucleate most of the cell’smicrotubules both in interphase and inmitosis. In plants and fungi, the centriolesand centrosome are replaced by spindle-pole bodies, but all of these microtubule-organizing centres have similar functionsand contain homologous proteins, showingthat they evolved from a single ancestor.

Each newborn cell inherits a single cen-trosome, containing a mother and a daugh-ter centriole. Before the cell reaches mitosis,the cycle of centriole replication must becompleted — mother and daughter centri-oles must separate from each other, andeach must direct the formation of a newcentriole. The daughter centriole pair mustthen acquire the ability to act as a centro-some and the two centrosomes mustmigrate to opposite sides of the nucleus,where they will act as the poles of themitotic spindle (Fig. 1).

This complex cycle was studied by label-ling centrin, a centriolar component, withgreen fluorescent protein (GFP; M. Born-ens, Inst. Curie, Paris). Because the mothercentriole can be distinguished from itsdaughter, their individual behaviour can bestudied in living cells. As cells complete ana-phase, the mother and daughter centriolessplit apart. The mother centriole is rela-tively static, whereas its daughter movesrapidly and irregularly around her. Depo-lymerization of microtubules causes thedaughter to swoop in graceful circles,whereas removal of actin filaments makes itjerk towards and away from the mother,indicating that the daughter can travelalong both filament systems, while remain-ing tethered to its mother by a flexible leash.During its travels, the daughter centriolecan encounter the midbody, the microtu-bule-rich remnant of the cytokinetic furrowthat connects a recently divided pair of cells.This meeting induces the severing of theconnection between the cell and the mid-body, freeing the two daughter cells tomigrate away from each other. This remark-

able finding implies that regulation of someevents in the cell cycle requires spatial prox-imity of interacting components, ratherthan long-range signal-transduction sys-tems that transmit global signals betweenspatially restricted events.

The differing motilities of mother anddaughter centrioles probably reflects theirdiffering interactions with actin filamentsand microtubules. The mother nucleatesmicrotubules and then retains their minusends, yielding the classical centrosome thatlies at the centre of an array of uniformlypolarized microtubules. During interphase,the daughter centriole pair can nucleate butnot bind microtubules, thus acting as asource of microtubules that are releasedinto the cytoplasm. Late in the cell cycle thedaughter centriole must acquire the abilityto retain microtubules so that it can form aspindle pole in mitosis and act as the cen-trosome in the daughter cell that inherits it.This maturation is associated with theappearance of spokes that radiate from theend of the centriole and contain the proteinninein, a candidate for binding to and stabi-lizing the minus ends of microtubules.

A new protein, C-NAP1, is likely to bepart of the intercentriolar leash (E. Nigg,Max Planck Institute, Martinsried). Inhibi-tion of this protein in tissue-culture cellscauses premature separation of the two cen-trioles during interphase, as does overex-pression of the protein kinase thatphosphorylates it, Nek-2A (a homologue ofthe fungal mitosis-inducing protein NimA).

Although centrioles organize the cen-trosome, female meiotic cells divide with-out centrioles. During the last year, avariety of studies have shown that spindlescan also form in mitotic cells that have beensurgically deprived of their centrosomes.These observations extend an emergingtheme in cell biology — there are oftenoverlapping mechanisms for convertinginitially isotropic systems into sophisti-cated structures. The centrosome-inde-pendence of mitosis turned out to belimited when video microscopy was used tofollow cells after microsurgical removal oftheir centrosomes (G. Sluder, Univ. Massa-chusetts, Worcester). The operated cellsentered mitosis and many appeared toform spindles and to segregate their chro-mosomes, but almost all of them failed toexecute complete cytokinesis. Surprisingly,in the next cell cycle, the surgically treated

cells failed to replicate their DNA or entermitosis. This defect may reflect the actionof a p53-dependent checkpoint that keepscells that have undergone aberrant mitosesfrom completing a subsequent cell cycle.Halting such cells helps to prevent cancer,as they already have abnormal numbers ofchromosomes and centrosomes, whichincreases the possibility of producing evenmore genetically abnormal progeny duringfurther divisions.

Genetic studies using Drosophila haveidentified several proteins that are needed

C

Figure 1 The centriole cycle in an animal cell. Each centriole is a cylinder composed of nine triplet microtubules that run parallel to its long axis. The centrioles replicate conservatively, with a new centriole forming near to one end of the mother centriole. The size of the centrioles is much exaggerated relative to that of the remainder of the spindle.

Mitotic spindle

Spindleassembly

Completecentriole replication

Initiation of centriole replication

Centrioleseparation

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meeting report

for the centrosome to assume its mitoticfunction. One of these is Aurora, a memberof a protein kinase family that plays a role inmitosis in many eukaryotes. In animal cells,one of the Aurora homologues (AIRK-1) isimportant for spindle assembly (C. Prigent,Univ. Rennes). Inhibition of this proteincauses the spindle to lose microtubules,even though the chromosomes stayattached to the remaining microtubules,indicating that the kinase may be importantfor crosslinking the microtubules of themitotic spindle. Another centrosome-asso-ciated protein, abnormal spindle (Asp), hasa more direct function in microtubulenucleation (D. Glover, Univ. Cambridge).Treating centrosomes with high salt con-centrations separates them into an insolu-ble matrix and a soluble fraction. Depletingthe latter of Asp destroys its ability torestore nucleating capacity to the salt-stripped centrosomes. Asp seems to be asubstrate for Polo, a protein kinase that hasseveral functions in mitosis, includinginducing the mitotic behaviour of centro-somes, stimulating the mitosis-inducingactivity of Cdk1, and switching on the ana-phase-promoting complex (APC), therebyinducing anaphase and exit from mitosis bycausing the destruction of mitotic cyclinsand the securins that regulate sister-chro-matid separation.

The Drosophila protein Pimples is thefunctional equivalent of the securins thathave been found in yeasts and vertebrates(C. Lehner, Univ. Bayreuth). Like thesecurin Cut2 from fission yeast, Pimplesseems to have two functions in chromo-some separation — it plays an unknownbut essential role in assembling themachinery that will induce sister separa-tion, and keeps that machinery inactive,allowing the spindle checkpoint to controlthe timing of sister separation by restrain-ing Pimples destruction. C. Lehner and S.Pines (Wellcome/CRC Institute, Cam-bridge, UK) also solved a long-standingmystery concerning the relationshipbetween cyclins A and B, which aredegraded at different times in mitosis, yetare both destabilized by the APC.Although both cyclin A and B contain adestruction box, a short sequence that isrequired for APC-dependent proteolysis atthe transition from metaphase to ana-phase, cyclin A also contains a larger andless well characterized motif that directsAPC-dependent destruction well beforethis important transition.

Plant and animal development dependson controlling three aspects of cell division— the relative sizes of the two daughters,the orientation of the division plane, andthe asymmetric segregation of moleculesthat influence future developmental deci-sions. In animals, all of these decisionsdepend on mitotic centrosomes, which usethe microtubules that point away from the

chromosomes (astral microtubules) toposition the spindle, which in turn directsthe assembly of the cleavage furrow that willbisect the dividing cell. In the first divisionof Caenorhabditis elegans, the spindle movesto the rear of the egg to produce an asym-metric cleavage. This process was studiedusing a combination of mutant analysis andRNA interference (RNAi) to investigate thefunction of all 2,299 genes on chromosomeIII during the first cell cycle (A. Hyman,EMBL, Heidelberg). A total of 128 geneshave discernable functions in this division,including zyg8, which encodes a microtu-bule-binding protein that is needed to limitthe posterior migration of the spindle. Cou-pling this type of systematic approach withmore conventional genetics provides acomplete listing of all the proteins thatcause even mild phenotypes in develop-mentally important cell cycles. The discov-ery that interfering with some genes inducesvisible abnormalities in the first cell divi-sion, but does not affect the final outcomeof development, indictates that mecha-nisms exist to buffer development fromsubstantial perturbations in the machinerythat controls cell division.

One well-studied series of cell divisionscomprises those that give rise to the senseorgans in larval and adult flies. By markingthe dividing cells with GFP it was possible tocorrect earlier errors in the description ofthese lineages (F. Schweisguth, Ecole Nor-male, Paris), and then to go on to determinehow the planes of the four divisions arespecified. Surprisingly, it seems that eachdivision is controlled by a different set ofgenes, which in each case set up sites on thecell cortex that capture microtubules fromone of the spindle poles, rotating the spindleinto the correct position.

Proper spindle positioning is particu-larly important in female meiosis, in whichtwo very asymmetric divisions producetwo tiny polar bodies and a big egg, thusconserving the vast majority of themother’s stockpiled proteins for her prog-eny. In mice this asymmetry depends onthe protein kinase Mos, which somehowdirects the meiotic spindle, which forms inthe centre of the egg, to migrate along actinfilaments to the cortex. Once the spindlereaches the cortex it induces a Mos-inde-pendent disappearance of microvilli in thisregion of the cell, and the cleavage furrowis restricted to this small area of the oocytessurface (M. Verlhac, Univ. Pierre et MarieCurie, Paris). Mos also has a conserved andimportant function in differentiating themeiotic cell cycle from the mitotic one bykeeping cells from entering interphase andreplicating their DNA between the twomeiotic divisions (T. Kishimoto, TokyoInst. Technology and N. Sagata, KyushuUniv.).

Having reached its appointed position,how does the spindle induce cleavage? The

INCENP protein is localized to chromo-somes at the beginning of mitosis, but asmitosis proceeds it relocates to the gapbetween the sister chromatids during met-aphase and then travels to the cell cortex atanaphase, thereby defining the regionwhere the cleavage furrow will form (W.Earnshaw, Univ. Edinburgh). Expression ofINCENP mutants perturbs cleavage, pre-venting the very final stages of cytokinesis,or causing the cleavage furrow to regresswhen it is roughly half-completed, depend-ing on the mutant. The identification of aDrosophila INCENP will bring sophisti-cated genetics to bear on the function of thisimportant protein.

One class of proteins that INCENPmay recruit is the septins, a group of fila-ment-forming, GTP-binding proteinsthat were first discovered by analysingcytokinesis-defective budding yeast (J.Pringle, Univ. N. Carolina). These pro-teins are found in animals and fungi andseem to have a function in membranedeposition at the leading edge of cleavagefurrows and at other sites of membranegrowth, such as developing spore walls. Infission yeast, however, mutation of allseptins fails to block cytokinesis, and theentire class of proteins seems to be absentfrom plants. These observations indicatethere may be two mechanisms of mem-brane deposition during cell division, onethat depends on the septins, and anotherthat is independent of them.

The meeting was dedicated to ProfessorYoshio Masui, whose pioneering workwith frog eggs identified key activities thatdefined the cell-cycle oscillator and pro-duced the cell-cycle extracts that haveplayed a crucial role in dissecting the cell-cycle of animal cells. He described studieson the mid-blastula transition (MBT), theloss of synchrony between neighbouringcells after 12 divisions of a fertilized Xeno-pus egg. Careful observation of the post-MBT cycles revealed that their lengthswere not continuously variable but weremultiples of 30 minutes, extending therange of organisms for which quantizedcell-cycle lengths have been reported.Mathematical models in fission yeast sug-gest that a similar quantization of cell-cycle lengths reflects regular oscillations inthe level of Cdk1/cyclinB activity, theamplitude of which varies stochasticallyand is often too small to trigger mitosis (A.Sveiczer, Budapest Univ. Technology andEconomics). Testing such models willdepend on the approach that is currentlyinvigorating cell biology — devising meth-ods to measure the activity and abundanceof key proteins in individual living cells.Andrew W. Murray is in the Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138, USA.email: amurray@mcb.harvard.edu

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