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PLC-γ in DT40 B lymphocytes completelyabrogated agonist-induced entry, evenwhen the cells were activated by an agoniststimulating a receptor that is coupled to adifferent PLC isozyme, PLC-β. Both thewild-type and lipase-inactive PLC-γ wereable to rescue agonist-induced entry in thePLC-γ-knockout cells. However, unlike theresults with overexpressed PLC-γ, neitherknock-down nor complete knock-out ofPLC-γ reduced calcium entry when activat-ed by store depletion with thapsigargin.

The findings of Patterson et al.6 clearlydemonstrate that PLC-γ is essential for ago-nist-induced calcium entry. However, afundamental conundrum seems to arisefrom the apparent lack of involvement ofPLC-γ in thapsigargin-activated entry (atleast with the knock-down and knock-outstrategies). Thapsigargin causes depletion ofcalcium stores and subsequent CCE, but thisprocess seems to be independent of PLC-γ.Activation of the physiological receptor alsocauses depletion of calcium stores and PLC-γ is not required for this depletion; yet PLC-γ is required for calcium entry. To addressthis issue, Patterson et al. considered thatdepletion of calcium stores with thapsigar-gin is probably more extensive and com-plete than with surface receptor activation.With sub-maximal depletion, they reason,additional signals may be required, andthese signals may be dependent on PLC-γ.However, previous work has establishedthat CCE is activated in a graded manner inresponse to degrees of calcium store deple-tion, even when the signal depleted is InsP3

(refs 8,9). Perhaps the key to understandingthe function of PLC-γ lies in two importantobservations. First, the catalytic activity ofPLC-γ is not required, indicating that itfunctions through its ability to associatewith other signalling molecules, rather thanthrough the generation of a signal.Consistent with this idea, SH2 domains(which help to target PLC-γ to its substrate)are not important, whereas an SH3 domain(which is involved in the interaction ofPLC-γ with the actin cytoskeleton10) isessential. Second, although PLC-γ is impor-tant for activation of what are thought to beclassical, store-operated channels, it is alsoimportant for activation of TRPC3 chan-nels, which depend on PLC activation, butdo not seem to depend on store depletion11.As Patterson et al. pointed out, thapsigarginprobably induces global depletion of stores,whereas signals from receptor activation arethought to be highly localized within thecell. It is well established that CCE channelsare regulated by a very small subset of thetotal cellular calcium stores12–14. Thus, onepossibility is that PLC-γ maintains localiza-tion of the receptor–PLC complex inappropriate juxtaposition to the critical cal-cium pools that regulate the store-operatedchannels. In this scenario, PLC-γ functionsupstream of calcium store depletion, thesine qua non of CCE7, rather than in paral-lel, as suggested by Patterson et al. Similarly,in the case of TRPC3, PLC-γ may localizethe signalling complex, which generatesmessengers for TRPC3 channels in appro-priate proximity to the channels.

Regardless of the precise explanation forits action, it is now clear that in addition toits well-established function in growth-factor-induced calcium release15, PLC-γhas a fundamental and previously unap-preciated role in signalling calcium entryacross the plasma membrane of cells. Thisfinding may provide a new lead in ourunderstanding of the regulation andmicro-organization of cellular signallingcomplexes, particularly for phospholipaseC-mediated pathways.James W. Putney Jr. is in the Calcium Regulation

Section, Laboratory of Signal Transduction,

National Institute of Environmental Health

Sciences, National Institutes of Health, Research

Triangle Park, NC 27709, USA

e-mail:Putney@niehs.nih.gov

1. Michell, R. H. Biochim. Biophys. Acta 415, 81–147 (1975).

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4. Acharya, J. K., Jalink, K., Hardy, R. W., Hartenstein, V. & Zuker,

C. S. Neuron 18, 881–887 (1997).

5. Broad, L. M. et al. J. Biol. Chem. 276, 15945–15952 (2001).

6. Patterson, R. L. et al. Cell 111, 529–541 (2002).

7. Putney, J. W. Jr. Capacitative Calcium Entry (Landes Biomedical

Publishing, Austin, TX 1997).

8. Parekh, A. B., Fleig, A. & Penner, R. Cell 89, 973–980 (1997).

9. Huang, Y. & Putney, J. W. Jr. J. Biol. Chem. 273, 19554–19559

(1998).

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(2000).

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Chem. 276, 33980–33985 (2001).

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21522–21528 (1996).

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(1995).

14. Gregory, R. B. et al. Biochem. J. 341, 401–408 (1999).

15. Rhee, S. G. & Bae, Y. S. J. Biol. Chem. 272, 15045–15048 (1997).

16. Clementi, E. & Meldolesi, J. Cell Calcium 19, 269–279 (1996).

Polar body formation: new rules forasymmetric divisions

Bernard Maro and Marie-Hélène Verlhac

Asymmetric cell divisions are pivotal throughout development and generate cell diversification within theembryo. The formation of polar bodies during oocyte meiotic maturation provides the most extreme caseof size difference between two daughter cells. New work in this issue indicates that formin-2, a microfila-ment-binding protein, is required for the eccentric positioning of the meiotic spindle that determines theseunequal divisions.

Asymmetric cell division can occurthrough two major cellular mecha-nisms, both of which generate daugh-

ter cells that can differ by either their sizeand/or by their content. In the first mecha-nism, the spindle is located eccentrically(not at the geometric centre), hence the twodaughter cells differ in size (Fig. 1a). In the

second mechanism, an axis of polarity isinitially established in the cell, along whichthe division spindle is positioned. Afterdivision, the two daughter cells will havedifferent contents (Fig. 1b). In both mecha-nisms, the spindle is crucial for the correctpositioning of the cleavage furrow.

Our view of the molecular mechanisms

that direct asymmetric cell division in ani-mals is derived mainly from work per-formed in genetic systems, such as thenematode Caenorhabditis elegans and thefruit fly Drosophila melanogaster. However,in both systems, the asymmetric cell divi-sions studied belong to the second catego-ry described above, where the spindle is

© 2002 Nature Publishing Group

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oriented according to an axis of polarityestablished before cell division1,2.

Asymmetric cell divisions also occurduring female meiosis to ensure that mostof the maternal stores are retained withinthe oocyte. This results in the formation ofdaughter cells of different sizes: the largeoocyte and the small polar bodies (seeFig. 2 for a summary of meiosis). In addi-tion, there are two successive M phaseswithout an intermediate S phase duringmeiosis to produce haploid gametes. Thefirst meiotic division is a unique reduction-al event that ensures homologous chromo-somes are segregated while the cohesionbetween sister chromatids is maintained.The second meiotic division is equational,with sister chromatids segregated as in amitotic division. During the first meioticdivision, asymmetric cell division occursonce the spindle, which has formed in thecentre of the oocyte, has reached the cell cor-tex. This triggers the metaphase–anaphasetransition and the first polar body is extrud-ed (see Fig. 2). It is only after fertilizationthat meiosis II will resume, resulting in thesecond asymmetric cell division and extru-sion of the second polar body. Two majorevents are needed to ensure these asymmet-rical cell divisions occur during meiosis:first, the meiotic spindle must migratetowards the cell cortex and second, a corticaldomain over the spindle (in which the cleav-age furrow is restricted) must be formed. Inaddition, during the second meiotic divi-sion, the spindle must rotate for cleavage totake place and for the second polar body tobe extruded (see Fig. 2).

Spindle migrations and rotations, suchas the one described above, are usuallyrelated to interactions between anchors onthe cell cortex and microtubules from thespindle asters2–4. However, spindles ofmouse oocytes are barrel-shaped, lack cen-trioles and are devoid of microtubuleasters5,6. Furthermore, oocytes incubated inmicrotubule depolymerising drugs do notform meiotic spindles, but chromosomesmigrate to the cortex7,8. The lack of astralmicrotubules connecting the spindle to thecortex and the migration of chromosomesto the cortex in the absence of microtubulessuggest that another mechanism is involvedin spindle migration. As oocytes incubatedin cytochalasin D, an inhibitor of microfila-ment formation, form meiotic spindles thatfail to migrate7,8, it seems that an interac-tion between the chromosomes and themicrofilament network could be involvedin this spindle migration. New work byLeader et al. in this issue seems to confirmthis view9. Leader et al. show that a micro-filament-binding protein, formin-2, is cen-tral to this process, as the spindle does notmigrate and the polar bodies are notextruded in oocytes from mice in which theformin-2 gene has been invalidated.

Formins are members of a conserved

family of actin nucleators that may directthe formation of straight actin microfila-ments10–12, in contrast to the Arp2/3 com-plex that nucleates branched filaments.Formins are known to be involved in theregulation of many cellular processes, suchas cell polarity, cytokinesis and cytoskeletalfunction10–12. The nucleation of a networkof straight microfilaments by formin-2 atthe cell cortex directed towards the centreof the oocyte (Fig. 3a) could interact withactin-binding proteins associated with thechromosomes (these actin-binding pro-teins remain to be identified). The forcesexerted on the chromosomes would begreater in the direction closest to the cellcortex, probably because interactionsbetween microfilaments and the chromo-somes will be more frequent. The directionof movement would be along the long axisof the spindle, where the resistance causedby the viscosity of the cytoplasm is weakest.This will increase the eccentricity of thespindle and, thus, favour the movement inthe same direction (Fig. 3b).

During meiosis, the chromosomeslocated under the cortex also seem to con-trol the formation of the overlying corticaldomains. During spindle migration inmeiosis I, an area devoid of microvilli andenriched in actin microfilaments forms inthe cortex overlying the spindle. In addi-tion, if meiotic chromosomes are artificial-ly dispersed, each group of chromosomeswill induce the formation of a domain rich

b

a

Figure 1 Mechanisms of asymmetric celldivision. a, An eccentrically located spin-dle is shown in green. After cell division,the two daughter cells differ in size. b,First, an axis of polarity is set-up to sepa-rate cellular contents (blue). Then the divi-sion spindle (green) is positioned alongthis axis. After cell division, the twodaughter cells have different contents.

a b c d e f

g h i j k l

Figure 2 Polar body formation in mouse oocytes. a, Oocytes are arrested in meioticprophase within the ovary, with a large nucleus called the germinal vesicle (GV; red). b,c, When meiosis I resumes, germinal vesicle breakdown (GVBD) occurs and the spindleforms in a central location (green). d, e, The spindle migrates along its long axis towardsthe nearest part of the cortex. Simultaneously, an area devoid of microvilli and enrichedin actin microfilaments begins to form in the cortex overlying the spindle (blue)7,17. f–h,When the spindle reaches the cortex, the metaphase–anaphase transition is triggeredand the first polar body forms in this actin-rich cortical domain. i, In metaphase II-arrested oocytes, the spindle is located parallel to the surface, under a cortical domainenriched in microfilaments and devoid of microvilli (blue)3. The cleavage furrow isrestricted to this region. j, After fertilization, two cortical bumps form in this corticaldomain over the two sets of anaphase chromosomes. k, One of these bumps enlargeswhile the other retract, resulting in a rotation of the spindle. l, When the spindle hascompletely rotated, cleavage takes place and the second polar body is extruded.

© 2002 Nature Publishing Group

in microfilaments and poor in microvil-li10,13. As the chromosomes are almost in thecentre of the cell at pro-metaphase I andthen under the cortex at the end ofmetaphase I, the existence of cortical differ-entiation in the membrane before spindlemigration could provide a polarity cue forthis migration. However, the microvilli-free

cortical domain appears progressively dur-ing spindle migration and, importantly,after the start of this movement8. Moreover,the cortex does not seem to influence theorientation of the spindle axis8. When thespindle migrates along its long axis, thechoice of its direction of migration seems tobe a consequence of a slight off-centre posi-tioning of the germinal vesicle, as it alwaysmigrates on the shortest distance, ratherthan migrating as a consequence of a pre-defined cortical site8. Therefore, the direc-tion of spindle migration results from aslight asymmetry in the positioning of thegerminal vesicle, and hence of the spindle.This asymmetry is further reinforced by theeffect of the chromosomes on the cortex.

Is formin-2 also involved in the chromo-some-dependent formation of the corticaldomain in which the cleavage furrow isrestricted? Chromosomes must movetowards the cortex first before they caninduce the cortical domain. This suggeststhat there is a maximum distance at whichthis cortical reorganization can be induced(Fig. 3c, d). Thus, as formin-2 is requiredfor spindle migration, it also central in thedifferentiation of the cortical domain. Inaddition, the stabilization of the interactionbetween the cortex and the chromosomescould concentrate formin-2 in this area,where it could be involved locally in thereorganization of the actin network and inthe recruitment of microfilaments from themicrovilli. This would result in a loss ofmicrovilli and enrichment in subcorticalmicrofilaments.

Formins are known to interact with theRho family GTPases11. Until now, there hasbeen no evidence to suggest that these regu-lators are involved in the control of asym-metric cell divisions during meiosis,although their role during cytokinesis is wellestablished15. However, the Mos/mitogen-activated protein kinase (MAPK) pathwayhas been involved in polar body forma-tion8,16. Oocytes from mos−/− mice form large

polar bodies. In these oocytes, the spindledoes not migrate before the metaphaseI–anaphase I transition and elongates atanaphase8. The chromosomes are still ableto induce their migration, but at a shorterdistance, and this would happen only at theend of anaphase when one set of chromo-somes is closer to the cortex, as a result ofspindle elongation. The cortex is still able torespond to the signal coming from the chro-mosomes, but again at a shorter distance.This would suggest that the Mos/MAPKpathway functions as an ‘amplifier’, increas-ing the range of action of this signal. Thus,it is possible that this pathway modulatesthe activity of formin-2. The identificationof a major factor in the control of polarbody formation opens the way for the char-acterization of the molecular mechanismsinvolved in the control of the female meiot-ic divisions in mammals.

Bernard Maro and Marie-Hélène Verlhac are in the

Biologie du Développement, UMR 7622 - CNRS -

Université Pierre et Marie Curie, 9 quai St Bernard,

75005 Paris, France

e-mail: maro@ccr.jussieu.fr

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Exp. Morph. 92, 11–32 (1986).

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Embryol. Exp. Morph. 81, 211–237 (1984).

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a b

c d

Figure 3 Putative role of formin-2 duringpolar body formation. a, b, A network ofstraight microfilaments (violet arrows) arenucleated from the cell cortex by formin-2(blue dots) and directed towards the cen-tre of the oocyte. These would interactwith actin-binding proteins (orange dots)associated with the chromosomes (red). c,d, Chromosomes can induce the formationof a cortical domain on the cell cortex ata limited distance (blue arrows). The inter-action between the cortex and the chro-mosomes, which increases during spindlemigration, could concentrate formin-2 (bluedots) in this area where it would locally re-organize the actin network (light blue).

News and Views contributionsThe News and Views section provides a forum in which newadvances in the field of cell biology, as reported in publishedpapers, can be communicated to a wide audience.Most News and Views pieces are linked to Articles that appearin Nature Cell Biology, but some may focus on papers ofexceptional significance that are published elsewhere.Unsolicited contributions will not normally be considered,although prospective authors are welcome to make proposalsto the Editor before the paper is published.As a general guideline, News and Views pieces should be

about 1,300 words, with one or two display items (figures,boxes and tables). They should make clear the advance (the‘news’) and communicate a sense of excitement, yet providea critical evaluation of the work in context of the rest of thefield. We encourage personal ‘views’, criticisms and predic-tions, but authors should not refer to their own work, exceptin passing.Detailed guidelines are available on request fromcellbio@nature.com and on Nature Cell Biology’s Web site(http://cellbio.nature.com).

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