Cortical domains and the mechanisms of asymmetric cell division

I

Cortical domains and -4 the m mechanisms ot asymmetric cell division

m

Asymmetric cell divisions are central to the generation of cell-fate diversity because factors that are present in a mother cell and distributed unequally at cell division can generate distinct daughters. The process of asymmetric cell division can be described as consisting of three steps: setting up an asymmetric cue in the mother cell, localizing factors with respect to this cue, and positioning the plane of cell division so that localized factors are partitioned

asymmetrically

between daughters. This review

describes how specialized cortical domains play a key role in each of these steps and discusses our current understanding

of the

molecular nature of cortical domains and the mechanisms by which they may orchestrate asymmetric cell divisions.

may generate differences between daughters’. More recent experiments have demonstrated that such a mechanism can indeed generate cell-fate diversity. Thus, in the developing Drosophila melanogasterperiphera1nervous system, a protein called NUMB is asymmetrically distributed in a sensory organ precursor and partitioned into only one of its daughter9. The two daughters give rise to different lineages and thus have distinct fates, say A and B. In the absence of NUMB, both daughters adopt fate A (Ref. 9). Conversely, when NUMB is overexpressed, they both adopt fate B (Ref. 8). Hence, the presence of NUMB in a daughter cell is both necessary and sufficient for the adoption of fate B, and NUMB can be referred to as a cell-fate determinantlO. Thus, the asymmetric distribution of a factor in a mother cell can result in cell-fate diversity among daughters. What are the mechanisms enabling a mother cell to partition factors like NUMB to only one daughter? We can envisage that there must be a trigger of the entire process, followed by the execution of three fundamental steps in the mother cell (Fig. 2). First, an initial asymmetric cue is set up. Second, this asymmetric cue is utilized to distribute factors to specific cellular locations. Third, the plane of cell division is positioned so that localized factors are segregated differentially to daughter cells. Here, we discuss each step in turn, focusing on recent evidence from C. elegans and Saccharomycescerevisiaethat illustrates possible underlying mechanisms. These examples reveal that specialized domains of the cell cortex, a thin actin-rich layer beneath the plasma membrane, play a key role in the execution of each step of asymmetric cell division. While cellular and molecular details may vary between systems, the central role of cortical domains is likely to be conserved and to contribute to the generation of cell diversity in many organisms. Setting

The authors are in the Cell Biology Programme, European Molecular Biology Laboratory, 69117 Heidelberg, Germany. E-mail: gonczy@ EMBL-Heidelberg. DE

382

Asymmetric cell divisions contribute to the generation of diverse cell types during development and differentiation in virtually all organisms1-3. There are several types of asymmetric cell divisions. Sometimes, daughters of an asymmetric cell division have distinct morphologies as well as fates. For instance, the Caenorhabditis eleguns one-cell embryo divides into a large and a small cell, each of which gives rise to distinct lineages415(Fig. 1). In other cases,daughters of an asymmetric cell division are morphologically identical, but nevertheless have distinct fates. For example, the fission yeast Schizosaccharomycespombedivides into two seemingly identical daughters, which differ in their capacity to switch mating type6. Finally, cell divisions in which initially equivalent daughter cells become distinct after subsequent cell signalling have also been referred to as asymmetric3. However, we focus here on the mechanisms underlying strictly asymmetric cell divisions, in which an asymmetry established in the mother cell results in daughters that are distinct from the moment they are born. Classical embryological experiments led to the hypothesis that factors distributed asymmetrically in a mother cell and partitioned unequally at cell division 0 1996

Elsevier

PII: SO962.8924(96)10035-O

Science

Ltd.

up an initial

asymmetric

cue

There are at least two ways in which the entire process of asymmetric cell division can be triggered. Often, the trigger is of external origin. For instance, the sperm entry site normally determines the anteroposterior (A-P) axis in the one-cell C. eleguns embryo since the initial asymmetric cue in the egg is established in response to components of the spermll. In other cases, the trigger may be internal and lie in the history of the mother cell. For example, the site of the previous cell division determines the location of the initial asymmetric cue during vegetative growth of haploid S. cerevisiae12. What is the nature of the initial asymmetric cue set up in the mother cell in response to the trigger (see Fig. 2b)? While this is not known in most instances, possible cues include a molecular gradient, a localized cytoplasmic signal or a site in the cell cortex. Part of the difficulty in identifying the initial asymmetric cue in a mother cell stems from ensuring that a candidate component is indeed part of the initial cue rather than a relay between that cue and subsequent steps of asymmetric cell division. This difficulty is illustrated by the analysis of the par (for partition defective) genes in C. elegans. In par mutant embryos, several aspects of asymmetric cell divisions are affected, trends

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including polarization of factors and positioning of the cleavage plane 13. Thus, the par genes are candidates for encoding components of the initial asymmetric cue. However, the molecular characterization of some of thepar genes and the distribution of their protein products reveals that the situation is more complex. For instance, par-l encodes a serine/threonine protein kinase that is localized asymmetrically to the posterior cortex of the one-cell embryo14. Although this distribution may have suggested that PAR-l is part of the initial asymmetric cue, this is probably not the case. Indeed, a mutation in a residue essential for kinase activity does not affect the asymmetric distribution of PAR-1 proteinr4. Since the mutation is likely to abolish PAR-1 activity completely, this indicates that the initial asymmetric cue is still present in the absence of PAR-l function. Therefore, instead of being part of the initial asymmetric cue, PAR-1 is probably a relay between the initial cue and subsequent steps of asymmetric cell division. There are six par genes, and one or a combination of the encoded proteins may still correspond to the initial asymmetric cue. par-3 encodes a protein of unknown biochemical function that localizes asymmetrically to the anterior cortex shortly after fertilization15. However, PAR-3 alone is not the initial asymmetric cue because PAR-3 distribution is affected in par-2 mutant embryos 15.Conversely, PAR-2 alone is not the initial asymmetric cue because PAR-2 distribution is disrupted in par-3 mutants15. Nevertheless, PAR-Z and PAR-3 together may still form the initial asymmetric cue. Thus, the cortical domain containing PAR-3 protein may be part of the initial asymmetric cue set up by sperm components in the C. elegans one-cell embryo. In S. cerevisiae, a small cortical domain probably constitutes the initial asymmetric cue, serving as a memory of the previous cell-division site. Cell division in this organism is asymmetric, generating a bud and a somewhat larger mother cell, which are distinct not only in size but also in their ability to switch mating tyP e16J7. Wild-type haploid cells divide with an axial pattern, the bud always emerging next to the previous cell-division site12.By contrast, bud3 or bud4 mutants bud at abnormal cellular locations, making these loci candidates for encoding components of the initial asymmetric cuer8. The cycle of Bud3p and Bud4p proteins suggests an elegant manner in which the initial asymmetric cue may be set during axial buddinglgJO (Fig. 3). Bud3p and Bud4p assemble at mitosis in a double-ring encircling the bud neck. The Bud3pBud4p double-ring splits into two at cytokinesis, yielding a single ring in each cell at the division site. Each Bud3pBud4p ring persists for a short period in the next cell cycle and is thought to recruit to a neighbouring cortical site the bud-site-selection and polarityestablishment proteins, which, together, ensure the emergence of the incipient bud. The assembly of the Bud3pBud4p double-ring at mitosis is itself dependent on the presence of septins, a set of proteins localized to the bud neck, which are required for cytokinesisZ1,22. In this manner, Bud3p and Bud4p recognize the site of the previous cell division marked by the septins to determine the axis of asymmetric cell division. trends

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FIGURE

1

Asymmetric factors. embryos

cell division containing

simultaneous anterior

can give

Live Caenorhabditis

elegans

fluorescently

Nomarski

optics

is to the left, posterior

asymmetrically

to give

cell and the smaller distributed posterior

asymmetrically cortex

of the University

one-cell labelled

to the right.

antibodies

differ

against

P, cell. In the two-cell between

the daughter

Boulder,

in size and distribution

images

P granules,

embryo

distinct embryo, kindly

viewed

with

in (b) and (d);

divides

cells -the

larger

the P granules

cells, and are found were

of

(c) and (d) stage

microscopy

The one-cell

morphologically

of P,. Bar, 10 pm. These of Colorado,

that

(a) and (b) and two-cell

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bud3 and bud4 mutants bud at abnormal cellular locations but do not affect the subsequent steps of asymmetric cell division. By contrast, par mutant embryos are defective in both the polarization of factors and the positioning of the cleavage plane. This raises the possibility that loci functionally analogous to BUD3 and BUD4 may yet have to be identified in C. elegans. Alternatively, the absence of an initial asymmetric cue may result in distinct consequences for the subsequent steps of asymmetric division in different organisms. In summary, the Bud3p and Bud4p cortical site represents an initial asymmetric cue in S. cerevisiae,and the PAR-3 cortical domain may be part of such a cue in C. elegans.This raises the possibility that specialized cortical domains provide a general means of establishing an initial asymmetric cue in the mother cell. Localizing

factors

How does an initial asymmetric cue result in the polarized distribution of factors (see Fig. Zc)? Early experiments with pharmacological agents indicated that polarization of cellular components in various cell types depends on the microtubule and actin networksZ3J4. However, the actual mechanisms by which the cytoskeleton polarizes factors have only just begun to emerge. These mechanisms suggest that 383

(a) Mother cell

+

(b) Initial asymmetric cue (step 1)

(4

Polarization of factors (step 2)

(d) Cleavage plane positioning

(step 3)

(e) Daughter

cells 0 w

Factors F-actin

-

Microtubules Cortical

domains FIGURE

2

The execution of three steps in the mother cell can ensure that factors like NUMB are partitioned to a single daughter in asymmetric cell divisions. (a) Mother cell, with uniformly distributed factors. (b) First step. An initial asymmetric cue is set, for instance in response to an external trigger. (c) Second step. Factors become polarized with respect to the initial asymmetric cue. Initially, the actin and microtubule cytoskeletons become polarized; then, the cytoskeleton polarizes factors; finally, factors become anchored stably at the cell cortex. (d) ihird itip. The plane of cell division is positioned so that factors are partitioned to only one daughter. Centrosomes have duplicated and migrated away from each other; in this cell, a rotation of the centrosome-nuclear-complex positions the centrosomes on the vertical axis. The cleavage plane (dashed line) forms equidistant from the spindle poles. (e) Daughter cells, only one of which contains the factors. Cortical domains play an important role in all three steps of asymmetric cell divisions. The three steps may be mediated by a single cortical site, whose molecular composition may differ at each step; the shape of the cortical site is drawn differently at each step to illustrate this possibility. Alternatively, distinct cortical sites may be involved at each step (not shown).

the localization of factors can be broken down into three phases. Initially, the cytoskeleton is polarized. Then, the cytoskeleton initiates the polarization of factors. Finally, the localization of factors is maintained by anchorage mechanisms. How does an initial asymmetric cue result in the first phase - the polarization of the cytoskeleton? A cascade of GTPases seems central to this process in a variety of systems.In S.certisiae, the actin cytoskeleton 384

becomes polarized as cortical actin patches accumulate at the incipient bud site and actin cables orient towards itz5. One of the proteins recruited by Bud3p and Bud4p to the incipient bud site is the GTPase Cdc42p26,27. Mutants in CDC42 and in a set of other polarity-establishment genes fail to polarize the actin cytoskeletonZ7. Cdc42p appears to be a key effector among polarity-establishment proteins since constitutively active Cdc42p promotes actin nucleation at the bud site in a permeabilized-cell assayz8.Experiments in other systems using constitutively active and dominant-negative versions of Cdc42p, as well as of related molecules such as Rat and Rho, suggest that a cascade of GTPases may generally transduce local cortical signals to modulate the polarization of the actin and microtubule cytoskeletons2g-31. In the second phase, the cytoskeleton initiates the polarization of factors. There seem to be two distinct mechanisms by which this can take place. First, factors bound to motor proteins can be transported in a directed manner along polarized actin or microtubule polymers. In this mechanism, factors become polarized because motor proteins recognize the intrinsic polarity of cytoskeletal polymers. Alternatively, factors can be moved towards a localized anchor by cytoplasmic streaming produced by the actin or microtubule cytoskeleton. In this mechanism, factors become polarized because of the restricted distribution of anchoring molecules. We discuss one example for each of these mechanisms. Motor-dependent movement may contribute to the polarization of factors such as the OSKARmRNA determinant to the posterior pole of the D. melanogaster oocyte. While OSKARmRNA is distributed uniformly in the oocyte during early stages of oogenesis, it becomes strictly localized to the posterior pole at stages 8-9 (Refs 32 and 33). Experiments in which the partial depolymerization of microtubules is induced indicate that the minus-ends of microtubules are located at the anterior of stage 8-9 oocytes, and the plus-ends at the posterior34. A fusion protein between the heavy chain of the plus-end-directed microtubule motor kinesin and P-galactosidase is transported to the posterior of stage 8-P oocytes35. Although there is no direct evidence for this, OXAR mRNA could be coupled similarly to a plus-end-directed motor and transported along polarized microtubule tracks to the posterior. Thus, in this first mechanism, the polarity inherent to microtubules can be translated into the polarization of factors by coupling with a motor protein (Fig. 4a). Cytoplasmic streaming mediated by the actin cytoskeleton may initiate the polarization of germ-linespecific factors called P granules in the C. elegnnsembryo. Cortical material, including actin foci, flows anteriorly during a short period in the one-cell embryo36,37.At the same time, cytoplasmic material, including P granules, streams towards the posterior of the ce114,36,38. All these movements are abolished by cytochalasin D, which prevents actin polymerizationz3J6. Taken together, these observations suggest that actin may drive the anterior cortical flow, which in turn could result in a volumetrically compensatory posterior cytoplasmic streaming36,37. Thus, in this trends

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second mechanism, cytoplasmic streaming generated by the cytoskeleton may initiate the polarization of factors (Fig. 4~). A third, maintenance, phase is required to anchor factors whose polarization was initiated by cytoplasmic streaming, as in the C. elegans embryo. Posteriorly located P granules are partitioned exclusively into I?,, the posterior daughter of the one-cell embryoz3 (Fig. lb). P granules remain associated with the posterior cortex of P, despite significant anterior cortical floods. Thus, there must be mechanisms that selectively anchor P granules at the posterior cortex (Fig. 4d). A maintenance phase can also be required to stably anchor factors initially polarized by motor proteins, as in the Drosophila oocyte. During stage 10 of oogenesis, vigorous cytoplasmic streaming takes place, and the kinesin heavy-chain+galactosidase fusion protein becomes distributed uniformly throughout the oocyte34,35. By contrast, OSKAR mRNA remains posteriorly localized32,33.Therefore, there must be mechanisms that selectively anchor OSKARmRNA at the posterior cortex (Fig. 4b). The product of OSKAR mRNA, OSKAR protein, is required for this process since anchorage is disrupted in oskar nonsense alleles3q. In summary, the localization of factors may occur in three phases and rely on specialized cortical domains. First, the initial asymmetric cue recruits to the cell cortex molecules such as Cdc42p, which polarize the actin and microtubule cytoskeleton. Second, the cytoskeleton initiates the polarization of factors by directed transport along polar cytoskeletal polymers or by cytoplasmic streaming. Third, anchoring mechanisms serve to maintain the localization of factors. P granules and OSKAR mRNA are anchored at the cell cortex in the C. elegans embryo and the Drosophila oocyte, respectively. In S. cerevisiae,cortically located She proteins are required for the asymmetric distribution of Ashlp, a cell-fate determinant that prevents mating-type switching in the daughter bud40-42. Finally, in the developing Drosophila nervous system, NUMB is localized in a crescent below the cell membrane of the sensory organ precursor cells. Taken together, these observations suggest that cortical anchors may be generally required for the asymmetric distribution of factors. Positioning

the cleavage

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Bud3plBud4p

FIGURE

6) October

Bud-site-selection and polarity-establishment proteins

The cycle of Bud3p and Bud4p determines the axis of asymmetric cell division during axial budding in Saccharomyces cerevisiae. (a) A cortical domain containing Bud3p and Bud4p accumulates in a double-ring around the bud neck late in the cell cycle. (b) The double-ring splits in two just before cell division, leaving a single ring of Bud3p and Bud4p in each cell. (c)The single ring is present transiently early in the cell cycle and recruits bud-site-selection and polarity-establishment proteins to a neighbouring cortical site. (d) The neighbouring cortical site becomes the site of the emerging bud.

(‘3

AB 0

1996

0

3

plane

How does a mother cell ensure that localized factors are correctly partitioned into only one daughter (see Fig. 2d)? The plane of cell division must be positioned accurately for this to be the case. In animal cells, the position of the cleavage plane is determined by that of the spindle poles during anaphase: the cleavage plane forms equidistant from the poles, at right angles to the spindle axis43.Thus, spindle poles must be positioned accurately to partition the factors correctly into daughter cells. Positioning of spindle poles and centrosomes appears to be achieved generally by interactions between microtubules nucleated at centrosomes and localized cortical anchors43. Centrosome rotation is fundamental for proper positioning of the cleavage plane in the I?, cell of the trends

m

Factors

FIGURE

F-actin

-

Microtubules Cortical

anchor

0

P,

Plus-end-directed motor protein

4

Two distinct mechanisms can polarize factors. (a) In the Drosophila melanogoster oocyte, factors such as OSKAR mRNA may be transported by plus-end-directed motors along polarized microtubule tracks towards the posterior (right in the figure) of stage 8-9 oocytes. (b) Polarized OXAR mRNA must then become anchored at the posterior cortex because it remains at that location in stage 9-l 0 oocytes despite vigorous cytoplasmic streaming (curved arrows). (c) In the Caenorhabditis elegans one-cell embryo, it is thought that the cortical flow of actin towards the anterior cell cortex generates the cytoplasmic streaming that may carry P granules towards the posterior (right in the figure). A transient pseudocleavage furrow bisects the one-cell embryo at this stage. (d) P granules must then become anchored at the posterior cortex because they remain at that location in P,, the posterior daughter of the one-cell embryo, despite the anterior flow of cortical material (curved arrows). Compensatory posterior cytoplasmic streaming also takes place in P, but is not represented here for the sake of clarity.

385

(4

AB

AB

P,

(b)

P granules

l

0

-

Cortical

Microtubules

anchor

Dynactin

Dynein FIGURE Analogous

models

for cleavage

pole body embryo. anterior

cortex

antero-posterior

is essential

complex,

complex

rotation

may

similarly pole body

tether

aligns

could

microtubules

cortex

capture

dynein,

dynactin

which

could

motility.

that the anaphase distributing

A spindle spindle

C. elegans a site in the on the

components

site. Perhaps, from

by minus-end-directed

of

dynactin

at

a centrosome

motility.

This could

No dynactin-containing

of AB, and rotation

cortical

stage

cortex,

are both

5

and spindle

the centrosomes

which cortical

does

not take place

located

in the vicinity

capture

microtubules

and pull this and the associated

minus-end-directed ensures

that

protein,

centrosome-nuclear-complex.

in the cortex

(b) In 5. cerevisiae,

(a) Two-cell

at the anterior

which

elegans

at the posterior

for the 90” rotation

the anterior

of the entire

assembles

cerevisiae.

and actin-capping

dynein,

and pull it towards induce

in Caenorhabditis

are anchored

are enriched

site tethers

positioning

in Saccharomyces P granules

axis. Actin

the dynactin this cortical

positioning

In P,, while

plane

nucleus

pole body is aligned

towards positioned

along

one set of chromosomes

in that

cell.

of the bud neck from

the spindle

the bud neck by at the bud

the budding

neck

axis, thus

to each daughter

cell.

C. elegans embryo 44,45.The cleavage plane in Pi bisects the A-P axis, thus partitioning the posteriorly located P granules to only one daughterz3. Therefore, the spindle poles must lie on the A-P axis during anaphase to ensure proper positioning of the cleavage plane. However, the centrosome pair initially lies perpendicular to the A-P axis-14,46. The correct orientation is only achieved after a 90” rotation, during which one centrosome moves towards a site in the anterior cell cortex, thus presumably inducing the rotation of the associated nucleus and the other centrosome44. Analyses with pharmacological agents and laserirradiation experiments indicate that microtubules located between the moving centrosome and the cortical site mediate rotation44,45. What is the nature of this cortical site? Dynactin, which binds to and stimulates the activity of the minus-end-directed microtubule motor dynein47, may be a component of the site. Indeed, actin and actin-capping protein, which are both part of the dynactin complex47, are enriched at the cortical site4*. Moreover, rotation is sensitive to cytochalasin D, indicating that the mechanism involved is actin-dependent44. Taken together, 386

these observations suggest the following model for rotation in I?, (Fig. 5a). Dynactin localized at the cortical site may tether dynein, which would then interact with the plus-end of microtubules and pull the associated centrosome towards the anterior cortex by minus-end-directed motility. Although the absence of mutants in dynein or dynactin components has thus far prevented a test of this model, the presence of the complex of actin and actin-capping protein does correlate with rotation. This complex does not assemble in the sister of Pi, AB, and rotation does not take place in that cell 44,48.Conversely, par-3 mutant embryos accumulate the complex in both AB and Pi, and rotation occurs in both cells in this case13,48.Thus, a localized cortical anchor for microtubules may direct positioning of the cleavage plane in C. elegans. Similar mechanisms seem to be required for spindle positioning in S. cerevisiae.Although the cleavage plane in S. cerevisiae is determined by the budding axis, with cytokinesis occurring at the bud neck, the spindle must be appropriately aligned with that axis to ensure that each daughter cell inherits one daughter nucleus. Spindle alignment is achieved by the positioning at the bud neck of one spindle pole body, the S. cerevisiae equivalent of centrosomes. Genetic analyses indicate that spindle pole body positioning requires not only astral microtubules and actin, but also dynein and the actin-related protein Arplp, another dynactin component47,4g-52. While the distribution of dynactin components has not been reported, a fusion protein between the dynein heavy chain and P-galactosidase can localize to cortical patchess3. Taken together, these observations suggest the following model for spindle pole body positioning (Fig. 5b). Cortical dynactin located in the vicinity of the bud neck could tether dynein, which in turn would capture astral microtubules and translocate them by minus-end-directed motility, thus positioning the spindle pole body at the bud neck. In summary, centrosome rotation in C. elegans and spindle pole body positioning in S. cerevisiae may both be executed by a microtubule-dependent motor protein tethered at a cortical site. This could prove to be a general mechanism for positioning the cleavage plane that ensures that polarized factors are partitioned correctly to only one daughter cell. The central

role of cortical

domains

In this review, we have discussed mechanisms by which factors such as NUMB can become localized asymmetrically in a mother cell and partitioned unequally to daughters. We have described how cortical domains play an important role in setting up the initial asymmetric cue, polarizing factors and positioning the cleavage plane. Why should cortical domains be so central to asymmetric cell division? We speculate that one reason may be that the trigger for the entire process is often external. This being the case, an initial asymmetric cue established in the cortex would probably result in a more rapid orientation of the mother cell than one located in the cytoplasm or the nucleus. Furthermore, an anchorage of localized factors at the trends

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cell cortex may ensure that they are partitioned strictly into one daughter at cell division. By contrast, factors localized in the cytoplasm may not be segregated in such an all-or-none fashion. Finally, cortical domains possibly provide a rigid reference point not found elsewhere in the cell to execute centrosome positioning. Whatever the reasons for such a central role, it will be essential to determine how specialized cortical domains are established in the mother cell. How do external triggers result in the initial asymmetric cue being set up in the cell cortex? What determines the restricted distribution of anchors for localized factors or that of dynactin components? An answer to such questions, along with a better molecular characterization of cortical domains, should reveal the origin of asymmetries that are fundamental for the generation of cell-fate diversity. References 1 WILSON, E. B. (1925) The Cell in Development and Heredity, Macmillan 2 STAIGER, C. (1993) Cm. Opin. Celi Biol. 5, 226-231 3 HORVITZ, H. R. and HERSKOWITZ, I. (1992) Cell68, 237-255 4 N&ON, V., GUERRIER, P. and MONIN, H. (1960) Bull. Biol. FL Be/g. 94, 131-202 5 SULSTON, J. E., SCHIERENBERC, E., WHITE, J. G. and THOMSON, J. N. (1983) Dev. Biol. 100, 64-l 19 6 MIYATA, H. and MIYATA, M. (1981) /. Cen. Appl. Microbial. 27, 365-371 7 DAVIDSON, E. (1986) in Gene Activity in Ear/y Development, pp. 409-524, Academic Press 8 RHYU, M. S., JAN, L. Y. and JAN, Y. N. (1994) Cell 76, 477-491 9 UEMURA, T., SHEPHERD, S., ACKERMAN, L., JAN, L. Y. and JAN, Y. N. (1989) Cell 58, 349-360 10 COHEN, S. and HYMAN, A. A. (1994) Curr. Biol. 4, 420-122 B. and HIRD, 5. N. (1996) Development 122, 11 GOLDSTEIN, 1467-1474 12 CHANT, J. and PRINCLE, J. R. (1995) 1. Cell Biol. 129, 751-765 13 KEMPHUES, K. J., PRIESS, J. R., MORTON, D. G. and CHENC, N. (1988) Cell 52, 31 I-320 14 GUO, S. and KEMPHUES, K. J. (1995) Cell 81, 61 l-620 15 ETEMAD-MOCHADAM, B., GUO, S. and KEMPHUES, K. J. (1995) Cell 83, 743-752 16 HARTWELL, L. H. and UNGER, M. W. (1977) I. Ceil Biol. 75, 422435 17 STRATHERN, J. N. and HERSKOWITZ, I. (1979) Cell 17, 371-381 18 CHANT, J. and HERSKOWITZ, I. (1991) Celi65, 1203-1212 19 CHANT, J., MISCHKE, M., MITCHELL, E., HERSKOWITZ, I. and PRINCLE, J. R. (1995) /, Cell Biol. 129, 767-778 20 SANDERS, 5. and HERSKOWITZ, I. (1996) /. Cell Biol. 134, 413-427 21 CHANT, J. (1996) Cell 84, 187-l PO 22 LONCTINE, M. S. et al. (1996) Curr. Opin. Cell Biol. 8, 106-l 19 23 STROME, S. and WOOD, W. B. (1983) Cell 35, 15-25 24 MAYS, R. W., BECK, K. A. and NELSON, W. J. (1993) Curr. Opin. Cell Biol. 6, 16-24 25 KILMARTIN, J. V. and ADAMS, A. E. (1984) 1, Cell Biol. 98, 26

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Acknowledgements We thank Farhah Assaad, Stephen Cohen, Marianna Giarre, Michael Glotzer, Eric Karsenti and Sigrid Reinsch for critical reading of the manuscript. Pierre Gonczy is supported by a. long-term fellowship from the European Molecular Biology Organization. Work in the laboratory of Anthony A. Hyman is supported by grants from the European Union and the Human Frontiers Science Program.

922-933 ZIMAN, M., PREUSS, D., MULHOLLAND, J., O’BRIEN, J. M., BOTSTEIN, D. and JOHNSON, D. I. (1993) Mol. Biol. Cell 4, 1307-l 316

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