Journal of Cell Science 111, 2077-2083 (1998) 2077 Printed in Great Britain © The Company of Biologists Limited 1998 JCS5004

COMMENTARY The role of nucleation in patterning microtubule networks

A. Hyman and E. Karsenti Cell Biology Programme, EMBL, Meyerhofstrasse 1, Heidelberg 69117, Germany (e-mail: [email protected]; [email protected])

Published on WWW 15 July 1998

SUMMARY

Control of microtubule nucleation is important for many we discuss the consequences of non-centrosome dependent microtubule dependent processes in cells. Traditionally, microtubule nucleation for formation of microtubule research has focused on nucleation of microtubules from patterns, concentrating on the assembly of mitotic spindles. centrosomes. However, it is clear that microtubules can nucleate from non-centrosome dependent sites. In this review Key words: Microtubule, Nucleation, Mitotic spindle, Centrosome

INTRODUCTION cell centrosomes. While text books generally show microtubules nucleated exclusively by centrosomes, in a variety of cells many If one looks at the pattern of microtubules in a typical tissue microtubules are not anchored at the centrosome. For instance culture cell, in general one will see microtubules distributed with in migrating newt lung cells, 80-90% of the microtubules are not their minus ends clustered together and their plus ends extending bound to the centrosome (Waterman-Storer and Salmon, 1997). out into the cytoplasm. The canonical model for how such arrays In epithelial cells microtubules form bundles parallel to the are generated is that microtubules, nucleated at centrosomes, apico-basal axis (Bacallao et al., 1989; Mogensen et al., 1989); grow through the cytoplasm. Such growth patterns create radial During myogenesis the centrosomes are eliminated during arrays of microtubules extending from the centrosome. In other formation of parallel bundles of microtubules in the developing situations microtubules form non-radial patterns. For instance in myotubes (Tassin et al., 1985). In most of these cells it is not mitotic spindles more microtubules extend from centrosomes clear how non-centrosome microtubules arise. towards chromosomes than away from chromosomes into the One possibility is that microtubules are centrosome cytoplasm. The discovery of dynamic instability of microtubules nucleated, but that their connections with the centrosome are in 1984 (Mitchison and Kirschner, 1984) suggested how then severed, allowing the microtubules to be organized into centrosome-nucleated microtubules could form patterns different patterns. This phenomenon, initially observed in (Kirschner and Mitchison, 1986). Dynamic instability describes Xenopus egg extracts (Belmont et al., 1990), has now been seen the stochastic behaviour of microtubules in which individual in tissue culture (Keating et al., 1997). The discovery of katanin microtubules transit between phases of growth and fast (McNally et al., 1996), a microtubule severing protein, suggests shrinkage. By such behaviour microtubules nucleating from that it will soon be possible to investigate the role of severing centrosomes were proposed to act as searching devices (Holy in generating microtubule patterns. Centrosome-free and Leibler, 1994; Kirschner and Mitchison, 1986). Upon microtubules can also arise from microtubule breakage contact with capture sites they would be stabilized, forming (Waterman-Storer and Salmon, 1997). In this case microtubules patterns. This idea of selective stabilization of microtubules are not severed at the centrosome but arise by breakage from nucleated from centrosomes was a major step forward in preexisting microtubules. Perhaps the clearest example of understanding the morphogenetic properties of microtubules. It microtubule severing is in the organization of microtubules in has been particularly useful in understanding how microtubules neurons. In these cells microtubules fill up the entire axon, while are stabilized by kinetochores during spindle formation, or how the centrosome is located in the cell body. How are the microtubule arrays are positioned in interphase by the use of microtubules in the axons formed? Recent studies have shown cortical sites. that microtubules are first nucleated at centrosomes, released, The exact nature of nucleating centers or centrosomes has and then carried down the axon by cytoplasmic dynein, perhaps been a matter of controversy. We prefer the definition as a pair anchored to the cell cortex (Ahmad et al., 1998; Ahmad and of centrioles surrounded by nucleating material (Fuller et al., Bass, 1995). Thus in this instance centrosomes act as a 1992; Stearns and Winey, 1997). There are other kinds of microtubule generator (Ahmad et al., 1998), and motors then nucleating centers, for example the spindle pole bodies in yeast, organize the microtubules into the correct distribution. which have structures that are very different from that of somatic Although non-centrosomal microtubules can be generated by 2078 A. Hyman and E. Karsenti severing or breaking of centrosomal-nucleated microtubules, a between 0-10 µM, that allows transient growth of number of recent papers have reminded us that microtubules microtubules. At this concentration, microtubules exhibit can also nucleate independently of centrosomes in cytoplasm. dynamic instability, transiting between phases of growth and Observation of free nucleation in Xenopus extracts (Gard and shrinkage. No assembly is possible in the absence of Kirschner, 1987), fragments of cells without nuclei (Karsenti et centrosomes or other nucleation sites. (2) A concentration in al., 1984b; Maniotis and Schliwa, 1991; McNiven and Porter, the order of 10-20 µM above which microtubules will grow 1988; Rodionov and Borisy, 1997) and normal tissue culture indefinitely from centrosomes. Nucleation in the absence of cells (Vorobjev et al., 1997; Yvon and Wadsworth, 1997) show centrosomes will still not take place. (3) A minimum that nucleation independent of centrosomes is a common concentration for free nucleation in the range of 20-40 µM (Bré occurrence. Nucleation of microtubules independently of and Karsenti, 1990; Fygenson et al., 1995; Mitchison and centrosomes raises three major questions: (1) what is the Kirschner, 1984) (Fig. 1a). Above the concentration for mechanism of microtubule nucleation in the absence of centrosome-independent nucleation, microtubules start to centrosomes? (2) how is the nucleation controlled so that appear through self nucleation (Fig. 1a), resulting in a mixture microtubules are nucleated only where they are needed? (3) how of centrosome and non-centrosome nucleated microtubules. are non-centrosomal microtubules organized into patterns? Pure tubulin nucleates at fairly high concentrations because elongation of the microtubule wall requires the formation of nucleation seed of about 12-15 dimers. At low concentrations MECHANISMS OF MICROTUBULE NUCLEATION the time required to form this stable nucleus is infinite and no elongation occurs (Fygenson et al., 1995; Mitchison and In order to understand why some microtubules are nucleated Kirschner, 1984). Above the minimum concentration, stable by centrosomes whereas others originate from apparently complexes form at a constant rate and the number of undefined cellular domains, it is necessary to understand the microtubules in the solution increases linearly as a function of mechanisms of microtubule nucleation. Microtubules are in time (Fygenson et al., 1995) until the polymer mass starts to dynamic exchange with a pool of soluble tubulin subunits. The deplete the free tubulin pool below the concentration required assembly characteristics of pure tubulin in vitro can be defined for formation of stable seed (Fig. 1b,c). by three ‘minimum concentrations’: (1) a concentration The mechanisms by which centrosomes nucleate

a)

[tub]dy < 10 µM 10 µM < [tub]ig < 20 µM 20 µM < [tub]fn

b)

[tub] < 10 µM 10 µM < [tub] < 20 µM Fig. 1. Concentration dependence of free versus dy ig 20 µM < [tub]fn centrosome induced nucleation in pure tubulin solutions. (a) The aster is nucleated by a centrosome. The given concentrations are roughly accurate. The length of microtubules are arbitrary, but given for infinite time. Above 10 µM they should have infinite length in the presence of an infinite supply of tubulin subunits. dy, dynamic instability regime; ig, infinite growth regime; fn, free nucleation regime. (b) Concentration dependent probability c) of assembly of a stable 12-15 mere nucleus supporting microtubule elongation in the absence of nucleating centers. (c) Microtubules form at a constant rate above the tubulin MT number concentration at which stable 12-15 meres can Time: 1 minute 2 minutes 3 minutes Time form ([tub] >20 µM). Microtubule nucleation 2079 microtubules at low tubulin concentration have recently been place. There are a number of observations suggesting that reviewed (Stearns and Winey, 1997). Much attention has nucleation can be controlled. For instance the assembly of focused on the role of γ-tubulin, a ubiquitous member of the microtubules in cytoplasts devoid of centrosomes increased tubulin family required for centrosome dependent nucleation dramatically when cells reached confluence, suggesting that in all species studied (Stearns and Winey, 1997). It is thought the level of free nucleation could be regulated by cell-cell to bind to centrosomes and form a stable nucleation template interactions (Karsenti et al., 1984b). Some insights into the for the addition of subsequent α-β dimers of tubulin, regulation of non-centrosome nucleation have come from the eliminating the requirement for the formation of the stable seed study of spindle assembly in the absence of centrosomes, of 12-15 α-β dimers which occurs only above about 20 µM which occurs during formation of female meiotic spindle in tubulin (Fig. 1). animal cells and all spindles in plants (Endow and Komma, The tubulin concentration in cells is generally below that 1997; Gard, 1992; Lambert, 1993; Matthies et al., 1996). The required for free nucleation of microtubules in vitro, steps with which a spindle forms without centrosomes in suggesting that non-centrosomal nucleation must be driven by Xenopus best illustrate how non-centrosomal nucleation must cellular factors. The mechanisms by which non-centrosomal be controlled in time and space for successful spindle microtubules nucleate fall into two classes: (1) nucleation sites assembly (Heald et al., 1997). (1) Control in time. During not located at centrosomes (see Fig. 2). In this mechanism, interphase, microtubules are nucleated both from centrosome dispersed nucleation sites provide a template to overcome the and non-centrosome sites. In mitosis, centrosome-dependent need for formation of a nucleation seed as with centrosomes. nucleation continues, but non-centrosome nucleation is shut The obvious candidate for this molecule is γ-tubulin, off, showing that non-centrosome nucleation is under control implicated in the nucleation process from centrosomes, and of the cell cycle (Verde et al., 1990). Cell cycle control of non- many cells have a large cytoplasmic pool of γ tubulin. It is not centrosome nucleation is seen in a number of embryonic known whether the cytoplasmic pool is active, but that it could systems for instance Caenorhabditis elegans embryos drive dispersed nucleation is suggested by the fact that over- (Albertson, 1984; Hyman and White, 1987). (2) Control in expression of γ tubulin in tissue culture cells drives the space. Although a mitotic extract has no non-centrosome formation of ectopic microtubule nucleation. (2) Microtubule nucleation, non-centrosomal nucleation can be triggered by stabilizing proteins could favour the formation of a nucleation the addition of chromatin. However, non-centrosomal seed. Microtubule associated proteins (MAPs) are traditionally nucleation is seen only in the vicinity of the chromatin, known as proteins which stabilize microtubules during growth nowhere else in the cytoplasm (Heald et al., 1997; Karsenti et (Mandelkow and Mandelkow, 1995). However, MAPs like tau al., 1984a). or MAP2 also reduce the tubulin concentration required for The mechanisms by which chromatin could stimulate nucleation in pure tubulin down to that required for nucleation nucleation remain obscure. One possibility is that the nucleation by centrosomes (Bré and Karsenti, 1990) (Fig. 3). activity of dispersed nucleation complexes, such as the γ-tubulin To date, we do not know whether non-centrosomal complex, is regulated. Thus, non-centrosome nucleation microtubules observed in vivo originate from spontaneous templates would be active in interphase, turned off in mitosis, but assembly due to the activity of MAPs or to nucleation by activated locally in the vicinity of chromatin. Another possibility dispersed nucleation sites. is that formation of a nucleation seed is favoured around chromatin, perhaps by the activation of MAPs (see Fig. 3). While nucleation may be controlled, another possibility is that CONTROL OF MICROTUBULE NUCLEATION nucleation is active throughout the cell cycle but in mitosis the nucleated microtubules are too unstable to grow. In this model, When microtubules nucleate in the absence of centrosomes, nucleation is uncontrolled, but microtubules would be stable in the cell needs a way to control where and when this takes interphase, unstable in mitosis and stabilized locally in the

a) b) c)

Centrosome -tubulin complex Minus end directed motor

Fig. 2. Nucleation by γ-tubulin templates. Nucleation focused by the association of γ-tubulin templates with centrioles (a), nucleation by randomly distributed γ-tubulin templates (b), and self organization of randomly nucleated microtubules by minus end directed motors (c). 2080 A. Hyman and E. Karsenti

[Tubulin] < [Tubulin] [Tubulin] < [Tubulin] [Tubulin] < [Tubulin] Fig. 3. The effect of MAPs and free nucleation free nucleation free nucleation microtubule destabilizing factors + + MAPs on centrosome dependent and MAPs free microtubule nucleation. In a + destabilizing factors tubulin solution that does not allow free nucleation, but centrosome dependent nucleation (left), adding MAPs like tau or MAP2 leads the appearance of - Centrosomes free microtubules in addition to centrosome-nucleated ones (center). It is expected that in some cells where there are MAPs like tau and MAP2, it is the presence of destabilizing factors that prevent the occurrence of + Centrosomes free microtubules (right).

vicinity of chromatin. We suspect that microtubule stability in ORGANIZATION OF RANDOMLY NUCLEATED vivo comes from the competing activities of microtubule MICROTUBULES destabilizing factors such as OP18/stathmin (Belmont and Mitchison, 1996; Marklund et al., 1996; Tournebize et al., 1997) While nucleation from centrosomes provides a built-in and XKCM1 (a microtubule-based motor) (Walczak et al., mechanism for focusing microtubules, the dispersed nucleation 1996), and the stabilizing activities of MAPs (Fig. 3). If of microtubules leaves a problem of microtubule organization. stabilizing factors were activated and destabilizing factors For instance it is easy to understand how a bipolar spindle is inactivated around chromatin, this would favour microtubule formed between two centrosomes. Before mitosis the growth (Hyman and Karsenti, 1996). Support for such a centrosome duplicates and each of the two centrosomes forms hypothesis has come from analysis of the activity of one one of the spindle poles. Microtubules nucleated from destabilizing molecule, OP18/stathmin, which is inactivated by centrosomes are captured by chromosomes and stabilized, chromatin (Andersen et al., 1997; Marklund et al., 1996). forming a mitotic spindle. However, if microtubule nucleation is Depletion of this destabilizing factor from Xenopus cytoplasmic dispersed, such as in plant spindles or meiotic animal spindles, extracts leads to enhanced microtubule nucleation in the vicinity the situation is complex. It seems that in these cases, of chromatin (Andersen et al., 1997). Further experiments on the microtubules are organized in space through the activity of role of MAPs and nucleating factors in spindle assembly will be motors. It has been known for a long time that the stabilization required to differentiate between these mechanisms. of microtubules by heavy water or taxol could lead to the

Fig. 4. The kinetic dominance of - centrosome centrosome dependent versus free nucleation during spindle assembly in - Taxol (observed) Xenopus egg extracts. In the absence of centrosomes, chromosomes stimulate free nucleation around them. These microtubules are then sorted by motors into a bipolar spindle (top). When one centrosome is present, it nucleates + 1 centrosome microtubules way before the appearance - Taxol of free microtubules, creating an array of (observed) microtubules with homogeneous polarity, plus ends towards the chromosomes. The free microtubules that appear later are automatically oriented and dragged towards the centrosome along the + 1 centrosome centrosomal microtubules by dynein. The + Taxol more microtubules in this pole, the (predicted) strongest the effect. This prevents the appearance of a second pole (middle). Both previous situations have been 5 minutes 15 minutes 30 minutes observed. This model predicts that if free microtubules appear in the same time and in similar numbers to the centrosomal ones, a bipolar spindle could still form in the presence of a single centrosome. This could be tested by assembling spindles in the presence of small amounts of taxol (bottom). Microtubule nucleation 2081

Merdes and Cleveland 1997), in other systems centrosomes seem to be required. For instance, in grasshopper spermatocytes, micromanipulated chromosomes require centrosomes to form a spindle. Inhibition of centrosome duplication in sea urchin eggs (resulting in the presence of only one centrosome per egg) results in the formation of a monopolar spindle (e.g. Mazia, 1984; Actin dependent Contraction Sluder and Rieder, 1985). These experiments suggested that centrosomes direct spindle bipolarity. Recent experiments in Xenopus egg extracts suggest that the role of centrosomes may be more complex. As stated above, in Xenopus extracts spindles can form in the absence of centrosomes by microtubule nucleation around chromatin (Heald et al., 1996). Spindles will also assemble in Xenopus extracts in the presence of

Microtubule reorganization centrosomes (Lohka and Maller, 1985). Here, preventing centrosome separation will force these microtubules to become -- organized into a monopolar array (Sawin et al., 1992). However, removing the centrosome allows the formation of bipolar arrays (Heald et al., 1997). Thus in Xenopus, centrosomes are not required for bipolarity, but when present they are dominant and their number will control the number of spindle poles (Heald et al., 1997). Therefore, the original sea urchin experiments did not show that two centrosomes are required to make a bipolar + + spindle. Rather they showed that when centrosomes are present their number can control the number of spindle poles. How can a single centrosome prevent the establishment of a Centriole Microtubule Dynein bipolar spindle in Xenopus extracts? We imagine two different Actin microfilament Tight and adherens junctions ways this could work. (1) Differences in nucleation rates. Non- centrosome nucleation is very slow to initiate in egg extracts Fig. 5. Establishment of a polarized array of microtubules in epithelial cells. Upon disruption of junctional complexes in cultures (several minutes) whereas centrosome dependent nucleation is epithelial cells, the centrioles move towards the nucleus from where almost immediate (Fig. 4). Why would the rate of nucleation a radial array of microtubules emanates (top). Following the from centrosomes be higher than that of free nucleation? This establishment of junctions, the centrioles split apart in a microtubule could be because the mechanisms of nucleation are different, and actin-dependent way (middle). We propose that dynein, anchored for example, non-centrosome nucleation is non templated but at the level of the junctions pulls onto the microtubules, thereby relies on the formation of a nucleation seed (Fig. 1). (2) Local pulling the centrioles apart. Probably, an actin-dependent contraction density of nucleating events. In this case, non-centrosome follows and in the same time, the dynein moves towards microtubule nucleation followed by elongation happens at random around minus ends, organizing the microtubules into apico-basal bundles chromatin. Therefore, the probability that enough microtubules and raising up the junctions. Although this is still entirely are cross-linked by motors to generate a pole and become hypothetical, this is just an extension of the self organization principle uncovered in the mechanism of mitotic spindle assembly. further stabilized together is low, leading to a time delay in the appearance of a visible pole (Fig. 4). By contrast, since the centrosome has many nucleating sites, it will nucleate many generation of microtubule asters in the absence of centrosomes microtubules in a concentrated spot even if each nucleation (De Brabander et al., 1981, 1986; Karsenti et al., 1984a). In the event is infrequent. In both cases, the centrosome breaks the absence of centrosomes, a set of randomly growing microtubules symmetry of microtubule distribution and all microtubules can become organized into an astral array by minus or plus-end nucleated from non-centrosome sites around the chromosomes directed motors (Nédélec et al., 1997; Urrutia et al., 1992; Verde would then become oriented by motors relative to these pre- et al., 1991) (Fig. 2c). This shows that an astral array of existing microtubules and moved towards the centrosome (Fig. microtubules can be generated by two independent pathways: 4). We would predict that the dominance of centrosomes could through nucleation from a fixed point, a centrosome, or through be abolished by reducing the asymmetry of microtubule growth the reorganization of randomly nucleated microtubules into an around chromosomes. For example small amounts of agents aster. Thus, formation of spindles in the absence of centrosomes which trigger nucleation, such as taxol, would favour the rate provides a nice example in which spindle assembly is triggered of free nucleation, perhaps triggering bipolar spindle formation by the control of nucleation around chromatin, followed by their in the presence of a centrosome (Fig. 4). In fact in some organization into spindles by motors. situations, preventing centrosome separation does not prevent formation of two poles (Wilson et al., 1997). This could be because the density of microtubules or the rate of nucleation is CENTROSOME VS NON-CENTROSOME SPINDLE higher in Drosophila embryos than in Xenopus extracts. POLE FORMATION In conclusion one of the interesting facts to emerge from the study of spindle assembly in meiosis is that whereas centrosomes While it is clear in some systems that spindles can form in the are dispensable, when present they provide dominant sites for absence of centrosomes (reviewed by Waters and Salmon, 1997; organization of microtubules. Thus they provide an added layer 2082 A. Hyman and E. Karsenti of regulation, allowing the determination of the position of the junctions. When the junctions are broken, the cells loose their spindle poles. This is essential in systems in which spindle polarity. Under such conditions, microtubules become positioning is part of the differentiation process that takes place organized in a radial array originating from an ill defined point during cell division. Thus the centrosome can be positioned by localized close to the nucleus (Fig. 5, top) (Bré et al., 1990). the microtubules it nucleates that act as sensors of spatial cues, If one allows the cells to re-establish junctions and polarize and then direct the organization of other microtubules in the cell again, microtubules reorganize so that they have a uniform from its location (Hyman and White, 1987). Most of the results orientation with their plus end facing the basolateral domain obtained on the role of centrosome vs non centrosome with some randomly oriented microtubules in the apical components in microtubule nucleation were obtained in meiotic domain (Fig. 5, bottom) (Bacallao et al., 1989; Mays et al., cells, and it is not clear to what extent the principles uncovered 1994). How could microtubules become oriented in such a in meiotic systems may also apply also during spindle assembly way? We envision two mechanisms by which they become in somatic cells. In at least one case bipolar spindles can assemble oriented in epithelial cells. in the absence of centrosomes in tissue culture cells (Debec et al., (1) The nucleating centers could be moved towards the apical 1982). However, as mentioned above, grasshopper spermatocytes domain of the cells, thereby orienting microtubule growth. This require a centrosome to make a mitotic spindle. It seems possible mechanism uses localization of nucleation sites to pattern the that the requirement for centrosomes reflects the ability of non- microtubule network. Actin may play an important role in this centrosome microtubules to nucleate in cytoplasm. When non- process (Buendia et al., 1990). γ-Tubulin has been localized centrosome microtubules can nucleate, centrosomes are not where it is expected to be if involved in microtubule nucleation required, although they influence the position of the spindle when in epithelial cells (Meads and Schroer, 1995; Mogensen et al., present. When non-centrosome microtubules cannot be formed 1997). However, there is no demonstration that the organization by nucleation, centrosomes are required as a generator of and orientation of microtubules is really defined by a microtubule polymer for spindle assembly. localization of nucleating material yet. (2) Minus end directed motors like dynein could be positioned on the junctions of the cells. In this way, PERSPECTIVES microtubules would be pulled away from the centrosome and moved, plus ends facing the basolateral domain of the cells In this review we have concentrated mainly on the role of (Karsenti et al., 1996). nucleation in building a mitotic spindle during meiosis. We are In summary, centrosomes provide an organization principle beginning to understand some of the basic mechanisms by different from free nucleation by two virtues: (a) one which microtubule distribution is controlled spatially and centrosome can nucleate many microtubules originating from temporally during mitotic spindle assembly. As discussed in one point, whereas free nucleation has no organizing power in the Introduction, there are many complex microtubule itself. (b) Centrosomes can nucleate under conditions where arrangements in differentiated cells which are clearly non- free nucleation is suppressed, providing dominant sites for centrosomal patterns, and at present we have little idea how microtubule organization. Nucleation in the absence of these arrangements are controlled. The challenge is to centrosomes requires organization principles such as motor understand how the principles elucidated from spindle interactions, but provides a potentially versatile system for assembly apply to these complex differentiated cell types. making such patterns. Given the vast array of different One of the important aspects will be to distinguish between microtubule patterns in diverse cell types, it is going to be centrosomes as microtubule generators, as in neurons, and non- important to understand the contribution of different forms of centrosome nucleation in the cytoplasm. During meiotic nucleation in generating microtubule patterns. spindle assembly, chromatin locally controls microtubule nucleation. In interphase, it is not clear what would control microtubule nucleation, but one possibility is the localization REFERENCES of enzymes which control the dynamics of microtubules to cellular sites as has been suggested for meiotic spindle Ahmad, F. G. and Bass, P. W. 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