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The Role of Nucleation in Patterning Microtubule Networks 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,
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