Curr. Issues Mol. Biol. (2003) 5: 99-112. Regulated Assembly of the Mitotic Spindle 99

Regulated Assembly of the Mitotic Spindle: A Perspective from Two Ends

Lynne Cassimeris and Robert V. Skibbens lattice has an intrinsic structural polarity due to the polarized arrangement of dimers within the lattice. The two ends of the microtubule differ in growth Dept. of Biological Sciences, 111 Research Dr., Lehigh rate, with the faster growing (plus) end localized away from University, Bethlehem, PA 18015, USA the . Spindle are organized into two overlapping arrays that form the bipolar spindle (see Figure 1). Here the fast growing plus ends Abstract originating at each centrosome meet in the spindle midzone (for more extensive reviews see Inoue and Salmon, 1995; segregration and division requires Wittmann et al., 2001). the regulated assembly of the mitotic spindle attach to the spindle by binding the plus apparatus. This mitotic spindle is composed of ends of microtubules. Before begins, each condensed chromosomes attached to a dynamic array chromosome is actually a pair of replicated sister of microtubules. The microtubule array is nucleated (reviewed in Salmon, 1989). These chromatids by and organized by associated are held together along the lengths of their arms by a structural and motor . Mechanical linkages complex of proteins called (below). At metaphase, between and microtubules are chromosomes are aligned in the center of the spindle, with critical for spindle assembly and chromosome sister chromatids facing the opposite poles. . Defects in either chromosome or attachment is mediated by the , a complex of centrosome segregation can lead to and DNA and proteins (shown in red in Figure 1B,C). The are correlated with cancer progression. In this review, of sister chromatids are organized on opposite we discuss current models of how centrosomes and sides of the chromosome complex. Based on the chromosomes organize the spindle for their equal organization of the sister kinetochores, the attachment of distribution to each daughter cell. a kinetochore to microtubules from one half spindle positions the opposite kinetochore toward the opposite Introduction pole. This organization ensures that replicated chromatids are attached to opposite poles and that each daughter cell A fundamental property of all cells is their ability to multiply will receive one, and only one, copy of each chromosome and reproduce. The replication and segregation of the as cells divide (reviewed in Salmon, 1989). must be performed with high fidelity, ensuring that The opposite ends of the microtubules, the minus ends, each daughter cell receives a full complement of DNA. For are focused near the centrosome, but not directly attached all eukaryotic cells, separation of the replicated genome is to it. The spindle microtubules are nucleated at the accomplished by the mitotic spindle during the M-phase of centrosome, but then released from the nucleation site. the . For a rapidly dividing mammalian cell, M- The minus ends of these free microtubules are then focused phase lasts for about an hour out of each 24 hour cell cycle. and anchored by several structural and motor proteins The spindle is assembled at M-phase and is a transient (discussed below). The centrosome is likely held to the structure, built to accurately move chromosomes and then minus ends of microtubules by additional proteins. This disassemble. In this review we discuss mechanisms centrosome anchoring mechanism would serve to link the responsible for assembling the mitotic spindle and attaching centrosome to the microtubules of the spindle and allow replicated chromosomes and centrosomes to this biological each daughter cell to inherit one, and only one, centrosome. machine. To provide a reference point, we begin by The majority of spindle microtubules do not form describing the structure of the spindle at metaphase - the attachments to chromosomes. These non-kinetochore midpoint of . We then describe the roles of microtubules turn over rapidly by dynamic instability, a novel centrosomes, microtubule associated proteins (MAPs), behavior where individual microtubules exist in persistent motor proteins and chromosomes in assemblying and phases of elongation or rapid shortening, with infrequent maintaining the mitotic spindle and anchoring and abrupt transitions between these phases. The transition chromosomes and centrosomes to this structure. from elongation to rapid shortening has been termed catastrophe, while the transition from rapid shortening to The Spindle at Metaphase elongation has been termed rescue (Walker et al., 1988).

The major structural components of the spindle are the Setting the stage: Microtubule reorganization at the microtubules, polymers of α and ß tubulin subunits. The G2/M transition

As the cell enters mitosis, a radial array of long *For correspondence. Email [email protected]. microtubules is disassembled to make way for the spindle.

© 2003 Caister Academic Press 100 Cassimeris and Skibbens

Dissolution of the interphase microtubule array occurs at assembly at tubulin concentrations insufficient to allow the G2/M transition and coincides with the brief period of spontaneous microtubule assembly. Within the time when the is broken apart (Zhai et centrosome, microtubules are nucleated by ring complexes al., 1996). Changes in the dynamic turnover of microtubules composed of γ tubulin and associated proteins (Oakley and likely play a major role in disassembly of the interphase Oakley, 1989; Zheng et al., 1995). The precise mechanism microtubules. During interphase, the frequency of rescue by which these γ tubulin ring complexes nucleate (the switch from a shortening state to a growth state) is microtubule assembly remains a controversial issue (Job relatively high. Frequent rescues allow microtubules to grow et al., 2003). to the longer lengths typical of interphase cells (~ 100 µm Each centrosome nucleates a starburst array of in a typical mammalian epithelial cell; Gliksman et al., microtubules, such that each interphase cell typically has 1993). At the G2/M transition, rescue frequency drops about a single microtubule array, and each mitotic cell, with its 8-fold (Rusan et al., 2002). This drop in rescue, combined replicated and separated centrosomes, forms a bipolar with a reduction in the amount of time microtubules spend spindle composed of two such arrays (Figure 1). Thus, neither growing nor shortening (paused), results in an centrosomes determine where microtubule assembly overall loss of microtubule polymer (Rusan et al., 2002). begins and can organize those microtubules into simple The removal of microtubules from the peripheral patterns depending on the placement of the centrosomes regions of cells is also partially accomplished by relative to each other. When separation of replicated microtubule bundling and -driven transport of these centrosomes fails at entry into mitosis, the resulting bundles (Figure 2). Bundles are moved to the minus ends monopolar spindle contains a single radial array of of microtubules and these bundles can be incorporated microtubules, which cannot support chromosome into the forming spindle (Rusan et al., 2002). This dynein- segregation. dependent clearing of microtubules from the The centrosome’s capacity to nucleate microtubules continues through and metaphase stages increases approximately 5-fold at mitosis (Synder and of mitosis (Rusan et al., 2002). Dynein-dependent transport McIntosh, 1975; Kuriyama and Borisy, 1981; Telzer and of free microtubule bundles has also been observed Rosenbaum, 1979), beginning at the G2/M transition (Piehl recently within mitotic spindles (Khodjakov et al., 2003). et al., 2002). This increase in nucleation rate correlates By late , growing microtubule ends are with an increase in the concentrations of γ tubulin and concentrated in the area surrounding the nuclear envelope. pericentrin associated with the centrosome (Khodjakov and These microtubules are stabilized by dynein/ Rieder, 1999; Dictenberg et al., 1998). It is not yet known associated with the nuclear envelope (Piehl and whether the recruitment of γ tubulin, or other proteins Cassimeris, 2003). Since dynein/dynactin specifically necessary for nucleation, is sufficient to generate the higher associates with the nuclear envelope at prophase (Busson mitotic rate of , or whether other et al., 1998), the motor complex provides local microtubule regulatory mechanisms, such as phosphorylation, stabilization during a brief window of time. The interaction contribute to centrosome maturation. A second pathway between nuclear envelope-bound dynein and microtubules independent of γ tubulin, may also contribute to microtubule allows dynein-dependent tearing of the nuclear envelope nucleation during mitosis (Hannak et al., 2002). The higher to facilitate breakup of the envelope (Salina et al. 2002; rate of nucleation during mitosis contributes signficantly to Beaudouin et al.2002; see Figure 2). the approximately 4 to 10-fold increase in the number of By the processes outlined above, both the interphase microtubules per cell. microtubule array and the nuclear envelope are broken Although microtubules are nucleated at the down. The stage is now set for spindle assembly. centrosome, spindle microtubules do not remain tethered Microtubules rapidly assemble into the nuclear area once to the centrosome. Instead, they are released, and the the nuclear envelope is broken apart. Since microtubule release rate is increased at mitosis (Belmont et al. 1990; assembly begins at the centrosome, in the next section reviewed in Compton, 2000; Bornens, 2002). It is not yet we discuss how centrosomes contribute to spindle clear whether , a microtubule severing assembly. localized to centrosomes, contributes to this microtubule release (Buster et al., 2002). The Centrosome: Microtubule nucleation and perhaps a bit more Centrosomes as depots for regulatory proteins

The centrosome contributes to spindle assembly and Studies over the last several years have pointed to an mitotic fidelity through several functions. The major function additional function of centrosomes as a depot for regulatory of the centrosome is to nucleate microtubule assembly. kinases and other potential upstream regulators of cell cycle Centrosomes can also serve as a scaffold to anchor progression. Multiple kinases and phosphatases have been regulatory proteins and they are necessary for cell cycle localized to centrosomes, and these are often associated progression, as discussed below. with scaffold proteins such as AKAP (reviewed by Zimmerman et al., 1999; Diviani and Scott, 2001; Doxsey, Centrosomes and microtubule nucleation 2001). Centrosome localization may contribute to the substrate specificity of some kinases and phosphatases Centrosomes are microtubule-organizing centers capable (see Diviani and Scott, 2001; Doxsey, 2001). of nucleating microtubules - they stimulate microtubule The centrosomal protein pericentrin, which is required Regulated Assembly of the Mitotic Spindle 101

Figure 2. Illustration of proposed steps of microtubule reorganization at the G2/M transition. Step 1, the long microtubules of the G2 interphase array (blue) are disassembled or bundled (shown as a red complex at the minus ends of the bundled microtubules) and moved towards the centrosome by dynein/dynactin. At this stage, dynein/dynactin also transiently associates with the nuclear envelope. Step 2, new microtubules are stabilized at the Figure 1. Microtubule organization in interpase (A) and mitotic (B) HeLa nuclear envelope by binding to dynein/dynactin. Step 3, dynein-dependent cells. (A) Microtubule (green) and chromosomes (blue) in an interphase pulling forces tear the nuclear envelope apart and microtubules now have cell. A single radial array of microtubules eminates from the single access to the chromosomes. See text for citations to relevant experiments. centrosome located near the nucleus (shown here only as the brightest green spot). (B) Microtubules (green) of the metaphase spindle are organized into two overlapping arrays. Kinetochore regions of chromosomes are shown in red. (C) Diagram of spindle organization at metaphase. Centrosomes are shown as violet circles, microtubules as black or blue lines and chromosomes as yellow ellipses. Microtubule minus ends are anchored to spindle poles by structural and motor proteins (shown as thick orange lines), while some plus ends make connections to the kinetochores of chromosomes (red). Overlapping anti-parallel microtubules are highlighted in blue. Cohesion factors, anchoring the chromatids together are shown in green. Each of these components is discussed in detail in the text.

Figure 3. Illustration of one model for pairing. association with requires the deposition complex (shown on the left). A subset of cohesins appears associated with chromatin prior to S-phase, suggesting stepwise assembly (not shown). To establish pairing, cohesin rings must either become catenated (yellow rings, shown on the right) or a single cohesin ring encircles both sister chromatids (not shown). The role of the establishment complex is unknown, but may facilitate ring catenation during DNA replication (shown in the center). 102 Cassimeris and Skibbens for microtubule nucleation and forms a structural lattice Microtubule Dynamics during mitosis with gamma tubulin at the centrosome (Dictenburg et al., 1998), is a member of the AKAP scaffold family (Diviani The above discussion focused on several possible and Scott, 2001). The sequence similarity between AKAPs functions for centrosomes, but their role as microtubule and pericentrin suggests that a dual scaffold is possible: nucleators is clearly the most important for spindle one that can both participate in microtubule nucleation and assembly. Once nucleated, these microtubules turn over anchor signaling molecules. at a fast rate, primarily by dynamic instability. Below we Several other regulatory proteins are also localized to discuss regulation of this dynamic turnover by the centrosomes including proteins of the ubiquitin degradation antagonistic activities of proteins able to stabilize or pathway and the tumor suppressor, (Ciciarello et al., destabilize microtubules. This dynamic turnover is 2001; reviewed by Zimmerman et al., 1999). The necessary for spindle assembly by allowing rapid localization of p53 to centrosomes may be related to its attachments to form between microtubules and function in cell cycle arrest (Ciciarello et al., 2001), but chromosomes (Hill et al., 1985; Kirschner and Mitchison, p53 also appears necessary for maintenance of normal 1986). centrosome numbers (reviewed by Tarapore and Microtubule assembly dynamics are regulated by the Fukasawa, 2002). cell cycle machinery, resulting in much more dynamic microtubules during mitosis. The ~ 10 fold faster turnover Centrosomes: spindle integrity and cell cycle of microtubules was initially observed using photobleaching progression methods almost 20 years ago (Saxton et al., 1984). Observations of individual microtubules in a number of Centrosomes are essential for spindle assembly in most, different experimental systems has shown that microtubule but not all, cells (Sluder and Rieder, 1985; Zhang and growth rate is faster in mitosis, although increased rates Nicklas, 1995). For example, spindles do not assemble in vary from a small 1.1 - 1.3 fold increase (Rusan et al., insect if centrosomes are removed at 2001; Belmont et al., 1990) to a near doubling in velocity prophase or early stages of mitosis (Zhang and Nicklas (Piehl and Cassimeris, 2003; Hayden et al., 1990). 1995). Surprisingly, laser ablation of centrosomes suggests Experiments in Xenopus egg extracts (Belmont et al., 1990) that some cells can assemble a spindle in the absence of suggested that the major change in microtubule dynamics centrosomes (Khodjakov et al., 1999). When centrosomes at mitosis is a large increase in catastrophe frequency. In are present, as is usually the case, they dominate spindle other systems, such as sea urchin egg extracts and assembly by nucleating microtubules (Heald et al., 1997). mammalian epithelial cells, rescue is regulated to a larger Once spindle assembly has progressed to metaphase/ degree (Glicksman et al., 1992; Rusan et al., 2002). These anaphase, centrosomes are no longer necessary for changes in catastrophe or rescue rates could each spindle maintenance (Mitchison and Salmon, 1992; Nicklas generate short microtubules with more rapid turnover rates. et al., 1989). Given that centrosomes do not appear necessary for Regulation of microtubule assembly dynamics by maintenance of microtubules and bipolar spindle structure associated proteins once cells progress to metaphase or anaphase, it is surprising that centrosomes are required for mitotic exit, The changes in microtubule assembly dynamics throughout completion of and entry into the next the cell cycle are regulated by the activation or inactivation (Khodjakov and Rieder 2001; Piel et al., 2001; reviewed of microtubule associated proteins (MAPs; reviewed in by Doxsey, 2001). These results suggest that centrosome Cassimeris, 1999). The cell cycle regulation of oncoprotein inheritence is important for cell cycle progression. We 18 (Op18), a microtubule destabilizer, is the best-studied suggest that during mitosis, it is necessary to attach example. Op18 is phosphorylated at entry into mitosis and centrosomes to spindle poles to ensure their delivery to all 4 serine residues must be phosphorylated to allow daughter cells. Centrosome inheritence may be necessary mitotic progression (Larsson et al., 1997). Phosphorylation not only to provide a microtubule organizing center, but preceeds sequentially: ser 25 and 38 are first also to ensure the proper localization of regulatory proteins, phosphorylated by CDK1, allowing subsequent such as those discussed above. phosphorylation of ser 16 and 63 by an unknown kinase(s) Defects in the number of centrosomes have been (Larsson et al., 1997; Marklund et al., 1996). Expression observed in a number of human tumors, including cells of non-phosphorylatable Op18 mutants prevents assembly from the early stages of tumor development (Pihan et al., of the mitotic spindle, presumably because microtubule 2001; Lingle et al., 2002; D’Assoro et al., 2002). In one assembly is severly compromised by the presence of case, expression of human papillomavirus E6 and E7 excess microtubule destabilizing activity. oncoproteins was sufficient to upset the normal coupling The idea that microtubule dynamics are regulated by between centrosome replication and the cell cycle, resulting a balance between stablizing and destabilizing MAPs was in extra centrosomes and anueploidy (Duensing et al., first proposed for XMAP215 (a stabilizer) and XKCM1 (a 2000). While the experiments with papillomavirus protein destablilizer). Depletion of one protein from Xenopus egg expression suggest a causitive role for increased extracts resulted in very long or very short microtubules, centrosome number in causing anueploidy, it is not yet depending on the protein removed. Depletion of both known whether abberant numbers of centrosomes causes proteins returned microtubule lengths and catastrophe the anueploidy associated with cancers, or results from frequency to near normal levels (Tournebize et al., 2000). that anueploidy. Regulated Assembly of the Mitotic Spindle 103

The counteracting activities of these associated proteins and disassembly at the minus end. Flux is likely driven by has also been demonstrated for their S. cerevisiae minus end directed motors, but the motor responsible has homologs (Severin et al., 2001), but whether the not been identified. The rate of flux differs considerably in mammalian homologs function to antagonize each other different systems, and thus the contribution of flux to is not yet clear. microtubule turnover rates can vary significantly (Maddox XMAP215 is phosphorylated by CDK1, which et al., 2002; Mitchison, 1989; Waterman-Storer et al., 1998). significantly reduces the ability of XMAP215 to stimulate microtubule growth rate (Vasquez et al., 1999). CDK1- Dynamic microtubules and motor proteins can self- phosphorylated XMAP215 still binds microtubules organize into a bipolar spindle (Vasquez et al., 1999) and protects microtubules from the destabilizing activity of XKCM1 (Tournebize et al. 2000; Dynamic microtubules nucleated by the centrosome are Popov et al., 2001). Therefore, mitotic phosphorylations sculpted into a fusiform spindle in large part by molecular can modify a protein’s activity without simply turning the motors. This is clearly observed for the focusing of protein “off”. In contrast to XMAP215, XKCM1 does not microtubule minus ends at the spindle pole. Microtubules appear regulated by phosphorylation. released from the centrosome are focused by two types of The antagonistic activities of XMAP215 and XKCM1 motors, cytoplasmic dynein and the Kin C class of are not the only balancing act able to regulate microtubule (Gaglio et al., 1996; Mountain et al., 1999; Walczak et al., assembly. The microtubule stabilizer MAP4 counteracts 1998). These motors are all minus end-directed motors the microtubule destabilizing activities of both XKCM1 and and would move along microtubules toward the spindle Op18 (Holmfeldt et al. 2002). It is not yet clear whether the pole. By binding and crosslinking adjacent microtubules, counter-acting activities of these proteins is important these motors pull the minus ends together. For cytoplasmic during mitosis. Op18 is turned off by mitotic phosphorylation dynein, crosslinking requires the associated complex (Larsson et al., 1997), as is the rescue-promoting activity dynactin and the structural protein NuMA (Merdes et al., of MAP4 (Ookata et al., 1995). Since XKCM1 (MCAK in 2000). The plus end directed Eg5 (bimC kinesin mammalian cells) counteracts the stabilizing activities of family) is also required for spindle pole organization, both XMAP215 and MAP4, mitotic phosphorylation of both although the precise function of this motor in spindle pole stabilizers may allow XKCM1/MCAK to gain the upper hand formation is not known (Gaglio et al., 1996; Sawin et al., and generate the more dynamic microtubules present 1992; Heck et al., 1993; Blangy et al., 1995; Wilson et al., during mitosis. 1997). Dissecting the functions of specific MAPs in mitotic The crosslinking of focused minus ends is critical for spindle assembly has not been straightforward since many spindle function. The presence of free minus ends at the proteins show complex binding patterns. The XMAP215 spindle poles is thought necessary for microtubule flux, family of MAPs is predominately localized to centrosomes while these minus ends also must be anchored to generate (Cullen et. al., 1999; 2001; Charrasse et al., 1998; Wang tension on chromosomes and allow chromosome and Huffaker, 1997), where they may contribute to movement. In the absence of proper anchorage, the microtubule nucleation (Lee et al., 2001; Popov et al., chromosomes would remain stationary and the poles and 2002). Genetic evidence also points to a function for this microtubules would be pulled toward them (Nicklas, 1989), protein family at microtubule plus ends or kinetochores as shown experimentally after inhibition of both NuMA and (Nabeshima et al., 1998; Garcia et al., 2001). Likewise, HSET, a kin C kinesin (Gordon et al., 2001). XKCM1 is likely necessary for the high rate of mitotic Plus and minus end directed kinesins also act microtubule turnover since this kinesin stimulates antagonistically to each other to generate a bipolar spindle. catastrophes (Walczak et al., 1996), but XKCM1 is also These antagonistic functions were first demonstrated in S. necessary for microtubule attachments to kinetochores cerevisae (Saunders and Hoyt, 1992) and have been (Walczak et al., 2002). Disruption of the mammalian shown subsequently in a wide range of experimental homolog, MCAK, also delays onset of anaphase (Maney systems (Sharp et al., 1999; Mountain et al., 1999). In the et al., 1998). absence of Eg5 or related plus end kinesins (bimC family), While dynamic microtubule turnover is required for centrosome separation fails and a monopolar spindle forms spindle assembly, subsets of microtubules become (Saunders and Hoyt, 1992; Sawin et al., 1992; Gaglio et differentially stabilized during mitosis. The most obvious al., 1996; Sharp et al., 1999; Mountain et al., 1999; Kapoor example of local stabilization occurs when microtubule plus et al., 2000). These results suggest that the plus end ends attach to kinetochores. By attaching to microtubule directed bimC kinesins push anti-parallel microtubules plus ends, kinetochores reduce the dynamic turnover of apart. Each bimC kinesin is a bipolar, tetrameric molecule these microtubules by approximately 7 fold (Cassimeris et making it capable of sliding antiparallel microtubules past al., 1990; Zhai et al., 1995). It is important to note that each other (Kashina et al., 1996). The plus end directed kinetochore binding to microtubule plus ends still allows sliding of antiparallel microtubules would push the spindle tubulin subunit addition and subtraction from these ends poles apart and would antagonize the tendency of minus (Mitchison et al., 1986), but the rates of addition and end directed motors to pull the centrosomes together. For subtraction are greatly reduced. all experimental systems tested, simultaneous inactivation Microtubules also turn over by flux, where the of the bim C (plus end directed) and Kin C (minus end microtubule polymer is pulled toward the microtubule minus directed) kinesins allows formation of a bipolar spindle. end (the poles), with concomitant assembly at the plus end Neither class of motors is absolutely required for spindle 104 Cassimeris and Skibbens assembly since bipolar spindles will assemble, provided 2001; Nachury et al., 2001), and the kinesin Eg5 (Wilde et both classes of motors are absent. al., 2001). Both TPX2 and NuMA are structural components Structural proteins, such as TPX2 and the XMAP215 necessary to organize spindle poles (reviewed in Compton, family, also contribute to spindle bipolarity (Gruss et al., 2000). TPX2 and NuMA are nuclear proteins during 2002; Garrett et al., 2002; Gergely et al., 2003). Whether interphase and therefore both are cargo for the nuclear these proteins function to cross-bridge anti-parallel import pathway, and have the sequences necessary to form microtubules or act in conjunction with motor proteins is a complex with the importins. -GTP can release these not yet known. proteins from importinß, freeing them to participate in spindle assembly (Gruss et al., 2001; Wiese et al., 2001; Chromosomes and spindle integrity Nachury et al., 2001). In tissue cells containing centrosomes, it is not known Microtubule dynamics, spindle assembly and maintenance whether a local environment favoring microtubule of the mitotic apparatus until anaphase onset are critically stabilization is present during assembly of the mitotic dependent not only on centrosomes, MAPs and motors, spindle, or whether such an environment is necessary. In but also on the chromosomes which bridge the two half- C. elegans embyros, Ran is required for spindle assembly, spindles. In this context, chromosomes represent both a but RCC1 is not (Askjaer et al., 2002). It is likely that chemical depot that generates a microtubule plus-end centrosomes dominate spindle assembly when they are stabilizing environment and a mechanical structure that present and can override any defects in a Ran-dependent can generate forces and link two half-spindles together. stabilization pathway (e.g., Faruki et al., 2002). The importance of chromosomes in spindle stability was For a local stabilizing gradient to function, the gradient elegantly demonstrated by Zhang and Nicklas (1995). would have to decay with dimensions approximating that Using micromanipulation to detach chromosomes and of the spindle. Odde has developed a model to estimate move them out into the cytoplasm, they showed that the hypothetical shape of a gradient of a phosphorylated spindles disassembled when the chromosomes were taken protein. This model incorporates a localized kinase, a away. uniform concentration of phosphatase, and free diffusion of the substrate. Based on assumptions of enzyme Chromosomes as chemical depots of microtubule phosphorylation and dephosphorylation rates, and protein regulatory factors. diffusion, he predicts that a gradient of phosphorylated substrate would decay exponentially over approximately Although most cells rely on centrosomes to organize a 10 µm (Odde, 2001). This 10 µm dimension is bipolar spindle, some meiotic and plant cells are approximately the length of a typical mammalian spindle, able to assemble a spindle in the absence of centrosomes. suggesting that “microtubule chemotaxis” toward In meiotic oocytes, or cell extracts derived from them, chromosomes is feasible. chromosomes play the central organizing role. By binding factors that act upstream to regulate microtubule stability, Chromosomes as mechanical bridges and force chromosomes mark the area where microtubule assembly producing organelles will occur (Kalab et al, 1999; Carazo-Salas et al., 1999; Ohba et al., 1999; Wilde and Zheng, 1999; Zhang et al., A centrosome and associated microtubule radial array 1999). Motor proteins, such as dynein/dynactin and Eg5, comprise a half-spindle, an organelle capable not only of along with structural proteins such as NuMA, and TPX2, force generation but motility. In large vertebrate cells, then organize and gather these microtubules into a bipolar premature centrosome separation during early prophase spindle shape (Walczak et al., 1998; Carazo-Salas et al., can result in two independent and free moving half-spindles 2001; Gruss et al., 2001, Wiese et al., 2001; Nachury et that “swim” throughout the cytoplasm (Bajer, 1982; Waters al., 2001). et al., 1993). Proper spindle assembly or fusion of the two Chromosomes are thought to create a local half-spindles into a stable mitotic apparatus requires that microtubule stabilizing environment in their vicinity because opposing centrosomes become mechanically tethered several regulatory proteins have been localized to mitotic through microtubule interactions. Three possible linkages chromatin, including RCC1, a guanine nucleotide exchange that can tether half-spindles together involve 1) anti-parallel factor for Ran (Carazo-Salas et al., 1999), and a Polo-like microtubule interactions (described above), 2) kinetochore kinase (Budde et al., 2001). Recent experiments have - microtubule attachments and 3) microtubule interactions demonstrated a local high concentration of Ran-GTP with kinesins localized to chromosome arms (termed around chromatin in meiotic Xenopus egg extracts (Kalab chromokinesins). et al, 2002), suggesting that downstream effectors may be One component of the mechanical linkage between regulated in this local environment. Each of these regulators two half-spindles involves interdigitation of opposing polar is thought to act upstream to regulate specific MAPs. For microtubules. As described above, crosslinking of example, local inactivation of a microtubule destabilizing microtubules from opposing spindle poles are in part protein (oncoprotein 18) near chromatin would favor produced by a variety of kinesin-like motor proteins. While microtubule assembly in this region of the cell (Anderson sufficient to initiate the association between two half et al., 1997; Budde et al., 2001). spindles, polar microtubule cross-linkages may not be Potential downstream targets of the Ran pathway sufficient to maintain a spindle structure. For instance, include TPX2, (Gruss et al., 2001), NuMA (Wiese et al., separated asters devoid of chromosomes initially form a Regulated Assembly of the Mitotic Spindle 105 bipolar spindle-like array, but this structure decays over quickly collapsed (Zeng et al., 1999; Meluh and Rose, 1990; time (Faruki et al., 2002). Thus, chromosomes remain an Endow et al., 1994). While the above observations reveal important contender in both spindle assembly and that opposing kinetochore microtubule attachments provide maintenance. a mechanical link that tethers together centrosomes of A second component linking two half spindles together opposing half-spindles, not all kinetochore defects result involves chromosome interactions with microtubules via in overt spindle defects but instead cause cell cycle arrest. the kinetochore. Kinetochores are unique protein This observation may be due to the intimate involvement complexes assembled onto the single DNA of the kinetochore-spindle assembly checkpoint system or region of each sister chromatid (Cheeseman et al., 2002; in partial abrogation of kinetochore structure. The reader Kitigawa and Hieter, 2001). Kinetochore-microtubule is directed to reviews on kinetochore structure and mitotic interactions provide, on average, a poleward pulling force checkpoints (Cheeseman et al., 2002; Hoyt, 2001; (Skibbens et al., 1993; Khodjakov and Rieder, 1996; Waters Kitagawa and Hieter, 2001; Amon, 1999). et al., 1996). This net pulling force occurs through coupled Several recent findings reveal that a third component microtubule plus-end depolymerization and of spindle assembly and maintenance involves activities (such as CENP-E, MCAK/XKCM1, and chromosome-microtubule interactions via chromokinesins cytoplasmic dynein) located at the kinetochore (Pfarr et (XKLP1, KLP38B and KID) - kinesins associated along al., 1990; Steuer et al., 1990; Yen et al., 1992; Wordeman chromosome arms. The chromokinesins KLP1 and and Mitchison, 1995; Walczak et al., 1996). These KLP38B are critical for establishing and maintaining spindle poleward-directed pulling forces act to counterbalance the integrity. For instance, XKlp1 depletion by antisense centrosome separation forces supplied by opposing polar oligonucleotides resulted in unstable spindles. Addition of microtubule-associated motors (Kapoor and Compton, XKlp1 to mitotic extracts containing preformed 2002). In the presence of chromosomes, half-spindles spindles resulted in spindle collapse and dissociation of remain independent up until microtubule plus-ends of one spindle microtubules. Likewise, defects in KLP38B function half-spindle are captured and stabilized by kinetochores or KLP38B depletion resulted in aberrant circular mitotic of chromosomes associated with the other half spindle. structures and abnormal anaphase (Vernos et al., 1995; Images obtained using high resolution video Wang and Adler, 1995; Molina et al., 1997; Ruden et al., and electron microscopy indicate that microtubule capture 1997; Walczak et al., 1998). Thus, microtubule association by a single opposing kinetochore is sufficient for two half- with one class of chromokinesins associated along the spindles to initiate fusion, effectively producing a mitotic chromosome arms appears central to spindle assembly that is stable over time (Bajer, 1982; and maintenance. Nicklas and Kubai, 1985; Rieder et al., 1990). These In contrast, KID-like kinesin family members are not observations (and below) reveal that chromosomes required for spindle assembly/maintenance but instead ultimately tether centrosomes together. appear to generate pushing or polar ejection forces that Amazingly, the kinetochore-microtubule attachment promote chromosome movement away from the spindle site continues to tether chromosomes to spindle poles pole. Typically, chromosomes reside at the half-spindle during tubulin addition and loss - allowing for chromosome periphery near microtubule plus-ends. Depletion of Kid from motion away and toward the centrosome while chromosomes within either monopolar or bipolar spindles mechanically linked to spindle poles (Rieder and Salmon, resulted in chromosome positioning close to the 1998). The role of the kinetochore-microtubule mechanical centrosomes and defects in chromosome congression to linkage is evident in the characterization of the metaphase (or II metaphase) plate (Antonio et kinetochore proteins such as the Duo1p, Dam1p, Dad1p al., 2000; Funabiki and Murray, 2000; Levesque and complex or Slk19p that associate with centromere DNA Compton, 2001; Perez et al., 2002). Intriguingly, spindles and microtubules. Loss of any one of these kinetochore remained predominantly intact under these conditions, proteins not only results in high rates of chromosome suggesting that the kid class of chromokinesins contribute missegregation, but also in a diversity of spindle defects. to chromosome positioning rather than spindle For mutations in either Duo1p, Dam1p, Dad1p, cells contain maintenance. aberrant spindles structures including bent spindles and partially elongated spindles that contain discontinuous or Sister chromatid cohesion - gluing together decreased microtubule in the spindle center kinetochore and chromokinesin functions (Cheeseman-Barnes et al., 2001; Enquist-Newman et al., 2001; Jones et al., 2001). It is likely that these cells contain Given the central role of chromosomes in both assembling two half spindles that are no longer joined together, similar and maintaining spindle integrity, it is not surprising that to the two separate prometaphase half spindles the stable association of two half spindles in part relies on occasionally observed in newt lung cells (Bayer, 1982; the glue that holds sister chromatids together. During S- Waters et al., 1993). In contrast, loss of Slk19p function phase, each chromosome is replicated to produce two resulted in cells that contained abnormally short, but still identical chromosomes - termed sister chromatids. Sister bipolar, spindles (Zeng et al., 1999). Cells harboring chromatids are paired along their entire length by an ill- mutations in both Slk19p and Kar3p (a kinesin-like defined interaction of three different cohesion complexes: microtubule minus-end destabilizing factor) were unable the structural cohesins Smc1p, Smc3p, Mcd1p/Scc1p, to assemble a bipolar structure and instead contained Scc3p/Irr1p, and Pds5p; the deposition complex comprised monopolar spindles. If allowed to pre-form, bipolar spindles of Scc2p and Scc4p; and the establishment factors Ctf7p/ 106 Cassimeris and Skibbens

Eco1p, Ctf18p/Chl12p, Ctf8p and Dcc1p (Figure 3) centrosome will not enter S phase (Khodjakov and Rieder, (Strunnikov et al. 1993; Kurlandzka et al. 1995; Guacci et 2001), suggesting that DNA replication requires that a cell al. 1997; Michaelis et al. 1997; Toth et al. 1999; Hartman have a centrosome. et al. 2000; Panizza et al. 2000; Ciosk et al., 2000; Furuya In normal circumstances, however, centrosomes are et al., 1998; Skibbens et al., 1999; Hanna et al., 2001; not produced de novo but duplicated in a highly regulated Mayer et al., 2001). Structural cohesion factors assemble fashion during S-phase under the control of CDK2 as rings structures that either encompass both sisters or complexed with E (Hinchcliffe et al., 1998; Lacey et one ring encircling each sister with the rings catentated al., 1999). For centrosome duplication, one target of active together (Carson and Christman; 2001, Melby et al., 1998; CDK2 is nucleophosmin, a component of the . Akhmedov et al., 1998; Campbell and Cohen-Fix, 2002; Phosphorylation likely regulates the activity or localization Ivanov et al., 2002). This pairing, or cohesion, sterically of nucleophosmin, since expression of a non- constrains sister chromatids so that sister kinetochores face phosphorylatable mutant prevented centrosome duplication away from each other, promoting proper chromosome bi- by blocking separation (Okuda et al., 2000). orientation. Cohesion also resists the poleward forces Similarly, sister chromatid pairing is intimately coupled generated at the kinetochore-microtubule interface and to the DNA replication machinery. For instance, numerous spindle poles that act to pull sister chromatids apart. Replication Factor C (RFC) complex subunits (Rfc2p, Rfc4p Tension resulting from poleward pulling forces and sister and Rfc5p) and at least one DNA polymerase (Polσ or chromatid cohesion is monitored by eukaryotic cells to Trf4p) are now known to play additional roles in cohesion ensure that each sister chromatid is properly associated (Wang et al., 2000; Mayer et al., 2001; Hanna et al., 2001; with spindle microtubules before initiating anaphase onset. Kenna and Skibbens, 2003). Ctf7p appears to be a critical Loss of cohesion is thus predicted to result both in an link between sister chromatid cohesion and DNA replication imbalance of forces, favoring chromosome-to-pole motion, (Figure 3). Ctf7p is required only during S-phase and and disruption of the mechanical coupling that links two associates with each of three Replication Factor C (RFC) half-spindles together. In both cases, the predictions are complexes containing Rfc2p-Rfc5 and one of three larger borne out by experimentation. Mechanical or laser-induced subunits: Rfc1p, Rad24p, or Ctf18p (Skibbens et al., 1999; severing between sister chromatids or ablating the Kenna and Skibbens, 2003). RFC complexes load sliding kinetochore itself results in movement of the intact sister clamps onto DNA and thus promote processive DNA chromatid(s) toward its associated spindle pole (Nicklas replication (Kelman, 1997). Two such sliding clamps have and Staehly, 1967; McNeill and Berns, 1981; Rieder et al., been identified. The homotrimeric PCNA sliding clamp 1986; Hays and Salmon, 1990; Skibbens et al., 1995;). functions during the bulk of DNA replication while the Similarly, yeast cells harboring mutations in the cohesion heterotrimeric Mec3p, Rad17p and Ddc1p sliding clamp is factors such as Ctf7p/Eco1p, Mcd1p/Scc1p and Pds5p a component of the DNA damage checkpoint mechanism have partially elongated and often bent spindles with (Green et al., 2000; Kondo et al., 1999; Longhese et al., separated but unequal masses of DNA (Guacci et al., 1997; 1997; Naiki et al., 2000; Paciotti et al., 1998). The Skibbens et al., 1999; Hartman et al., 2000). Cohesion- association of the cohesion establishment factor Ctf7p with deficient cells delay in mitosis by activating the kinetochore/ all three RFC complexes suggests that each is capable of spindle checkpoint. Coupled with the observation that these participating in cohesion establishment. spindles are only partially elongated (i.e., not true It is likely that cross-talk exists between the processes anaphase) with broken spindles or spindles with decreased required to activate centrosome duplication during S-phase microtubule intensity in the mid-region (i.e., half-spindle and those that couple cohesion establishment to DNA separation), we suggest that defects in sister chromatid replication. Toward this end, recent experiments in yeast cohesion result in the inability of kinetochore poleward cells reveal a direct physical interaction between regulators pulling forces to counteract the forces produced by of chromatid cohesion and the duplication microtubule motors generating centrosome separation machinery (L. Antoniacci, M. Kenna, P. Uetz, and R. V. forces. Thus, integrity of the spindle apparatus must in part Skibbens, manuscript in preparation). Our understanding rely on maintaining cohesion between sister chromatids. of the mechanisms coupling DNA replication, cohesion establishment and centrosome replication are likely to In the beginning: links between centrosome expand in the next few years. It will be interesting to identify duplication and establishment of sister chromatid the feedback controls that monitor each replication process, cohesion and how it communicates to the other system when problems arise. Such a feedback loop would ensure that The geometry of two separated centrosomes, each DNA and centrosome replication remain in synchrony, and nucleating a radial array of microtubules, is central to bipolar contribute to mechanisms to prevent anueploidy. spindle assembly and proper chromosome segregation during mitosis. Typically, a single centrosome is passed Concluding remarks along to each daughter cell and replicated prior to division. Under experimental conditions, centrosomes can form de Numerous studies have detailed the critical roles novo if the original centrosomes are first experimentally centrosomes and chromosomes play in spindle assembly destroyed. Cells apparently have a mechanism to sense (while a few have suggested that each structure may not the absence of centrosomes and assemble new ones only be necessary at all times!). Given the importance of if necessary (Khodjakov et al., 2002). Cells lacking a centrosomes for subsequent cell cycle progression and Regulated Assembly of the Mitotic Spindle 107 for the localization of various signaling molecules (e.g. p53), 14: 25-34. we suggest that centrosomes attach themselves to the Budde, P.P., Kumagai, A., Dunphy, W.G. and Heald, R. minus ends of microtubules to ensure their transport to 2001. Regulation of Op18 during spindle assembly in daughter cells, while chromosomes attach themselves to Xenopus egg extracts. J. Cell Biol. 153. 149-157. the plus ends of microtubules for the same goal. Busson, S., Dujardin, D., Moreau, A., Dompierre, J., De Centrosomes have staked themselves at homeplate, the Mey, J.R. 1998. Dynein and dynactin are localized to astral separated chromatids run to join them, giving each microtubules and at cortical sites in mitotic epithelial cells. daughter the right stuff to continue on and on. Curr. Biol. 8, 541-544. Buster D., McNally, K., McNall,y F.J. 2002. Katanin inhibition prevents the redistribution of gamma-tubulin at mitosis. Acknowledgements J. Cell Sci. 115:1083-92. Campbell, J. and Cohen-Fix, O. 2002. Chromosome Thanks to the editors of CIMB for inviting us to submit this cohesion: ring around the sisters? Trends Biochem. Sci. review. Thanks to Meg Kenna and Michelle Piehl for helpful 27: 492 discussions. We aimed at a more comprehensive review Carazo-Salas, R.E., Guarguaglini, G., Gruss, O.J., Segref, than is typical, and have left out citations to many excellent A., Karsenti, E. and Mattaj, I.W. 1999. Generation of GTP- studies. We apologize to our colleagues whose work we bound Ran by RCC1 is required for chromatin-induced could not include. Supported by grants from NIH GM58025 mitotic spindle formation. Nature. 400: 178-181. (LC) and NSF 0212323 (RVS). Carazo-Salas, R.E., Gruss, O.J., Mattaj, I.W., Karsenti, E. 2001. Ran-GTP coordinates regulation of microtubule nucleation and dynamics during mitotic-spindle assembly. References Nat. Cell Biol. 3: 228-234. Carson, D. R., and M. F. Christman. 2001. Evidence that Akhmedov, A.T., C. Frei, M. Tsai-Pflugfelder, B. Kemper, replication fork components catalyze establishment of S.M. Gasser, and R. Jessberger. 1998. Structural cohesion between sister chromatids. Proc. Natl .Acad. maintenance of chromosomes protein C-terminal Sci. USA. 98: 8270-8275. domains bind preferentially to DNA with secondary Cassimeris, L. 1999. Accessory Protein Regulation of structure. J. Biol. Chem. 273: 24088-24094. Microtubule Assembly Dynamics Throughout the Cell Amon, A. 1999. The spindle checkpoint. Curr Opin. Gen. Cycle. Cur. Opin. Cell Biol. 11: 134-141. Devel. 9: 69-75. Cassimeris, L., Rieder, C.L., Rupp, G. and Salmon, E.D. Andersen, S.S.L., Ashford, A.J., Tournebize, R., Gavet, O., 1990. Stability of Microtubule Attachment to Metaphase Sobel, A., Hyman, A.A. and Karsenti, E. 1997. Mitotic Kinetochores in PtK1 Cells. J. Cell Sci. 96: 9-15. chromatin regulates phosphorylation of /Op18. Charrasse, S., Schroeder, M., Gauthier-Rouviere, C., Ango, Nature. 389: 640 – 643. F., Cassimeris, L., Gard, D. L. and Larroque, C. 1998. Antonio, C., Ferby, I., Wilhelm, H., Jones, M., Karsenti, E., The TOGp protein is a new human microtubule- Nebreda, A.R., and Vernos, I. 2000. Xkid, a chromokinesin associated protein homologous to the Xenopus required for chromosome alignment on the metaphase XMAP215. J. Cell Sci. 111: 1371-1383. plate. Cell. 102: 425-435. Cheeseman-Barnes, I.M, Enquist-Newman, M., Muller- Askjaer, P., Galy, V., Hannak, E., and Mattaj, I.W. 2002. Reichert, T., Drubin, D.G., and Barnes, G. 2001. Mitotic Ran GTPase cycle and importins alpha and beta are spindle integrity and kinetochore function linked by the essential for spindle formation and nuclear envelope Duo1p/Dam1p complex. J. Cell Biol. 152: 197-212. assembly in living embryos. Mol. Cheeseman, I.M., Drubin, D.G., Barnes, G. 2002. Simple Biol. Cell. 13: 4355-4370. centromere, complex kinetochore: linking spindle Bajer, A.S. 1982. Functional autonomy of monopolar microtubules and centromeric DNA in budding yeast. J. spindle and evidence for oscillatory movement in mitosis. Cell Biol. 157: 199-203. J. Cell Biol. 93: 33-48. Ciciarello, M, R Mangiacasale, M Casenghi, M Zaira Beaudouin, J., Gerlich, D., Daigle, N., Eils, R. and Limongi, M D’Angelo, S Soddu, P Lavia, and E Cundari. Ellenberg, J. 2002. Nuclear envelope breakdown 2001. p53 displacement from centrosomes and p53- proceeds by microtubule-induced tearing of the lamina. mediated G1 arrest following transient inhibition of the Cell. 108: 83-96. mitotic spindle. J. Biol. Chem. 276: 19205-13. Belmont, L.D., Hyman, A.A., Sawin, K.E. and Mitchison, Ciosk, R., M. Shirayama, A. Shevchenko, T. Tanaka, A. T.J. 1990. Real time visualization of cell cycle dependent Toth, A. Shevchenko, and K. Nasmth. 2000. Cohesin’s changes in microtubule dynamics in cytoplasmic extracts. binding to chromosomes depends on a separate complex Cell. 62:579-589. consisting of Scc2 an Scc4. Mol. Cell 5: 243-254. Blangy, A., Lane HA, d’Herin, P, Harper, M, Kress, M and Compton, D. 2000. Spindle assembly in animal cells. Ann. Nigg EA. 1995. Phosphorylation by p34cdc2 regulates Rev. Biochem. 69: 95-114. spindle association of human Eg5, a kinesin-related motor Cullen, C.F., Deak, P., Glover, D.M., Ohkura, H. 1999. mini essential for bipolar spindle formation in vivo. Cell. 83: spindles: A encoding a conserved microtubule- 1159-1169. associated protein required for the integrity of the mitotic Bornens, M. 2002. Centrosome composition and spindle in Drosophila. J. Cell Biol. 146:1005-18. microtubule anchoring mechanisms. Curr. Opin. Cell Biol. Cullen, C.F., Ohkura, H. 2001. Msps protein is localized to 108 Cassimeris and Skibbens

acentrosomal poles to ensure bipolarity of Drosophila 2002. hTPX2 is required for normal spindle morphology meiotic spindles. Nat. Cell Biol. 3: 637-42. and centrosome integrity during vertebrate . D’Assoro, A.B., Barrett, S.L, Folk, C., Negron, V.C., Curr. Biol. 12: 2055-2059. Boeneman, K., Busby, R., Whitehead C., Stivala, F., Gergely, F., Draviam, V.M., Raff, J.W. 2003. The ch-TOG/ Lingle, W.L., and Salisbury, J.L. 2002. Amplified XMAP215 protein is essential for spindle pole organization centrosomes in breast cancer: a potential indicator of in human somatic cells. Dev. 17: 336-341. tumor aggressiveness. Breast Cancer Res. Treat. 75: 25- Gliksman, N.R., Skibbens, R.V., and Salmon, E.D. 1993. 34. How the transition frequencies of microtubule dynamic Dictenberg, J.B., Zimmerman, W., Sparks, C.A., Young, instability (nucleation, catastrophe and rescue) regulate A., Vidair, C., Zheng, Y., Carrington, W., Fay, F.S., Doxsey, microtubule dynamics in interphase and mitosis: analysis S.J. 1998. Pericentrin and gamma-tubulin form a protein using a Monte Carlo computer simulation. Mol. Biol. Cell. complex and are organized into a novel lattice at the 4: 1035-1050. centrosome. J. Cell Biol. 141:163-74. Gliksman, N.R., Parsons, S.F., Salmon, E.D. 1992. Okadaic Diviani, D. and JD Scott. 2001. AKAP signaling complexes acid induces interphase to mitotic-like microtubule at the . J. Cell Sci. 114: 1431-1437. dynamic instability by inactivating rescue. J. Cell Biol. Doxsey, S. 2001. Centrosomes as command centres for 119:1271-1276. cellular control. Nat. Cell Biol. 3: E105-E108. Gordon, M.B., Howard, L., Compton, D.A. 2001. Duensing, S., Lee, L.Y., Duensing, A., Basile, J., Chromosome movement in mitosis requires microtubule Piboonniyom, S., Gonzalez, S., Crum, C.P., and Munger, anchorage at spindle poles. J. Cell Biol. 152:425-34. K. 2000. The human papillomavirus type 18 E6 and E7 Green, C. M., H. Erdjument-Bromage, P. Tempst, and N. oncoproteins cooperate to induce mitotic defects and F. Lowndes. 2000. A novel Rad24 checkpoint protein genomic instability by uncoupling centrosome duplication complex closely related to replication factor C. Curr Biol. from the cell cycle. Proc. Natl. Acad. Sci. USA. 97: 10002- 10: 39-42. 10007. Gruss, O.J., 2001. Ran induces spindle assembly by Endow, S.A., Kang, S.J., Satterwhite, L.L., Rose, M.D., reversing the inhibitory effect of importin alpah on TPX2 Skeen, V.P., Salmon, E.D. 1994. Yeast Kar3 is a minus- activity. Cell. 104: 83-92. end microtubule motor protein that destabilizes Gruss, O.J., Wittmann, M., Yokoyama, H., Pepperkok, R., microtubules preferentially at the minus ends. EMBO J. Kufer, T., Sillje, H., Mattaj, I.W. and Vernos, I. 2002. 13: 2708-2713. Chromosome-induced microtubule assembly mediated Enquist-Newman, M., Cheeseman, I.M., Van Goor, D., by TPX2 is required for spindle formation in HeLa cells. Drubin, D.G., Meluh, P, and Barnes, G. 2001. Dad1p, Nat. Cell Biol. 4: 871-879. third component of the Duo1p/Dam1p complex involved Guacci, V., Koshland, D. and Strunnikov, A. 1997 A direct in kinetochore function and mitotic spindle integrity. Mol. link between sister chromatid cohesion and the Biol. Cell. 12: 2601-2613. chromosomes condesnation revealed throughthe analysis Faruki, S., Cole, R.W., and Rieder, C.L. 2002. Separating of MCD1 in S. cerevisiae. Cell. 91: 47-57. centrosomes interact in the absence of associated Hanna, J. S., E. S. Kroll, V. Lundblad, and F. A. Spencer. chromosomes during mitosis in cultured vertebrate cells. 2001. Saccharomyces cerevisiae CTF18 and CTF4 are Cell Motil. Cytoskel. 52: 107-121. required for sister chromatid cohesion. Mol. Cell Biol. 21: Funabiki, H. and Murray, A.W. 2000. The Xenopus 3144-3158. chromokinesin Xkid is essential for metaphase Hannak, E., Oegema, K., Kirkham, M., Gonczy, P., chromosome alignment and must be degraded to allow Habermann, B., Hyman, A.A. 2002. The kinetically anaphase chromosome movement. Cell. 102: 411-424. dominant assembly pathway for centrosome asters in Furuya, K., K. Takahashi, and M. Yanagida. 1998. Faithful Caenorhabditis elegans is gamma-tubulin dependent. J. anaphase is ensured by Mis4, a sister chromatid cohesion Cell Biol. 157: 591-602. molecule required in S phase and not destroyed in G1 Hartman, T., K. Stead, D. Koshland, and V. Guacci. 2000. phase. Genes Dev 12: 3408-3418. Pds5p is an essential chromosomal protein required for Gaglio, T., Saredi, A., Bingham, J.B., Hasbani. M.J., Gill, both sister chromatid cohesion and condensation in S.R., Schroer, T.A., Compton, D.A. 1996. Opposing motor Saccharomyces cerevisiae. J. Cell Biol 151: 613-26. activities are required for the organization of the Hayden, J.H., Bowser, S.S., and Rieder, C.L. 1990. mammalian mitotic spindle pole. J. Cell Biol. 135: 399- Kinetochores capture astral microtubules during 414. chromosome attachment to the mitotic spindle: Direct Gaglio, T., Dionne, M.A., Compton, D.A. 1997. Mitotic visualization in live newt lung cells. J. Cell Biol. 111: 1039- spindle poles are organized by structural and motor 1045. proteins in addition to centrosomes. J. Cell Biol. 138: Hays, T. S., Salmon, E. D. 1990. Poleward force at the 1055-66. kinetochore in metaphase depends on the number of Garcia,, M.A., Vardy, L., Koonrugsa, N., and Toda, T. 2001. kinetochore microtubules. J. Cell Biol. 110: 391-404. yeast ch-TOG/XMAP215 homologue Alp14 Heald, R., Tournebize, R., Habermann, A., Karsenti, E., connects mitotic spindles with the kinetochore and is a and Hyman, A. 1997. Spindle assembly in Xenopus egg component of the Mad2-dependent spindle checkpoint. extracts: Respective roles of centrosomes and EMBO J. 20 3389-3401. microtubule self-organization. J. Cell Biol. 138: 615-628. Garrett, S., Auer, K., Compton, D.A., and Kapoor, T.M. Heck, M.M.S., Pereira, A., Pesavento, P., Yannoni, Y., Regulated Assembly of the Mitotic Spindle 109

Spradling, A.C., and Goldstein, L.S.B. 1993. The kinesin- enhance the fidelity of cytokinesis in vertebrates and are like protein KLP61F is essential for mitosis in Drosophila. required for cell cycle progression. J. Cell Biol. 153: 237- J. Cell Biol. 123: 665-679. 242. Hill, T.L. 1985. Theoretical problems related to the Khodjakov, A., Cole, R.W., Oakley, B.R., and Rieder, C.L. attachment of microtubules to kinetochores. Proc. Natl. 1999. Centrosome-independent mitotic spindle formation Acad. Sci. USA. 82: 4404-4408. in vertebrates. Curr. Biol. 10: 59-67. Hinchcliffe, E.H., Li C., Thompson E.A., Maller, J.A., Sluder, Khodjakov, A., Rieder, C.L., Sluder, G., Cassels, G., Sibon, G. 1998. Requirement of Cdk2- activity for O., Wang, C.L. 2002. De novo formation of centrosomes repeated centrosome reproduction in Xenopus egg in vertebrate cells arrested during S phase. J. Cell Biol. extracts. Science. 283: 851-854. 158: 1171-1181. Holmfeldt, P., Brattsand, G., and Gullberg, M. 2002. MAP4 Khodjakov, A, Copenagle, L., Gordon, M.B., Compton, D.A. counteracts microtubule catastrophe promotion but not and Kapoor, T.M. 2003. Minus-end capture of preformed tubulin sequestering activity in intact cells. Curr. Biol. 12: kinetochore fibers contributes to spindle morphogenesis. 1-20. J. Cell Biol. 160: 671-683. Hoyt, M.A. 2001. A new view of the spindle checkpoint. J. Kirschner, M, and Mitchison, T. 1986. Beyond self Cell Biol. 154: 909-911. assembly: from microtubules to morphogenesis. Cell. 45: Inoue, S. and Salmon, E.D. 1995. Force generation by 329-342. microtubule assembly/disassembly in mitosis and related Kitagawa, K. and Hieter, P. 2001. Evolutionary conservation movements. Mol. Biol. Cell. 6: 1619-1640. between budding yeast and human kinetochores. Nat. Ivanov, D., A. Schleiffer, F. Eisenhaber, K. Mechtler, C. H. Rev. Mol. Cell Biol. 2: 678-687. Haering, and K. Nasmyth. 2002. Eco1 is a novel Kondo, T., K. Matsumoto, and K. Sugimoto. 1999. Role of acetyltransferase that can acetylate proteins involved in a complex containing Rad17, Mec3, and Ddc1 in the yeast cohesion. Curr. Biol. 323-328. DNA damage checkpoint pathway. Mol. Cell Biol. 19: Job, D., Valiron, O., and Oakley, B. 2003. Microtubule 1136-1143. nucleation. Curr. Opin. Cell Biol. 15: 111-117. Kuriyama, R. and Borisy, G.G. 1981. Microtubule- Jones, M.H., He, X., Giddings, T.H., and Winey, M. 2001. nucleating activity of centrosomes in Chinese hamster Yeast Dam1p has a role at the kinetochore in assembly ovary cells is independent of the centriole cycle but of the mitotic spindle. Proc. Natl. Acad. Sci. USA. 98: coupled to the mitotic cycle. J. Cell Biol. 91:822-6 13675-13680. Kurlandzka, A., J. Rytka, R. Gromadka, and M. Murawski. Kalab, P., Ru, R.T. and Dasso, M. 1999. The ran GTPase 1995. A new essential gene located on Saccharomyces regulates mitotic spindle assembly. Curr. Biol. 9: 481-484. cerevisiae chromosome IX. Yeast. 11: 885-90. Kalab, P., Weis, K., and Heald, R. 2002. Visualization of a Lacey, K.R., Jackson, P.K,. and Stearns, T. 1999. Cyclin- Ran-GTP gradient in interphase and mitotic Xenopus egg dependent kinase control of centrosome duplication. Proc. extracts. Science. 295: 2452-2456. Natl. Acad. Sci. USA. 96: 2817-2822. Kapoor, T.M., Mayer, T.U., Coughlin, M.L., Mitchison, T. Larsson, N., Marklund, U., Melander Gradin, H., Brattsand, 2000. Probing spindle assembly mechanisms with G., and Gullberg, M. 1997. Control of microtubule monastrol, a small molecule inhibitor of the mitotic kinesin, dynamics by oncoprotein 18: Dissection of the regulatory Eg5. J. Cell Biol. 150: 975-988. role of multisite phosphorylation during mitosis. Mol. Cell Kapoor, T.M. and Compton, D.A. 2002. Searching for the Biol. 17: 5530-5539. middle ground: mechanisms of chromosome alignment Lee, M.J., Gergely, F., Jeffers, K., Peak-Chew, S., and Raff, during mitosis. J. Cell Biol. 157: 551-556. JW. 2001. Msps/XMAP215 interacts with the centrosomal Kashina, A.S., Baskin, R.J., Cole, D.G., Wedaman, K.P., protein D-TACC to regulate microtubule behavior. Nat. Saxton, W.M., Scholey, J.M. 1996. A bipolar kinesin. Cell Biol. 3: 643-649. Nature. 379: 270-272. Levesque, A.A. and Compton, D.A. 2001. The Kelman, Z. 1997. PCNA: structure, functions and chromokinesin Kid is necessary for chromosome arm interactions. Oncogene. 14: 629-640. orientation and oscillation, but not congression, on mitotic Kenna, M.A. and Skibbens, R.V. 2003. A mechanical link spindles. J. Cell Biol. 154: 1135-1146. between cohesion establishment and DNA replication: Lingle, W.L., Barrett, S.L., Negron, V.C., D’Assoro, A.B., Ctf7p/Eco1p, a cohesion establishment factor, associates Boeneman, K., Liu, W., Whitehead, C.M., Reynolds, C., with three different RFC Complexes. Mol. Cell Biol. (in Salisbury, J.L. 2002. Centrosome amplification drives press). chromosomal instability in breast tumor development. Khodjakov, A., and Rieder, C. L. 1996. Kinetochores moving Proc. Natl. Acad. Sci. USA. 99: 1978-1983. away from their associated pole do not exert a significant Longhese, M. P., V. Paciotti, R. Fraschini, R. Zaccarini, P. pushing force on the chromosome. J. Cell Biol. 135: 315- Plevani, and G. Lucchini. 1997. The novel DNA damage 327. checkpoint protein ddc1p is phosphorylated periodically Khodjakov, A. and Rieder, C.L. 1999. The sudden during the cell cycle and in response to DNA damage in recruitment of gamma-tubulin to the centrosome at the budding yeast. EMBO J. 16: 5216-5226. onset of mitosis and its dynamic exchange throughout Maddox, P., Desai, A., Oegema, K., Mitchison, T.J., and the cell cycle, do not require microtubules. J. Cell Biol. Salmon, E.D. 2002. Poleward microtubule flux is a major 146: 585-596. component of spindle dynamics and anaphase a in mitotic Khodjakov, A., and Rieder, C.L. 2001. Centrosomes Drosophila embryos. Curr. Biol. 12:1670-4. 110 Cassimeris and Skibbens

Marklund, U., Larsson, N., Melander Gradinm H., DNA damage checkpoints throughout the cell cycle in Brattsandm G., and Gullbergm M. 1996. Oncoprotein 18 Saccharomyces cerevisiae. Mol. Cell Biol. 20: 5888-5896. is a phosphorylation-responsive regulator of microtubule Nicklas, R. B. and Staehly, C. A. 1967. Chromosome dynamics. EMBO J. 15: 5290-5298. micromanipulation. I. The mechanics of chromosome Maney, T., Hunter, A.W., Wagenbach, M., Wordeman, L. attachment to the spindle. Chromosoma. 21: 1-16. 1998. Mitotic centromere-associated kinesin is important Nicklas, R.B. and Kubai, D.F 1985. Microtubules, for anaphase chromosome segregation. J. Cell Biol. 142: chromosome movement, and reorientation after 787-801. chromosomes are detached from the spindle by Mayer, M. L., S. P. Gygi, R. Aebersold, and P. Hieter. 2001. micromanipulation. Chromosoma. 92: 313-324. Identification of RFC (Ctf18p, Ctf8p, Dcc1p): An alternate Nicklas, R.B. 1989. The motor for chromosome movement RFC complex required for sister chromatid cohesion in in anaphase is in or near the kinetochore. J. Cell Biol. S. cerevisiae. Mol. Cell 7: 959-970. 109: 2245-2255. McNeill, P.A. and Berns, M.W. 1981. Chromosome behavior Nicklas, R.B, Lee, G.M., Rieder, C.L., and G Rupp, G. 1989. after laser microirradiation of a single kinetochore in Mechanically cut mitotic spindles: clean cuts and stable mitotic PtK2 cells. J. Cell Biol. 88: 543-553. microtubules. J. Cell Sci. 94: 415-423. Melby, T.E., C.N. Ciampaglio, G. Briscoe, and H.P. Oakley, C.E., and Oakley, B.R. 1989. Identification of Erickson. 1998. The symmetrical structure of structural gamma-tubulin, a new member of the tubulin superfamily maintenance of chromosomes (SMC) and MukB proteins: encoded by mipA gene of Aspergillus nidulans. Nature. long, antiparallel coiled coils, folded at a flexible hinge. J. 338: 662-624. Cell Biol. 142: 1595-1604. Ohba, T., Nakamura, M., Nishitani, H., Nishimoto, T. 1999. Meluh P.B., and Rose, M.D. 1990. KAR3, a kinesin-related Self-organization of microtubule asters induced in gene required for yeast nuclear fusion. Cell. 60: 1029- Xenopus egg extracts by GTP-bound Ran. Science. 284: 1041. 1356-1358. Merdes, A., Heald, R., Samejima, K., Earnshaw, W.C., and Odde, D. 2001. Mathematical model foor microtubule Cleveland, D.W. 2000. Formation of spindle poles by chemotaxis. Mol. Biol. Cell. 12: 173a. dynein/dynactin-dependent transport of NuMA. J. Cell Okuda, M., Horn, H.F., Tarapore, P., Tokuyama, Y., Smulian, Biol. 149: 851-861. A.G., Chan, P.K., Knudsen, E.S., Hofmann, IA, Snyder, Michaelis, C., R. Ciosk, and K. Nasmyth. 1997. Cohesins: J.D., Bove, K.E., Fukasawa, K. 2000. Nucleophosmin/ chromosomal proteins that prevent premature separation B23 is a target of CDK2/cyclin E in centrosome of sister chromatids. Cell. 91: 35-45. duplication. Cell. 103: 127-140. Mitchison, T., Evans, L., Schulze, E., Kirschner, M. 1986. Ookata K., Hisanaga, S., Bulinski, J.C., Murofushi, H., Sites of microtubule assembly and disassembly in the Aizawa, H.T. Itoh, J., Hotani, H., Okumura, E., Tachibana, mitotic spindle. Cell. 45: 515-527. K., and Kishimoto, T. 1995. interaction with Mitchison, T.J. 1989. Polewards microtubule flux in the microtubule-associated protein 4 (MAP4) targets p34cdc2 mitotic spindle-evidence from photoactivation of kinase to microtubules and is a potential regulator of M- fluorescence. J. Cell Biol. 109: 637-652. phase microtubule dynamics. J. Cell Biol. 128: 849-862. Mitchison, T.J. and Salmon, E.D. 1992. Poleward Paciotti, V., G. Lucchini, P. Plevani, and M. P. Longhese. kinetochore fiber movement occurs during both 1998. Mec1p is essential for phosphorylation of the yeast metaphase and anaphase-A in newt lung cell mitosis. J. DNA damage checkpoint protein Ddc1p, which physically Cell Biol. 119: 569-582. interacts with Mec3p. EMBO J. 17: 4199-4209. Molina, I., Baars, S., Brill, J.A., Hales, K.G., Fuller, M.T., Panizza, S., T. Tanaka, A. Hochwagen, F. Eisenhaber, and and Ripoll, P. 1997. A chromatin-associated kinesin- K. Nasmyth. 2000. Pds5 cooperates with cohesin in related protein required for normal mitotic chromosome maintaining sister chromatid cohesion. Curr. Biol. 10: segregation in Drosphila. J. Cell Biol. 139: 1361-1371. 1557- 1564. Mountain, V., Simerly, C., Howard, L., Ando, A., Schatten, Perez, L.H., Antonio, C., Flament, S., Vernos, I., and G., Compton, D.A. 1999. The kinesin-related protein, Nebreda, A.R. 2002. Xkid chromokinesin is required for HSET, opposes the activity of Eg5 and cross-links the meiosis I to meiosis II transition in Xenopus laevis microtubules in the mammalian mitotic spindle. J. Cell oocytes. Nat. Cell Biol. 4: 737-742. Biol. 147:351-66. Pfarr, C.M., Coue, M., Grisson, P.M., Hays, T.S., Porter, Nachury, M.V., Maresca, T.J., Salmon, W.C., Waterman- M.E., and McIntosh, J.R. 1990. Cytoplasmic dynein is Storer, C.M., Heald, R., Weis, K. 2001. Importin beta is a localized to kinetochores during mitosis. Nature. 345: 263- mitotic target of the small GTPase Ran in spindle 265. assembly. Cell. 104: 95-106. Piehl, M., Tulu, S., Wadsworth, P., and L. Cassimeris. 2002. Nabeshima, K., Nakagawa, T., Straight, A.F., Murray, A., Functional Maturation of the Centrosome: Cell Cycle Chikashige, Y., Yamashita, Y.M., Hiraoka, Y., Yanagida, Dependent Changes in Microtubule Nucleation. Mol. Biol. M. 1998. Dynamics of during metaphase- Cell. 13: 43a. anaphase transition in fission yeast: Dis1 is implicated in Piehl, M. and Cassimeris, L. 2003. Organization and force balance in metaphase bipolar spindle. Mol. Biol. dynamics of growing microtubule plus ends during early Cell. 9: 3211-3225. mitosis. Mol. Biol. Cell. 14: 916-925. Naiki, T., T. Shimomura, T. Kondo, K. Matsumoto, and K. Piel, M., Nordberg, J., Euteneuer, U. and Bornens, M. 2001. Sugimoto. 2000. Rfc5, in cooperation with rad24, controls Centrosome-dependent exit of cytokinesis in animal cells. Regulated Assembly of the Mitotic Spindle 111

Science. 291: 1550-1553. embryos. Nat. Cell Biol. 1: 51-54. Pihan, G.A., Purohit, A., Wallace, J., Malhotra, R., Liotta, Skibbens, R.V., Skeen, V.P., and Salmon, E. D. 1993. L., Doxsey, S.J. 2001. Centrosome defects can account Directional instability of kinetochore motility during for cellular and genetic changes that characterize prostate chromosome congression and segregation in mitotic newt cancer progression. Cancer Res. 61:2212-9 lung cells: a push-pull mechanism. J. Cell Biol. 122: 859- Popov, A.V., Pozniakovsky, A., Arnal, I., Antony, C., Ashford, 875. A.J., Kinoshita K., Tournebize, R., Hyman, A.A, Karsenti, Skibbens, R.V., Rieder, C.L., Salmon, E.D. 1995. E. 2001. XMAP215 regulates microtubule dynamics Kinetochore motility after severing between sister through two distinct domains. EMBO J. 20: 397-410. centromeres using laser microsurgery: evidence that Popov, A.V., Severin, F., Karsenti, E. 2002. XMAP215 is kinetochore directional instability and position is regulated required for the microtubule-nucleating activity of by tension. J. Cell Sci. 108: 2537-2548. centrosomes. Curr. Biol. 12:1326-30. Skibbens, R. V., L. B. Corson, D. Koshland, and P. Hieter. Rieder, C.L., Davison, E.A., Jensen, L.C.W., Cassimeris, 1999. Ctf7p is essential for sister chromatid cohesion and L., and Salmon, E.D. 1986. Oscillatory Movements of links mitotic chromosome structure to the DNA replication Monooriented Chromosomes and Their Position Relative machinery. Genes Dev. 13: 307-319. to the Spindle Pole Result from the Ejection Properties Sluder, G., and Rieder, C.L. 1985. Experimental separation of the and Half-spindle. J. Cell Biol. 103: 581-591. of pronuclei in fertilized sea urchin eggs: chromosomes Rieder, C.L., Alexander, S.P., and Rupp, G. 1990. do not organize a spindle in the absence of centrosomes. Kinetochores are tranported poleward along a single J. Cell Biol. 100: 897-903. astral microtubule during chromosome attachment to the Synder, J.A. and McIntosh, J.R. 1975. Initiation and growth spindle in newt lung cells. J. Cell Biol. 110: 81-95. of microtubules from mitotic centers in lysed mammalian Rieder, C.L. and Salmon, E.D. 1998. The vertebrate cell cells. J. Cell Biol. 67: 744-760. kinetochore and its roles during mitosis. Trends Cell Biol. Steuer, E.R., Wordeman, L., Schroere, T.A., and Sheetz, 8: 310-318. M.P. 1990. Localization of cytoplasmic dynein to mitotic Ruden, D.M., Cui, W., Sollars, V., and Alterman, M. 1997. spindles and kinetochores. Nature. 345: 266-268. A Drosophila kinesin-like protein, Klp38B, functions during Strunnikov, A. V., V. L. Larionov, and D. Koshland. 1993. meiosis, mitosis, and segmentation. Dev. Biol. 191: 284- SMC1: an essential yeast gene encoding a putative head- 296. rod-tail protein is required for nuclear division and defines Rusan, N.M., Fagerstrom, C.J., Yvon, A-M. C., and a new ubiquitous protein family. J. Cell Biol 123: 1635- Wadsworth, P. 2001. Cell cycle-dependent changes in 1648. microtubule dynamics in living cells expressing green Tarapore, P. and Fukasawa, K. 2002. Loss of p53 and fluorescent protein-alpha tubulin. Mol. Biol. Cell. 12: 971- centrosome hyperamplification. Oncogene. 21: 6234-40. 980. Telzer, B.R., and Rosenbaum, J.L. 1979. Cell cycle- Rusan, N.M., Tulu, U.S., Fagerstrom, C., Wadsworth, P. dependent, in vitro assembly of microtubules onto 2002. Reorganization of the microtubule array in pericentriolar material of HeLa cells. J. Cell Biol. 81: 484- prophase/prometaphase requires cytoplasmic dynein- 97. dependent microtubule transport. J. Cell Biol. 158: 997- Toth, A., R. Ciosk, F. Uhlmann, M. Galova, A. Schleiffer, 1003 and K. Nasmyth. 1999. Yeast cohesin complex requires Salina, D., Bodoor, K., Eckley, D.M., Schroer, T.A., Rattner, a conserved protein, Eco1p(Ctf7), to establish cohesion J.B. and Burke, B. 2002. Cytoplasmic dynein as a between sister chromatids during DNA replication. Genes facilitator of nuclear envelope breakdown. Cell. 108: 97- Dev. 13: 320-333. 107. Tournebize, R., Popov, A., Kinoshita, K., Ashford, A.J., Salmon, E.D. 1989. Microtubule dynamics and Rybina, S., Pozniakovsky, A., Mayer, T.U., Walczak, C.E., chromosome movement. In: Mitosis Molecules and Karsenti, E., and Hyman, A.A. 2000. Control of Mechanisms. J. Hyams and B. Brinkley, eds. Academic microtubule dynamics by the antagonistic activities of Press, Inc. San Diego CA. pp. 119-181. XMAP215 and XKCM1 in Xenopus egg extracts. Nature Saunders, W and MA Hoyt. 1992. Kinesin-related proteins Cell Biol. 2: 13-19. required for structural integrity of the mitotic spindle. Cell. Vasquez, R.J., Gard, D.L. and Cassimeris, L. 1999. 70: 451-458. Phosphorylation by CDK1 regulates XMAP215 function Sawin, K.E., LeFullec, K., Philippe, M. and Mitchison, T.J. in vitro. Cell Motil. Cytoskel. 43: 310-321. 1992. Mitotic spindle organization by a plus-end-directed Vernos, I., Raats J., Hirano T., Heasman J., Karsenti E., microtubule motor. Nature. 359: 540-543. and Wylie C. 1995. Xklp1, a chromosomal Xenopus Saxton, W.M., Stemple, D.L, Leslie, R.J., Salmon, E.D., kinesin-like protein essential for spindle organization and Zavortink, M., McIntosh, J.R. 1984. Tubulin dynamics in chromosome positioning. Cell. 81: 117-127. cultured mammalian cells. J. Cell Biol. 99: 2175-2186. Walczak, C.E., Mitchison, T.J., Desai, A. 1996. XKCM1: A Severin, F., B. Habermann, T. Huffaker and T Hyman. 2001. Xenopus kinesin-related protein that regulates Stu2 promotes mitotic spindle elongation in anaphase. microtubule dynamics during mitotic spindle assembly. J. Cell Biol. 153: 435-442. Cell. 84: 37-47. Sharp, D.J., Yu, K.R., Sisson, J.C., W Sullivan, W. and Walczak, C.E, Vernos, I., Mitchison, T.J., Karsenti, E. and Scholey, J.M. 1999. Antagonistic microtubule-sliding Heald, R. 1998. A model for the proposed roles of different motors position mitotic centrosomes in Drosophila early microtubule-based motor proteins in establishing spindle 112 Cassimeris and Skibbens

bipolarity. Curr. Biol. 8: 903-913. spindle assembly by altering microtubule dynamics and Walczak, C.E., Gan, E.C., Desai, A., Mitchison, T.J. and the balance of motor activities. Nat. Cell Biol. 3: 221-227. Kline-Smith, S.L. 2002. The microtubule-destabilizing Wilson, P.F., Fuller, M.T. and Borisy, G.G. 1997. Monastral kinesin XKCM1 is required for chromosome positioning bipolar spindles: implications for dynamic centrosome during spindle assembly. Curr. Biol. 12: 1885-1889. organization. J. Cell Sci. 110: 451-464. Walker, R.A., O’Brien, E.T., Pryer, N.K., Soboeiro, M.F., Wittmann T, Hyman A, Desai A. 2001. The spindle: a Voter, W.A., Erickson, H.P. and Salmon, E.D. 1988: dynamic assembly of microtubules and motors. Nat. Cell Dynamic instability of individual, MAP-free microtubules Biol. 3: E28-E34. analyzed by video light microscopy: Rate constants and Wordeman, L. and Mitchison, T.J. 1995. Identification and transition frequencies. J. Cell Biol. 107: 1437-1448. partial characterization of mitotic centromere-associated Wang, S.Z. and Adler, R. 1995. Chromokinesin: a DNA- kinesin, a kinesin-related protein that associates with binding, kinesin-like nuclear protein. J. Cell Biol. 128: 761- centromeres during mitosis. J. Cell Biol. 128: 95-105. 768. Yen, T.J., Li, G., Schaar, B.T., Szilak, I., and Cleveland, Wang, Z., I. B. Castano, A. De Las Penas, C. Adams, and D.W. 1992. CENP-E is a putative kinetochore motor that M. F. Christman. 2000. Pol kappa: A DNA polymerase accumulates just before mitosis. Nature. 359: 536-39. required for sister chromatid cohesion. Science. 289: 774- Zeng X, Kahana JA, Silver PA, Morphew MK, McIntosh 779. JR, Fitch IT, Carbon J, Saunders WS. 1999. Slk19p is a Wang, P.J. and Huffaker, T.C. 1997. Stu2p: A microtubule- centromere protein that functions to stabilize mitotic binding protein that is an essential component of the yeast spindles. J. Cell Biol. 146: 415-425. spindle pole body. J. Cell Biol. 139: 1271 – 1280. Zhai Y., Kronebusch, P.J., and Borisy, G.G. 1995. Waterman-Storer, C.M., Desai, A., Bulinski, J.C., and Kinetochore microtubule dynamics and the metaphase- Salmon, E.D. 1998. Fluorescent speckle microscopy, a anaphase transition. J. Cell Biol. 131: 721-724. method to visualize the dynamics of protein assemblies Zhai, Y., Kronebusch, P.J., Simon, P.M., and Borisy, G.G. in living cells. Curr. Biol. 8: 1227-1230. 1996. Microtubule dynamics at the G2/M transition: Abrupt Waters, J.C., Cole, R.W., and Rieder, C.L. 1993. The force- breakdown of cytoplasmic microtubules at nuclear producing mechanism for centrosome separation during envelope breakdown and implications for spindle spindle formation in vertebrates is intrinsic to each aster. morphogenesis. J. Cell Biol. 135: 201 – 214 J. Cell Biol. 122: 361-372. Zhang, D. and Nicklas, R. 1995. The impact of Waters, J.C., Skibbens, R.V., and Salmon, E. D. 1996. chromosomes and centrosomes on spindle assembly as Oscillating mitotic newt lung cell kinetochores are, on observed in living cells. J. Cell Biol. 129: 1287-1300. average, under tension and rarely push. J. Cell Sci. 109: Zhang, C., Hughes, M., Clarke, P.R. 1999. Ran-GTP 2823-2831. stabilises microtubule asters and inhibits nuclear Wiese, C., Wilde, A., Moore, M.S., Adam, S.A., Merdes, assembly in Xenopus egg extracts. J. Cell Sci. 112: 2453- A., and Zheng, Y. 2001. Role of importin-beta in coupling 2461. Ran to downstream targets in microtubule assembly. Zheng, Y., Wong, M.L., Alberts, B., Mitchison, T. 1995. Science. 291: 653-656. Nucleation of microtubule assembly by a gamma-tubulin- Wilde, A. and Zheng, Y. 1999. Stimulation of microtubule containing ring complex. Nature. 378: 578-83. aster formation and spindle assembly by the small Zimmerman, W., Sparks, C.A. and Doxsey, S.J. 1999. GTPase Ran. Science. 284: 1359-1362. Amorphous no longer: the centrosome comes into focus. Wilde, A., Lizarraga, S.B., Zhang, L, Wiese, C., Gliksman, Curr. Opin. Cell Biol. 11: 122-128. N.R., Walczak, C.E. and Zheng, Y. 2001. Ran stimulates Further Reading

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