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Mitosis: Spindle assembly and chromosome motion
Mitosis: spindle assembly chromosome motion Patricia University
and
Wadsworth
of Massachusetts,
Amherst,
Massachusetts,
USA
New studies on mitosis demonstrate the complexity of interactions that contribute to chromosome motion and spindle assembly. Genetic and immunological approaches reveal the requirement for kinesin-related proteins during cell division in diverse cells. Observations of the dynamic behavior of microtubules demonstrate that their disassembly can produce sufficient force to move chromosomes in vitro, that their poleward movement, or flux, contributes to anaphase motion, and that the direction of anaphase motion can be reversed by induction of kinetochore microtubule elongation. Current
Opinion
in Cell Biology 1993, 5:123-128
Introduction
Mitosis is the portion of cell cycle when the previously replicated chromosomes are equipartitioned into two daughter cells. Chromosome segregation is accomplished by the mitotic spindle, which is assembled at the beginning and disassembled at the completion of mitosis. Determining the forces and interactions that regulate spindle morphogenesis from a dynamic array of microtubules (MTs), and elucidating how chromosomes are moved - throughout mitosis - in association with spindle MTs are key areas in current research. The purpose of this review is to focus on recent papers that examine spindle assembly and function during mitosis. For a more complete treatment of mitosis many excellent reviews are available [ l-51. Chromosome
motion during
mitosis
During prophase the interphase MT array is disassembled, and replaced by the shorter and more dynamic MT arrays characteristic of mitosis. These dynamic astral MTs, nucleated from the previously replicated centrosomes, mediate the motion of the centrosomes toward opposites sides of the still intact nuclear envelope, thus forming the two spindle poles. At nuclear envelope breakdown chromosomes are released into this MT-rich environment, initiating spindle formation. In many cases chromosomes initially move toward one spindle pole; with time, however, these unstable mono-oriented chromosomes re-orient until each chromosome achieves a bipolar orientation [ 51. Bipolar-orientated chromosomes are connected to each pole by a kinetochore fiber (k-
fiber), which forms as astral MTs are captured by the kinetochore [ 1,6]. Although not all the MTs of the mature k-fiber extend the entire distance from kinetochore to pole, the fiber nonetheless forms a mechanical link between chromosome and pole [7.1. Bipolar-oriented chromosomes move back and forth along the spindle axis, or congress, until a stable position mid-way between the poles is attained. With time, the magnitude and velocity of these congression motions are greatly reduced; at metaphase, only oscillatory motions of chromosomes toward and away from the poles are observed [ 1,5]. Finally, the chromatids separate and the slow (0.5-2.0 pm/min), relatively synchronous motion of the chromosomes to the poles ensues [2]. Changes in the level of free calcium modulate the rate of anaphase, and the assembly state of spindle MTs in living cells, suggesting that this ion regulates anaphase motion [@I. Additional separation of the chromosomes is achieved by pole-to-pole separation, or anaphase B [9]. Microtubule
assembly and disassembly
Spindle formation and function requires a dynamic array of MTs [10-l 21. Measurements of the rate of forward incorporation of fluorescent tubulin and of the steady state rate of fluorescence recovery after photobleaching reveal an average half-time for non-kinetochore spindle MTs of approximately 30 seconds [10,11,13]; k-fiber MTs turn over more slowly [4,13,14], presumably due to the interaction of the MT plus ends with the kinetochore (but see [ 151). In addition to the dynamic turnover of spindle MTs, k-fiber MTs lengthen and shorten as chromosomes move during prometaphase, metaphase and anaphase
Abbreviations AP oscillation-oscillation k-fiber-kinetochore
from the poles; CEN DNA-centromeric DNA; DI-dynamic fiber; KHC-kinesin heavy chain; KRP-kinesin-related protein; MKLP-mammalian kinase-like protein; MT-microtubule. away
@ Current
Biology
Ltd ISSN 0955674
instability;
123
124
Cytoplasm
and cell motility
[4]. These length changes occur for the’most part by tubulin subunit addition and loss at the kinetochore proximal end of the MTs [16-IS], although subunit loss at the spindle pole (flux) also contributes [ 19,20**].
In pioneering studies of living mitotic cells, moue [ 21,221 postulated that controlled assembly and disassembly of the spindle fibers themselves might provide the force for chromosome motion. Recently, in vitro experiments have revealed that MT assembly and disassembly reactions are in fac? capable of generating force. Two experiments are especially noteworthy. Koshland and co-workers [23] examined the interaction of MTs and isolated chromosomes in z&-o. When MT disassembly was induced by dilution of the tubulin subunit pool, the distance between a marked region on the MT and the kinetochore decreased as MTs disassembled. Thus, a depolymerizing MT can both maintain an attachment to the kinetochore and be moved toward it under these conditions. More recently, MTs of uniform polarity have been nucleated from Tetrdytnena cortices and mixed with chromosomes in z&-o. Again, dilution was used to induce rapid MT shortening, and chromosomes moved to the attached MT end as the MTs disassembled [24*-l. Motion occurred in the absence of detectable levels of ATP, and in the presence of inhibitors of dynein [ 240.1. The discovery that MT disassembly can produce forces sufftcient to move chromosomes in vitro supports theoretical predictions [ 251. However, it should be noted that in both experiments an appreciable fraction of chromosome-MT complexes dissociate or are stabilized upon dilution [23,24**], and the rate of chromosome motion [24**] exceeds that of anaphase in zCo. Additional information concerning the MT-kinetochore interaction is clearly needed to determine how energy derived from polymer assembly/disassembly might be coupled to chromosome translocation in zk,o. The contribution of MT assembly and disassembly to chromosome oscillations in living cells has also been recently examined using high resolution optical tracking methods (RV Skibbens, ED Salmon, abstract, J Cell Biof 1991, 115347). These observations have revealed that chromosomes oscillate at constant velocity toward and away from the poles (AR) [ 31, on monopolar, bipolar and anaphase spindles [ 26,271. Oscillations are characterized by abrupt switches in direction; brief pauses in chromosome motion are also observed. The abrupt and stochastic nature of the transitions is strikingly similar to the dynamic instability (DI) behavior of individual MTs [28], suggesting that k-fiber MTs may undergo coordinated dynamic instability behavior (ED Salmon, personal communication). Although the mechanism(s) that regulate oscillatory behavior are not understood, modification of kinetochore components [4,29] and regulation of k-fiber MT DI behavior [30**,31] may influence the direction of chromosome motion. In addition, because AP motions are observed in cases where no opposing k-fiber is present, the data clearly reveal that both directions of motion can be generated within the half spindle [ 1,26,27]. Finally, analysis of chromosome morphology reveals that AR motion can be accompanied by a deformation of the kinetochore region and therefore is not solely due to ejection forces generated by a dynamic array
of MTs acting along the entire length of the chromosome [ 11. Rather, AR motion is due, at least in part, to forces generated at or near the kinetochore [26,30**]. Changes in chromosome position have also been observed in living cells injected with biotin-labeled tubulin. In these experiments, high, but not low, concentrations of biotin-tubulin induced elongation of k-fiber MTs and the transient reversal of chromosome motion in anaphase cells (30*-l. Immunolocalization of the injected biotin-tubulin demonstrated that the elongation of k-fiber MTs occured by subunit addition at the MT plus ends, proximal to the kinetochore. Chromosome reversal was observed in early to mid-, but not late, anaphase, and deformation of the kinetochore region was also observed. These results suggest that tub&n concentration may regulate the direction, and perhaps rate, of chromosome motion in zpirlo[30**]. This possibility is consistent with MT dynamic instability behavior: both the rate of MT elongation and the frequency of catastrophe depend on tubulin concentration when assayed in zfitro [31]. These observations [30**] reveal that k-fiber MT elongation is intimately coupled to plus end directed chromosome motion; whether polymerization provides the force, activates plus end motors within the kinetochore or performs a combination of these functions remains to be determined.
Poleward
microtubule
flux
An additional feature of spindle MTs, and perhaps MTs in general, has recently been revealed from photoactivation of fluorophores bound to tubulin [ 191. In these experiments, cells are injected with a non-fluorescent caged tubulin, and later this tubulin, incorporated into MTs, is locally photoactivated. The activated regions are bright against a dark background and are therefore more readily detected than regions generated by photobleaching [ 11,13,16]. The results of these experiments demonstrate that a slow poleward motion of the photoactivated region occurs during metaphase and anaphase [ 19,20**]. Flux occurs at approximately 0.5 um/min, slower than the rate of chromosome motion in the same cells [ 20**]. Comparison of anaphase chromosome motion with the motion of the photoactivated regions reveals that during early anaphase 75 % of chromosome-to-pole shortening occurs by loss of subunits proximal to the kinetochore, while the remaining 25 % results from subunit flux. As anaphase progresses, a greater proportion of motion results from flux rather than subunit loss at the kinetochore [ 20**]. Similarly, the incorporation of MTs into the k-fiber 132,331 and the kinetochore proximal incorporation of biotin-tubulin [30**] are also reduced as cells progress through mitosis, revealing time-dependent modification of the kinetochore-MT interaction in lklo. Finally, examination of flux in spindles assembled in htro [34] reveals that flux occurs in the absence of k-fibers and is suppressed by inhibitors of plus end directed motors [35-l. The observation that both flux and subunit loss at the kinetochore occur during mitosis suggests that the fidelity of chromosome segregation may be ensured by the existence of multiple mitotic mechanisms.
Mitosis:
Cytoplasmic
dynein
and mitotic
motions
While MT assembly and disassembly are clearly necessary for spindle formation and function, recent experiments demonstrate that motor enzymes are also required for various aspects of mitosis. Among the candidate motor molecules is cytoplasmic dynein, a complex composed of 400kD heavy chains and subunits of 74, 59, 57, 55, 53 kD; additional associated proteins also co-purify with the dynein complex [36]. Dynein heavy chains bind and hydrolyze ATP, thus providing energy for motile events; the hmction of the other components are less well understood, but probably include binding of dynein to specific subcellular structures and regulation of dynein enzymatic activity [ 2,36,37]. Several approaches are beginning to provide information concerning the contribution of dynein to mitotic motion. For example, during the initial attachment of chromosomes to the spindle during prometaphase, chromosomes are often observed to move rapidly toward the nearest spindle pole. This phenomenon has been directly observed in highly flattened newt lung epithelial cells using correlative light and electron microscopy. These observations reveal that poleward motion (2&50 um/min) occurs via a lateral interaction of the corona region of the kinetochore with a single MT emanating from the spindle pole; motion occurs in the absence of MT disassembly [SS] . The characteristics of particle and chromosome motions in these cells are remarkably similar and consistent with force production by cytoplasmic dynein [39]. This hypothesis is Further strengthened by the observation that anti-dynein antibodies stain the kinetochores in mitotic cells [40,41] and on isolated chromosomes [42]. How might cytoplasmic dynein contribute to other aspects of mitosis? The recent observation that the motor for anaphase is located at or near the kinetochore [29,43,44] is consistent with a role for kinetochoreassociated dynein in anaphase motion. If this is the case, then k-fiber MT disassembly might modulate the rate at which the motor operates [ 11; alternatively, a motor with different kinetic characteristics could be involved [45**]. Dynein may also be involved in generating the pole-directed forces that are detected throughout mitosis [2,5] and a requirement for cytoplasmic dynein in aster assembly and maintenance in cell extracts has recently been demonstrated [46*]. These processes may be mediated by dynein bridging MTs to other MTs, to a spindle matrix, to centrosome components [ 2,7] and/or to membranous elements in the spindle (471, consistent with the immunolocalization of dynein along the k-fibers and at the poles of mitotic cells [40,41]. Kinesin-related
proteins
Despite its attractiveness as a motor for plus end directed motions during mitosis, there is no evidence at present that conventional, kinesin heavy chain (KI-IC) plays a role in chromosome motion: KHC mutants display normal mitosis [48]; KHC is localized to vesicles, including vesicles in the mitotic apparatus, but not to spindle MTs or kinetochores [49,50]; and monoclonal
spindle
assembly
and chromosome
motion
Wadsworth
antibodies to conventional kinesin do not interfere with mitosis in living cells (see Skoufias and Scholey, this issue, pp 95-104). However, analysis of mitotic and meiotic mutants has revealed a superfamily of genes that share significant homology with the motor, but not the tail, domain of KHC ( [ 51-53,54**,55**,56-581; reviewed In [ 591) and participate in the events of mitosis. Recent analysis of several mitotic mutants clearly reveals a role for kinesin-related proteins (KRPs) in spindle assembly. For example, in Rspergillus niduhns bimC and Schizosacchromyces pombe cut7 mutants, spindle assembly is defective [52,53]. In Succharomyces cere vi&e, several KRPs have been identified that appear to play functionally redundant roles: double, but not single mutants, are inviable. Again, the arrested cells contain duplicated but unseparated spindle poles [ 54**,55**]. When the function of these proteins (Cin8p and Kiplp) is eliminated following spindle assembly, the separated poles collapse back to a side-by-side position In the nuclear envelope. In contrast, elimination of function after entry into anaphase has no detectable effect on mitosis [6O**]. Finally, the collapse of the spindle poles could be partially supressed by mutations in yet another kinesin-related gene, KAIU [ 571. Thus, the activity of KRPs is required to effect pole separation; the opposing forces that maintain spindle structure may also be generated by different members of the kinesin superfamily [ bO**] . A role for a Xenopus KRP, Eg5, in the assembly of bipolar spindles in vitro has also recently been demonstrated [61**]. Spindle formation in these extracts is characterized by the initial formation of half spindles, which later fuse to form bipolar spindles. Addition of antibodies to the stalk domain of Eg5, or immunodepletion of Eg5 from extracts, prevents bipolar spindle formation. Instead, half spindles with broad, or splayed, poles were observed; with time these hised to form enlarged aggregates (rosettes); few bipolar spindles were detected. Analysis of the Eg5 protein reveals that it is a slow (2um/min), plus end directed motor. The data suggest that Eg5 functions to maintain the integrity of the spindle pole; however, the similiarity of the Eg5 sequence to KRPs that function in pole separation [52,53,54°*,55m*] further suggests that Eg5 may also mediate interactions between antiparallel MTs [61**] during spindle assembly. Two mammalian KRPs have recently been identiIied using antibodies previously demonstrated to block m&osis in living cells [62**,63**]. One of these proteins, CENP-E, localizes to kinetochores during prometaphase and metaphase and to interzonal MTs in anaphase cells. Pulse-labeling experiments demonstrate that this protein accumulates during Gz-M and is degraded following completion of mitosis [62**]. Mammalian kinesinlike protein 1 (MKLP-I), the antigen recognized by the previously described CHOl antibody, localizes to the interphase nucleus and the interzonal MTs, but not to the kinetochore [63**]. When added to in vitro preparations of MTs nucleated from Tetrabymena cortices [24**], MKLP-1 induces the ATP-sensitive formation of MT bundles between adjacent cortices. Moreover, when axonemes are added to this preparation, the axonemes were observed to move, with their plus ends trail@, to-
125
126
Cytoplasm
and cell motility
ward the plus ends of the nucleated MTs. This is the first demonstration of antiparallel MT sliding mediated by a KRP, although bundle formation by a sea urchin KRP complex had been previously noted [64*]. These data stron@y suggest that MKLP-1 mediates anaphase B spindle elongation; however given that micro-injection of antibodies to MJUP-1 blocks cells in a metaphase-like configuration, a role for this protein in events before anaphase B is also possible. Finally, a protein coAplex, CBF3, that binds centromeric DNA (CENDNA) has been identified from yeast. To ex&nIne *the interaction of this complex with MTs in vitro, biotinylated CENDNA was bound to fluorescent strepavidin beads and then CBF3 was added. The bead-DNAXBF3 complexes were observed to move on taxol-stabilized MTs in z&-o, motion (at an average velocity of 4 timin) was ATP-dependent and minus end directed [45**]. Although the component of the CBF complex responsible for motility is not yet known, the ability of the complex to utilize ATP analogs for motility suggests that it is a minus end directed kinesin-like motor [45-l. Taken together, the range of phenotypes observed in various KRP mutants [ 51-53,540*,55**,57,58], the ability of some KRPs to function as minus end directed motors [65,66], the immunolocalization of KRPs to kinetochores, interzone MTs and spindle poles [61==-63**], the diversity of KRP tail domains [ 54*=,55=~,59,61~*-63**] and the existence of more than one KRP in a given cell type, all strongly suggest that multiple members of this superfamily contribute to the process of mitosis in diverse cells.
ification of MT DI behavior [ 15,311. Finally, kinetochore motors could function in attaching the chromosome to the disassembling k-fiber MTs. In the case of plus end directed motion when chromosomes are attatched endon to the k-fiber MTs, MT assembly and motion must be coordinated because both the motors and chromosome are already at or near the distal end of the k-fiber MTs. Some observations suggest that polymerization may provide the force, but the activation of motors as MTs assemble cannot yet be ruled out [ 26,30==,4,62=*]. Thus, in the special case when chromosomes are attatched end-on to k-fiber MTs, the activity of mitotic motors and MT assembly/disassembly are necessarily coupled; elucidating the contributions of each of these processes to chromosome motion is a key piece in the mitotic puzzle. Acknowledgements The author thanks L Cacsimeris and J Scholey and ED Salmon ported by NSF.
for her comments on this for sharing unpublished
References and recommended Papers review.
. ..
of particular interest, have been highlighted of special interest of outstanding interest
published as:
1.
SAIMON ED: Microtubule ment. In Milaris: Molecules JH and Brinkley BR. New
2.
MCINTOSH
manuscript, data. Sup.
reading
within
the
annual
period
of
Dynamics and Chromosome Moveand Mecbcmistns Edited by Hyams York: Academic Press; 1989:11~181.
JR, PFARR CM:
Mitotic
Motors.
.I Cell
Rio/
1991.
115:577-585.
Conclusions
3.
PICKE-IT~HEAPS 128:63-108.
Experimental investigations continue to provide new, exciting and somewhat unexpected pieces to the puzzle of mitosis. As discussed herein, genetic and immunological approaches provide strong evidence for the contribution of multiple kinesin-related motors to spindle assembly and elongation [ 52,53,54**,55**,6@*-63**]. In addition, cytoplasmic dynein and some KRP motors are located at the kinetochore where they may contribute to chromosome motion [40-42,62=*]. These data suggest that the fidelity of chromosome segregation is achieved by the collective contributions of multiple motor proteins.
4.
MITCHKIN
Finally, it is important to note that where motor proteins have been demonstrated to function in mitosis, the interaction between the motor and the MT is lateral and is apparently not directly influenced by the assembly state of the MT end. For example, the poleward motion of attaching chromosomes in newt cells and the MKIP-l-mediated sliding of antiparallel MTs occur via lateral interactions [38,63**]. What is not yet clear is how motors contribute to chromosome motion when the kinetochore-MT interaction is end-on, and k-fiber length changes are coupled to motion. Under these conditions, minus end directed motors at the kinetochore could provide the force for chromosome-to-pole motion [40-42,45**], and MT disassembly govern motor activity [l]. Alternatively, motors might contribute indirectly, by prompting kdbre MT disassembly [ 251, perhaps by mod-
Function
J: Ceil
Division
in Diatoms.
TJ: in
Microtubule Mitosis. At~nu
RB: Mitosis.
Adv
Rev Cell
Dynamics Cell Biol Biol
1971,
/,?I Ret, QIo/ and 1988,
1991,
Kinetochore 41527-49.
5.
NICKLG
2~225-299.
6.
HA~QEN JH, BO~SER SS, RIEDEH CL: Kinetochores Capture Astral Microtubules During Chromosome Attatchment to the Mitotic Spindle: Direct Visualization in Live Newt Lung Cells. / Cell Biol 1990, 111:1039-1045.
MCDONAID KL. O’Too~e ET, MAS’~-RONARDE DN, MCINTOSH JR: Kinetochore Microtubules in PtK Cells. J Cell Rio/ 1992, 118:369-383. The three.dimensional arrangement of chromosomal libers of PtK cells was reconstnrcted from serial thin sections. The k-fiber MTs were observed to maintain a clustered arrangement; a characteristic spacing between non-k-fiber MTs and k-fiber MTs was not detected, suggesting that interactions between these two sorts of microtubules are weak. 7. .
8.
ZHANC DH, WADSWORTH P, HEI’IER PK: Modulation of Anaphase Spindle Microtubule Structure in Stamen Hair Cells of Trudescuntfa by Calcium and Related Agents. / Cell Sci 1992, 102:79-89. The effect of calcium on spindle microtubules in living cells previously injected with fluorescent tubulin was examined. Injection of calcium at levels previously demonstrated to effect the rate of chromosome motion revealed a distinct decay in spindle fluorescence, supporting the view that calcium ions facilitate anaphase via KMT depolymerlzation. .
9.
HOGAN CJ, CANDE Spindle
1990, 10.
Formation
WZ: Antiparallel and Anaphase
Microtubule B. Cell Moril
Interactions: Qioskeleion
1699-103.
SAXON WM, M, MCINTOSH Cells. J Cell
ST’I~MIXE DL, LESLIE RJ, SI\L~~ON JR: TubuUn Dynamics in Cultured Biol 1984, 99:2175-2186.
ED,
ZAVORTINK Mammalian
Mitosis: 11.
12.
SALVON ED, LESUE RJ, S~XT’ON Spindle Microtubule Dynamics Cell Biol 1984, 99:2165-2174.
WM, in
JORDAN m THROWER D, WIISON Podophyllotoxin and Nocodazole plications for the Role of Microtubule J Cell Sci 1992, 102:401-416.
mow Sea
ML, Urchin
MCINTOSH Embryos.
JR: J
L: Effects of Vinblastine. on Mitotic Spindles: ImDynamics in Mitosis.
13.
WAIXWORTH P, SAIMON ED: Analysis of the Treadmilling Model During Metaphase of Mitosis Using Fluorescence Recovery After Photobleaching. J Cell Riol 1986, 102:1032-1038.
14.
CAWMEIUS L, RI!XXH CL, RLIPI’ G, kIMON ED: Stability crotubule Attatchment to Metaphase Kinetochores Cells. J Cell Sci 1990, 969-15.
15.
HYAIAN AA, Stability by 110:1607-1616.
MITCI-IISON Kinetochores
TJ:
Modulation in Vitro.
J
GORUSKY GJ, SAME 91, Bo~lss GG: ics and Chromosome Motion Visualized Cells. J Cd Biol 1988, 106:1185-1192.
Microtubule in Living
17.
NICKIAS RR: The Motor for Poleward ment in Anaphase is in or Near the Riot 1989, 109:224%2255.
Chromosome Kinetochore.
18.
19.
assembly
and chromosome
motion
Wadsworth
27.
RIEDER CL DAVISON EA, JENSEN LCW, CASSIMERIS L, SALMON Oscillatory Movements of Monooriented Chromosomes their Position Relative to the Spindle Pole Result from Ejection Properties of the Aster and Half-Spindle. J Cell 1986, 103:581-591.
28.
MITCHISON TJ, KIRXXINER tubule Growth. Nafure
29.
HYhlAN Motor Nature
MW: 1984,
AA, MITCHISON TM: Two Activities with Opposite 1991, 35 I:20621 1.
Dynamic 312:237-242. Different Polarities
Instability
127
ED: and the Biol
of Micro-
Microtubule-based in Kinetochores.
SHELDEN E, WADSWORTH P: Microinjection of Biotin-Tubulin into Anaphase Cells Induces Transient Elongation of Kinetochore Microtubules and Reversal of Chromosome-to-pole Motion. J CeN Biol 1992, 116:1409-1420. Anaphase cells were injected with biotin-labeled tubulin to examine the relationship between chromosome motion and MT assembly. At high, but not low, concentrations of injected Nbulin, subunit incorpowtion wds detected at the kinetochore proximal ends of KMTs and was accompanied by a transient reversal of chromosome-to-pole motion. Thus, kinetochores retain the ability to mediate plus end dependent assembly of KMTs and plus end directed chromosome motion in anaphase cells. The results suggest that chromosome motion may be regulated by the concentration of tubulin.
30. ..
of Miin PtKl
of Microtubule Cell Riol 1990,
16.
spindle
DynamAnaphase
MoveJ Cdl
31.
WISE D, CASSl~lElUS L, Rlene~ CL. WADSWORIH P, S/\L\ION ED: Chromosome Fiber Dynamics and Congression Oscillations in Metaphase PtK2 Cells at 23 C. Cell Mofil Qtmkeleton 1991. 18:131-142.
WAU~ER RA, O’BRIEN ET, PRYER NK, SOBEIRO MF. VOTER Wq ERICKSON HP, SALCION ED: Dynamic Instability of Individual Microtubules Analyzed by Video Light Microscopy: Rate Constants and Transition Frequencies. J Cell Eiol 1988, 107~1437-1448.
32.
MITCHISON TJ: Polewards Spindle: Evidence from Cell Biol, 1989 109r637-652.
WADSWORTH P, SHE~DEN E. RUPP G. RIED~R CL: Biotin-Tubulin Incorporates into Kinetochore Fiber Microtubules During Early but not Late Anaphase. J Cell Bioll989, 109~2257-2265.
33.
GORBSKY GJ, BORISY GG: Microtubules Fiber Turnover in Metaphase but Biol 1989, 109:653462.
Microtubule Photoactivation
Flux of
in the Mitotic Fluorescence.
J
20. ..
not
of the Kinetochore in Anaphase. J CeiI
MITCHISON TJ, SAI~\ION ED: Poleward Kinetochore Fiber Movement Occurs During Both Metaphase and Anaphase-A in Newt Lung Cell Mitosis. J Cell Biol 1992, 119569-582. Local photoacdvadon of MT fluorescence was used to examine pole. wards motion or flux in metaphase and anaphase. Comparison of chromosome-to-pole motion and polewards Hux revealed that MT depolymerization at the kinekxhore accounted for 63%. and depolymerization at the pole 37 %, of the rate of chromosome motion. Depolymerization at the pole also occurred in metaphase. The results are significant in that they reveal thar flux occurs throughout mitosis and that more than one mechanism contributes to chromosome motion.
SAWIN KE, MITCHISON TJ: Poleward Microtubule Flux in Spin. dles Assembled in Vitro. J Cell Riot 1991, 112:941-954. Poleward flux of microtubules was examined in spindles assembled in Xejzoplrsextracts using photoactivation methods. Poleward flux was ob.se~ed in these spindles that lack kinetochore fibers, suggesting that flux is a general property of MTs. Further, flux was inhibited by AMP-PNP, but not vanadate. suggesting that kinesin-like motors may contribute.
21.
36.
VAI~I! Based
37.
GILL SR. SCHROER TA, SZIIAK I, S~ELIER ER, actin. A Conserved, Ubiquitously Expressed an Activation of Vesicle Motility Mediated Dynein. J Cell Biol 1991, 115:1639-1650.
38.
RIEDER CL, AIE~ANDIZR SP: Kinetochores are Transported Poleward Along a Single Astral Microtubule During Chromosome Attachment to the Spindle in Newt Lung Cells. J Cell Biol 1990, 1 lO:Sl-95.
39.
AIEXANDER SP, RIEDER CL Chromosome Motion During tatchment to the Vertebrate Spindle: Initial Saltatory-like Behavior of Chromosomes and a Quantitative Analysis Force Production by Nascent Kinetochore Fibers. J Cell 1990, 113:805-815.
lNOr$ S, SATO H: CeU of Molecules. The Nature their Role in Chromosome
Motility by of Mitotic Movement.
Labile Spindle .I Get1
Association Fibrcs and PlyGo/ 1966,
50~259-292. 22.
INOlle
S: CeU
Division
and
the
Mitotic
Spindle.
J CeN
34.
KOSHIAND DE, MIIXHISON TJ. KIKXXNEK MW: mosome Movement Driven by Microtubule tion in Vitro. Nature 1988, 331:499-5Oq.
Rio/
Polewards ChroDepolymeriza-
24.
COLIE M, LOLIHIIJJO VA, MCINTOS~I JR: Microtubule Depolymerization Promotes Particle and Chromosome Movement in Vitro. J Cell Biol 1991, 112:1165-1175. Chromosome motion ~3s examined in an in vitro system consisting of MTs nucleated from 7ii/r&)~f)zetzu cortices and isolated chromosomes. Chromosomes were observed to move to the attached MT ends when MT disassembly wds induced by dilution. Motion occurred when the level of ATP was very low and in the presence of orthovanadate, sug gesting that MT depolymerization provided the free energy for motion. ..
25.
HIU. TL KII~CHNER MW: Bioenergetics and Kinetics tubule and Actin Filament Assembly-Disassembly. Q/o/ 1982, 78:1-125.
26.
BAJER AS: Functional Autonamy of Evidence for Oscillatory Movement 1982, 93:33X!.
Monopolar in Mitosis.
of MicroIn! /&L,
Spindle J CX/
and Riot
in
TJ: Mitotic Spindle Vitro. J Cell Riol
Assembly by Two 1991. 112:925-940.
35.
1981. 91:1315-1475. 23.
SAWIN KE, MITCHISON Different Pathways
RB: Cytoplasmic Motility. Tren&
Dynein: Cell Biol
Advances in 1991, 1:25-29.
Microtubule
SHEETZ M: DynComponent of by Cytoplasmic
40.
PFARR CM, COLIC M, GRISSON PM, HAYS Ts, MCINTOSH JR: Cytoplasmic Dynein is Localized chores During Mitosis. Nafrrre 1990, 345:263-265.
41.
S’I’IXI~R ER, WORDE~MAN L, SCHROER TA, tion of Cytoplasmic Dynein to Mitotic tochores. N&Ire 1930, 3451266268.
42.
WORDELIA~ Subdomains CHO Mitotic
Atof Bid
PORTER ME, to Kineto-
SHEETZ MP: LocalizaSpindles and Kine-
L, STLXIER E. SHEE~Z M. MITCHISON Within the Kinetochore Domain Chromosomes. J Cell Biol 1991,
T: Chemical of Isolated 114:285-294.
128
Cytoplasm
and cell motility
43.
GOIU%Y GJ, SAMMAK PJ, BORING GG: Microtubule ics and Chromosome Motion Visualized in Living Cells. / Cell Biol 1988, 106:1185-1192.
44.
NICK~AS RB: The Motor for Poleward Chromosome ment in Anaphase is in or Near the Kinetochore. Bid 1988, 109~2245-2255.
DynamAnaphase
found to perform overlapping and essential iclentilied as a component of the spindle.
roles in mitosis.
CIN8 W;IS
56.
tiGuaurc R, PARIS J, COIJTTIIRIER A, ROCHI C, PHIUI’PB M: Cloning by Differential Screening of a Xenopus cDNA that Encodes a Kinesin-related Protein. r\!o/ Ce// Biol 1991, 11:3395-3398.
HYMAN M, MIDDLFTON K, CENI-OIA M, MITCHI~ON TJ, CARI~ON J: Microtubule-motor Activity of a Yeast Centromere-binding Protein Complex. Nature 1992, 359:533-536. A complex of proteins, CpF3, that binds to yeast centromeric DNA was found fo link CEN DNA to MTs in vitra Further, the complex contains minus end directed motor activity. While the identity of motor protein in the complex is not yet available, the ability of the complex to utilize ATP analogs suggests that it is kinesin-like.
57.
MBLLII~ PB, ROSE MD: KAR3, A Kinesin-related Gene quired for Yeast Nuclear Fusion. Ceil 1990, 60:102+1041
58.
ZHANG P, KNOWLES BA, GOLDSTEIN ISB, HA~IIY RS: A Kinesin-like Protein Required for Distributive Segregation in Dmsophila. Cell 1990, 62:1053-1062.
59.
BLOOM
46. .
60.
’
MoveJ Ce//
45. ..
VERDE F, B~RREZ J-M, ANIONS C, KNlseh7’1 E: Taxol-induced Asters in Mitotic Extracts of Xenopus Eggs: Requirement for Phosphorylated Factors and Cytoplasmic Dynein. J Ce// Bid 1991. 112:1177-1187. Tax01 wbs used to ‘induce aster assembly in mitotic Xetroplts extracts. A$er formation occurred by a reorganization of MTs in the extract. Aster formation and maintenance required dy&in activity and phosphotylated factors. 47.
HEPIER PK: Membranes in the Mitotic Apparatus. In Mitosis Molecules and Mecbankms Edited by Hyams JS and Brinkley BR New York:
Academic
Press;
1989:241-269.
48.
SAXION WM, HICKS J, f&X.DWilN ISB, GAFF EC: Kinesin Heavy Chain is Essential for Viability and Neuromuscular Functions in Dmsopbilia, but Mutants Show No Defects in Mitosis. Cell 1991, 64:10931102.
49.
WRIGHT BD, HENSON JH, WEDA~IAN KP, WIILY PJ, MORAND JN, ScHOI.IZY JM: Subcellular Localization and Sequence of Sea Urchin Kinesin Heavy Chain: Evidence for its Association with Membranes in the Mitotic Apparatus and Interphase Cytoplasm. J CL?//Biol 1991, 113~817-833.
50.
PFI~TER KK, WAGNER MC, STENOII%N ST, Blu~v ST AND BIXX)M GS: A Battery of Monoclonal Antibodies to Kinesin Heavy and Light Chains Stain Vesicle-like Structures, but not Microtubules in Cultured Cells. J Cell Rio/ 1989. 108:1453-1463.
51.
ENDOW Meiotic
SA, I~ENIKOFF S, SOIEH-NIEDZIELA L: Mediation of and Early Mitotic Chromosome Segregation in Dmsopbila by a Protein Related to Kinesin. Nature 1990, 345:81--83.
52.
ENOS AP, MORRIS RN: Mutation of a Gene Kinesin-like Protein Blocks Nuclear Division Cell 1990, 60:101+1027.
53.
that Encodes a in A nidulans
HAGAN I, YANAGIDA M: Kinesin-related Cut7 Protein ciates with Mitotic and Meiotic Spindles in Fission Nature 1992, 356:74-76.
AssoYeast.
54. ..
ROOF DM, MELUH PB, ROSE MD: Kinesin-related Proteins Required for Assembly of tbe Mitotic Spindle. J Cell Biol 1992, 118:95-108. The existence of multiple kinesin-related proteins in S. cerevisiae is reported. Double mutants for IWO of these genes, KIpl and C/N& are inviable and arrest with duplicated but unseparated spindle poles. lmmunocytochemistry reveals that KIPl is associated with the spindle. These results reveal that these kinesin-related proteins are functionally redundant. HOYT MA, HE L, LOO KK, .. cenwisiue Kinesin-related totic Spindle Assembly. Genetic screens for chromosome to identify kinesin-related genes. work are very similar to [%a*]: 55.
SAUNDERS WS: Two Sacchammyces Gene Products Required for MiJ Cell Biol 1992, 118:109-120. instability in S. cerervkiae were used The results and significance of this CfN8 and K/P1 gene products are
GS: Motor
Proteins
for
Cytoplasmic
Re-
Microtubules.
Curs Opin Cell Biol 1992, 4:66-73. SALIN~~R~ WS, HOYT MA: Kinesin-related Proteins required for Structural Integrity of the Mitotic Spindle. Cc// 1992, 70:451358. Experiments were perfomied to test the role of CINS/KlPl folknving spindle assembly. When hmction was eliminated, the previously sepa. rated poles were drawn together, suggesting that acti\iry is required to resist opposing forces. Loss of hmction of another kinesin-related gene, w, partially suppresscul the spindle collapse, further suggesting that opposing forces are generated hy kinesin-related pro’eins.
..
61. ..
SAU;IN KE, IIGLI~LIIC K, PHII.II~I~~ M, M~~CHISON TJ: Mitotic Spindle Organization by a Plus-end Directed Microtubule Motor. Nature 1992, 359:54Cbj43. The contribution of Eg5, a Xenoplts kinesin related protein, to spindle assembly in vifro was examined. Antibodies to Eg5, or immunodeple tion of Eg5. prevented bipolar spindle formation and disrupted fomied spindles. Eg5 was further demonstrated to be a slow, plus end directed motor irr vitm, 62. ..
YEN TJ. LI G, SCHAAR BT. S%IIAK I, C~~EIAND DW: CENP-E is a Putative Kinetochore Motor that Accumulates Just Before Mitosis. Nature 1992, 359:536-539. CENP-E, previously localized to kinetochores during prometaphase and metaphase and fo interzonal MTs during late anaphase. was identified as a member of the kinesin superfamily. Pulse-labeling experiments reveal thar CENP-E accumulates and is lost in a cell cycle dependent manner. NISI.• W C, LoMniLIX) VA, KLIHN,~~IA R. MCINTOSH JR: A Plusend Directed Motor Enzyme that Moves Antiparallel Microtubules in Vitro Localizes fo the Interzone of Mitotic Spindles. Nature 1992. 359543-547. Identification of the CHOI antigen as MKLP-I. a member of the kinesin superfamily. Demonstrates that MKLP-1 mediates the ATP-sensitive formation of antiparallel MT bundles and the sliding of antipardllel MTs 63. ..
in vilro. 64.
COLE DC. CANDE WZ. B*KIN
RJ, SKOLIFW DA, HOGAN CJ, SCHOIIY JM: Isolation of a Sea Urchin Kinesin-related Protein Using Peptide Antibodies. J Cell Sci 1992, 101:291-301. Polyclonal antibodies to synthetic peptides corresponding to conserved regions of kinesin were used to identify kinesin-related proteins in sea urchin eggs and embryos. The antibodies recognize a complex of three polypeptides that bind to MTs in an ATP-sensitive manner and bundle MTs in c~itro.These results demonstrate that this kinesin.related complex mediates MT-MT interactions in mirotic cells. .
65.
WAU~ER RA, SAL~ION ED, ENDOW SA: The Drosophila Claret Segregation Protein is a Minus End-directed Motor Molecule. Nature 1990, 347~780-782.
66.
MCDONALII like Ned Microtubule
HB, STEWART RJ, GOLDS~:IN LSD: Tbe KinesinProtein of Drosophila is a Minus End-directed Motor. Ce// 1990, 651159-l 165.
P Wadswonh, Department of Biology, Morrill Science Center, of Massachusetts, Amherst, Massachusetts 01003, USA
University
and
Wadsworth
of Massachusetts,
Amherst,
Massachusetts,
USA
New studies on mitosis demonstrate the complexity of interactions that contribute to chromosome motion and spindle assembly. Genetic and immunological approaches reveal the requirement for kinesin-related proteins during cell division in diverse cells. Observations of the dynamic behavior of microtubules demonstrate that their disassembly can produce sufficient force to move chromosomes in vitro, that their poleward movement, or flux, contributes to anaphase motion, and that the direction of anaphase motion can be reversed by induction of kinetochore microtubule elongation. Current
Opinion
in Cell Biology 1993, 5:123-128
Introduction
Mitosis is the portion of cell cycle when the previously replicated chromosomes are equipartitioned into two daughter cells. Chromosome segregation is accomplished by the mitotic spindle, which is assembled at the beginning and disassembled at the completion of mitosis. Determining the forces and interactions that regulate spindle morphogenesis from a dynamic array of microtubules (MTs), and elucidating how chromosomes are moved - throughout mitosis - in association with spindle MTs are key areas in current research. The purpose of this review is to focus on recent papers that examine spindle assembly and function during mitosis. For a more complete treatment of mitosis many excellent reviews are available [ l-51. Chromosome
motion during
mitosis
During prophase the interphase MT array is disassembled, and replaced by the shorter and more dynamic MT arrays characteristic of mitosis. These dynamic astral MTs, nucleated from the previously replicated centrosomes, mediate the motion of the centrosomes toward opposites sides of the still intact nuclear envelope, thus forming the two spindle poles. At nuclear envelope breakdown chromosomes are released into this MT-rich environment, initiating spindle formation. In many cases chromosomes initially move toward one spindle pole; with time, however, these unstable mono-oriented chromosomes re-orient until each chromosome achieves a bipolar orientation [ 51. Bipolar-orientated chromosomes are connected to each pole by a kinetochore fiber (k-
fiber), which forms as astral MTs are captured by the kinetochore [ 1,6]. Although not all the MTs of the mature k-fiber extend the entire distance from kinetochore to pole, the fiber nonetheless forms a mechanical link between chromosome and pole [7.1. Bipolar-oriented chromosomes move back and forth along the spindle axis, or congress, until a stable position mid-way between the poles is attained. With time, the magnitude and velocity of these congression motions are greatly reduced; at metaphase, only oscillatory motions of chromosomes toward and away from the poles are observed [ 1,5]. Finally, the chromatids separate and the slow (0.5-2.0 pm/min), relatively synchronous motion of the chromosomes to the poles ensues [2]. Changes in the level of free calcium modulate the rate of anaphase, and the assembly state of spindle MTs in living cells, suggesting that this ion regulates anaphase motion [@I. Additional separation of the chromosomes is achieved by pole-to-pole separation, or anaphase B [9]. Microtubule
assembly and disassembly
Spindle formation and function requires a dynamic array of MTs [10-l 21. Measurements of the rate of forward incorporation of fluorescent tubulin and of the steady state rate of fluorescence recovery after photobleaching reveal an average half-time for non-kinetochore spindle MTs of approximately 30 seconds [10,11,13]; k-fiber MTs turn over more slowly [4,13,14], presumably due to the interaction of the MT plus ends with the kinetochore (but see [ 151). In addition to the dynamic turnover of spindle MTs, k-fiber MTs lengthen and shorten as chromosomes move during prometaphase, metaphase and anaphase
Abbreviations AP oscillation-oscillation k-fiber-kinetochore
from the poles; CEN DNA-centromeric DNA; DI-dynamic fiber; KHC-kinesin heavy chain; KRP-kinesin-related protein; MKLP-mammalian kinase-like protein; MT-microtubule. away
@ Current
Biology
Ltd ISSN 0955674
instability;
123
124
Cytoplasm
and cell motility
[4]. These length changes occur for the’most part by tubulin subunit addition and loss at the kinetochore proximal end of the MTs [16-IS], although subunit loss at the spindle pole (flux) also contributes [ 19,20**].
In pioneering studies of living mitotic cells, moue [ 21,221 postulated that controlled assembly and disassembly of the spindle fibers themselves might provide the force for chromosome motion. Recently, in vitro experiments have revealed that MT assembly and disassembly reactions are in fac? capable of generating force. Two experiments are especially noteworthy. Koshland and co-workers [23] examined the interaction of MTs and isolated chromosomes in z&-o. When MT disassembly was induced by dilution of the tubulin subunit pool, the distance between a marked region on the MT and the kinetochore decreased as MTs disassembled. Thus, a depolymerizing MT can both maintain an attachment to the kinetochore and be moved toward it under these conditions. More recently, MTs of uniform polarity have been nucleated from Tetrdytnena cortices and mixed with chromosomes in z&-o. Again, dilution was used to induce rapid MT shortening, and chromosomes moved to the attached MT end as the MTs disassembled [24*-l. Motion occurred in the absence of detectable levels of ATP, and in the presence of inhibitors of dynein [ 240.1. The discovery that MT disassembly can produce forces sufftcient to move chromosomes in vitro supports theoretical predictions [ 251. However, it should be noted that in both experiments an appreciable fraction of chromosome-MT complexes dissociate or are stabilized upon dilution [23,24**], and the rate of chromosome motion [24**] exceeds that of anaphase in zCo. Additional information concerning the MT-kinetochore interaction is clearly needed to determine how energy derived from polymer assembly/disassembly might be coupled to chromosome translocation in zk,o. The contribution of MT assembly and disassembly to chromosome oscillations in living cells has also been recently examined using high resolution optical tracking methods (RV Skibbens, ED Salmon, abstract, J Cell Biof 1991, 115347). These observations have revealed that chromosomes oscillate at constant velocity toward and away from the poles (AR) [ 31, on monopolar, bipolar and anaphase spindles [ 26,271. Oscillations are characterized by abrupt switches in direction; brief pauses in chromosome motion are also observed. The abrupt and stochastic nature of the transitions is strikingly similar to the dynamic instability (DI) behavior of individual MTs [28], suggesting that k-fiber MTs may undergo coordinated dynamic instability behavior (ED Salmon, personal communication). Although the mechanism(s) that regulate oscillatory behavior are not understood, modification of kinetochore components [4,29] and regulation of k-fiber MT DI behavior [30**,31] may influence the direction of chromosome motion. In addition, because AP motions are observed in cases where no opposing k-fiber is present, the data clearly reveal that both directions of motion can be generated within the half spindle [ 1,26,27]. Finally, analysis of chromosome morphology reveals that AR motion can be accompanied by a deformation of the kinetochore region and therefore is not solely due to ejection forces generated by a dynamic array
of MTs acting along the entire length of the chromosome [ 11. Rather, AR motion is due, at least in part, to forces generated at or near the kinetochore [26,30**]. Changes in chromosome position have also been observed in living cells injected with biotin-labeled tubulin. In these experiments, high, but not low, concentrations of biotin-tubulin induced elongation of k-fiber MTs and the transient reversal of chromosome motion in anaphase cells (30*-l. Immunolocalization of the injected biotin-tubulin demonstrated that the elongation of k-fiber MTs occured by subunit addition at the MT plus ends, proximal to the kinetochore. Chromosome reversal was observed in early to mid-, but not late, anaphase, and deformation of the kinetochore region was also observed. These results suggest that tub&n concentration may regulate the direction, and perhaps rate, of chromosome motion in zpirlo[30**]. This possibility is consistent with MT dynamic instability behavior: both the rate of MT elongation and the frequency of catastrophe depend on tubulin concentration when assayed in zfitro [31]. These observations [30**] reveal that k-fiber MT elongation is intimately coupled to plus end directed chromosome motion; whether polymerization provides the force, activates plus end motors within the kinetochore or performs a combination of these functions remains to be determined.
Poleward
microtubule
flux
An additional feature of spindle MTs, and perhaps MTs in general, has recently been revealed from photoactivation of fluorophores bound to tubulin [ 191. In these experiments, cells are injected with a non-fluorescent caged tubulin, and later this tubulin, incorporated into MTs, is locally photoactivated. The activated regions are bright against a dark background and are therefore more readily detected than regions generated by photobleaching [ 11,13,16]. The results of these experiments demonstrate that a slow poleward motion of the photoactivated region occurs during metaphase and anaphase [ 19,20**]. Flux occurs at approximately 0.5 um/min, slower than the rate of chromosome motion in the same cells [ 20**]. Comparison of anaphase chromosome motion with the motion of the photoactivated regions reveals that during early anaphase 75 % of chromosome-to-pole shortening occurs by loss of subunits proximal to the kinetochore, while the remaining 25 % results from subunit flux. As anaphase progresses, a greater proportion of motion results from flux rather than subunit loss at the kinetochore [ 20**]. Similarly, the incorporation of MTs into the k-fiber 132,331 and the kinetochore proximal incorporation of biotin-tubulin [30**] are also reduced as cells progress through mitosis, revealing time-dependent modification of the kinetochore-MT interaction in lklo. Finally, examination of flux in spindles assembled in htro [34] reveals that flux occurs in the absence of k-fibers and is suppressed by inhibitors of plus end directed motors [35-l. The observation that both flux and subunit loss at the kinetochore occur during mitosis suggests that the fidelity of chromosome segregation may be ensured by the existence of multiple mitotic mechanisms.
Mitosis:
Cytoplasmic
dynein
and mitotic
motions
While MT assembly and disassembly are clearly necessary for spindle formation and function, recent experiments demonstrate that motor enzymes are also required for various aspects of mitosis. Among the candidate motor molecules is cytoplasmic dynein, a complex composed of 400kD heavy chains and subunits of 74, 59, 57, 55, 53 kD; additional associated proteins also co-purify with the dynein complex [36]. Dynein heavy chains bind and hydrolyze ATP, thus providing energy for motile events; the hmction of the other components are less well understood, but probably include binding of dynein to specific subcellular structures and regulation of dynein enzymatic activity [ 2,36,37]. Several approaches are beginning to provide information concerning the contribution of dynein to mitotic motion. For example, during the initial attachment of chromosomes to the spindle during prometaphase, chromosomes are often observed to move rapidly toward the nearest spindle pole. This phenomenon has been directly observed in highly flattened newt lung epithelial cells using correlative light and electron microscopy. These observations reveal that poleward motion (2&50 um/min) occurs via a lateral interaction of the corona region of the kinetochore with a single MT emanating from the spindle pole; motion occurs in the absence of MT disassembly [SS] . The characteristics of particle and chromosome motions in these cells are remarkably similar and consistent with force production by cytoplasmic dynein [39]. This hypothesis is Further strengthened by the observation that anti-dynein antibodies stain the kinetochores in mitotic cells [40,41] and on isolated chromosomes [42]. How might cytoplasmic dynein contribute to other aspects of mitosis? The recent observation that the motor for anaphase is located at or near the kinetochore [29,43,44] is consistent with a role for kinetochoreassociated dynein in anaphase motion. If this is the case, then k-fiber MT disassembly might modulate the rate at which the motor operates [ 11; alternatively, a motor with different kinetic characteristics could be involved [45**]. Dynein may also be involved in generating the pole-directed forces that are detected throughout mitosis [2,5] and a requirement for cytoplasmic dynein in aster assembly and maintenance in cell extracts has recently been demonstrated [46*]. These processes may be mediated by dynein bridging MTs to other MTs, to a spindle matrix, to centrosome components [ 2,7] and/or to membranous elements in the spindle (471, consistent with the immunolocalization of dynein along the k-fibers and at the poles of mitotic cells [40,41]. Kinesin-related
proteins
Despite its attractiveness as a motor for plus end directed motions during mitosis, there is no evidence at present that conventional, kinesin heavy chain (KI-IC) plays a role in chromosome motion: KHC mutants display normal mitosis [48]; KHC is localized to vesicles, including vesicles in the mitotic apparatus, but not to spindle MTs or kinetochores [49,50]; and monoclonal
spindle
assembly
and chromosome
motion
Wadsworth
antibodies to conventional kinesin do not interfere with mitosis in living cells (see Skoufias and Scholey, this issue, pp 95-104). However, analysis of mitotic and meiotic mutants has revealed a superfamily of genes that share significant homology with the motor, but not the tail, domain of KHC ( [ 51-53,54**,55**,56-581; reviewed In [ 591) and participate in the events of mitosis. Recent analysis of several mitotic mutants clearly reveals a role for kinesin-related proteins (KRPs) in spindle assembly. For example, in Rspergillus niduhns bimC and Schizosacchromyces pombe cut7 mutants, spindle assembly is defective [52,53]. In Succharomyces cere vi&e, several KRPs have been identified that appear to play functionally redundant roles: double, but not single mutants, are inviable. Again, the arrested cells contain duplicated but unseparated spindle poles [ 54**,55**]. When the function of these proteins (Cin8p and Kiplp) is eliminated following spindle assembly, the separated poles collapse back to a side-by-side position In the nuclear envelope. In contrast, elimination of function after entry into anaphase has no detectable effect on mitosis [6O**]. Finally, the collapse of the spindle poles could be partially supressed by mutations in yet another kinesin-related gene, KAIU [ 571. Thus, the activity of KRPs is required to effect pole separation; the opposing forces that maintain spindle structure may also be generated by different members of the kinesin superfamily [ bO**] . A role for a Xenopus KRP, Eg5, in the assembly of bipolar spindles in vitro has also recently been demonstrated [61**]. Spindle formation in these extracts is characterized by the initial formation of half spindles, which later fuse to form bipolar spindles. Addition of antibodies to the stalk domain of Eg5, or immunodepletion of Eg5 from extracts, prevents bipolar spindle formation. Instead, half spindles with broad, or splayed, poles were observed; with time these hised to form enlarged aggregates (rosettes); few bipolar spindles were detected. Analysis of the Eg5 protein reveals that it is a slow (2um/min), plus end directed motor. The data suggest that Eg5 functions to maintain the integrity of the spindle pole; however, the similiarity of the Eg5 sequence to KRPs that function in pole separation [52,53,54°*,55m*] further suggests that Eg5 may also mediate interactions between antiparallel MTs [61**] during spindle assembly. Two mammalian KRPs have recently been identiIied using antibodies previously demonstrated to block m&osis in living cells [62**,63**]. One of these proteins, CENP-E, localizes to kinetochores during prometaphase and metaphase and to interzonal MTs in anaphase cells. Pulse-labeling experiments demonstrate that this protein accumulates during Gz-M and is degraded following completion of mitosis [62**]. Mammalian kinesinlike protein 1 (MKLP-I), the antigen recognized by the previously described CHOl antibody, localizes to the interphase nucleus and the interzonal MTs, but not to the kinetochore [63**]. When added to in vitro preparations of MTs nucleated from Tetrabymena cortices [24**], MKLP-1 induces the ATP-sensitive formation of MT bundles between adjacent cortices. Moreover, when axonemes are added to this preparation, the axonemes were observed to move, with their plus ends trail@, to-
125
126
Cytoplasm
and cell motility
ward the plus ends of the nucleated MTs. This is the first demonstration of antiparallel MT sliding mediated by a KRP, although bundle formation by a sea urchin KRP complex had been previously noted [64*]. These data stron@y suggest that MKLP-1 mediates anaphase B spindle elongation; however given that micro-injection of antibodies to MJUP-1 blocks cells in a metaphase-like configuration, a role for this protein in events before anaphase B is also possible. Finally, a protein coAplex, CBF3, that binds centromeric DNA (CENDNA) has been identified from yeast. To ex&nIne *the interaction of this complex with MTs in vitro, biotinylated CENDNA was bound to fluorescent strepavidin beads and then CBF3 was added. The bead-DNAXBF3 complexes were observed to move on taxol-stabilized MTs in z&-o, motion (at an average velocity of 4 timin) was ATP-dependent and minus end directed [45**]. Although the component of the CBF complex responsible for motility is not yet known, the ability of the complex to utilize ATP analogs for motility suggests that it is a minus end directed kinesin-like motor [45-l. Taken together, the range of phenotypes observed in various KRP mutants [ 51-53,540*,55**,57,58], the ability of some KRPs to function as minus end directed motors [65,66], the immunolocalization of KRPs to kinetochores, interzone MTs and spindle poles [61==-63**], the diversity of KRP tail domains [ 54*=,55=~,59,61~*-63**] and the existence of more than one KRP in a given cell type, all strongly suggest that multiple members of this superfamily contribute to the process of mitosis in diverse cells.
ification of MT DI behavior [ 15,311. Finally, kinetochore motors could function in attaching the chromosome to the disassembling k-fiber MTs. In the case of plus end directed motion when chromosomes are attatched endon to the k-fiber MTs, MT assembly and motion must be coordinated because both the motors and chromosome are already at or near the distal end of the k-fiber MTs. Some observations suggest that polymerization may provide the force, but the activation of motors as MTs assemble cannot yet be ruled out [ 26,30==,4,62=*]. Thus, in the special case when chromosomes are attatched end-on to k-fiber MTs, the activity of mitotic motors and MT assembly/disassembly are necessarily coupled; elucidating the contributions of each of these processes to chromosome motion is a key piece in the mitotic puzzle. Acknowledgements The author thanks L Cacsimeris and J Scholey and ED Salmon ported by NSF.
for her comments on this for sharing unpublished
References and recommended Papers review.
. ..
of particular interest, have been highlighted of special interest of outstanding interest
published as:
1.
SAIMON ED: Microtubule ment. In Milaris: Molecules JH and Brinkley BR. New
2.
MCINTOSH
manuscript, data. Sup.
reading
within
the
annual
period
of
Dynamics and Chromosome Moveand Mecbcmistns Edited by Hyams York: Academic Press; 1989:11~181.
JR, PFARR CM:
Mitotic
Motors.
.I Cell
Rio/
1991.
115:577-585.
Conclusions
3.
PICKE-IT~HEAPS 128:63-108.
Experimental investigations continue to provide new, exciting and somewhat unexpected pieces to the puzzle of mitosis. As discussed herein, genetic and immunological approaches provide strong evidence for the contribution of multiple kinesin-related motors to spindle assembly and elongation [ 52,53,54**,55**,6@*-63**]. In addition, cytoplasmic dynein and some KRP motors are located at the kinetochore where they may contribute to chromosome motion [40-42,62=*]. These data suggest that the fidelity of chromosome segregation is achieved by the collective contributions of multiple motor proteins.
4.
MITCHKIN
Finally, it is important to note that where motor proteins have been demonstrated to function in mitosis, the interaction between the motor and the MT is lateral and is apparently not directly influenced by the assembly state of the MT end. For example, the poleward motion of attaching chromosomes in newt cells and the MKIP-l-mediated sliding of antiparallel MTs occur via lateral interactions [38,63**]. What is not yet clear is how motors contribute to chromosome motion when the kinetochore-MT interaction is end-on, and k-fiber length changes are coupled to motion. Under these conditions, minus end directed motors at the kinetochore could provide the force for chromosome-to-pole motion [40-42,45**], and MT disassembly govern motor activity [l]. Alternatively, motors might contribute indirectly, by prompting kdbre MT disassembly [ 251, perhaps by mod-
Function
J: Ceil
Division
in Diatoms.
TJ: in
Microtubule Mitosis. At~nu
RB: Mitosis.
Adv
Rev Cell
Dynamics Cell Biol Biol
1971,
/,?I Ret, QIo/ and 1988,
1991,
Kinetochore 41527-49.
5.
NICKLG
2~225-299.
6.
HA~QEN JH, BO~SER SS, RIEDEH CL: Kinetochores Capture Astral Microtubules During Chromosome Attatchment to the Mitotic Spindle: Direct Visualization in Live Newt Lung Cells. / Cell Biol 1990, 111:1039-1045.
MCDONAID KL. O’Too~e ET, MAS’~-RONARDE DN, MCINTOSH JR: Kinetochore Microtubules in PtK Cells. J Cell Rio/ 1992, 118:369-383. The three.dimensional arrangement of chromosomal libers of PtK cells was reconstnrcted from serial thin sections. The k-fiber MTs were observed to maintain a clustered arrangement; a characteristic spacing between non-k-fiber MTs and k-fiber MTs was not detected, suggesting that interactions between these two sorts of microtubules are weak. 7. .
8.
ZHANC DH, WADSWORTH P, HEI’IER PK: Modulation of Anaphase Spindle Microtubule Structure in Stamen Hair Cells of Trudescuntfa by Calcium and Related Agents. / Cell Sci 1992, 102:79-89. The effect of calcium on spindle microtubules in living cells previously injected with fluorescent tubulin was examined. Injection of calcium at levels previously demonstrated to effect the rate of chromosome motion revealed a distinct decay in spindle fluorescence, supporting the view that calcium ions facilitate anaphase via KMT depolymerlzation. .
9.
HOGAN CJ, CANDE Spindle
1990, 10.
Formation
WZ: Antiparallel and Anaphase
Microtubule B. Cell Moril
Interactions: Qioskeleion
1699-103.
SAXON WM, M, MCINTOSH Cells. J Cell
ST’I~MIXE DL, LESLIE RJ, SI\L~~ON JR: TubuUn Dynamics in Cultured Biol 1984, 99:2175-2186.
ED,
ZAVORTINK Mammalian
Mitosis: 11.
12.
SALVON ED, LESUE RJ, S~XT’ON Spindle Microtubule Dynamics Cell Biol 1984, 99:2165-2174.
WM, in
JORDAN m THROWER D, WIISON Podophyllotoxin and Nocodazole plications for the Role of Microtubule J Cell Sci 1992, 102:401-416.
mow Sea
ML, Urchin
MCINTOSH Embryos.
JR: J
L: Effects of Vinblastine. on Mitotic Spindles: ImDynamics in Mitosis.
13.
WAIXWORTH P, SAIMON ED: Analysis of the Treadmilling Model During Metaphase of Mitosis Using Fluorescence Recovery After Photobleaching. J Cell Riol 1986, 102:1032-1038.
14.
CAWMEIUS L, RI!XXH CL, RLIPI’ G, kIMON ED: Stability crotubule Attatchment to Metaphase Kinetochores Cells. J Cell Sci 1990, 969-15.
15.
HYAIAN AA, Stability by 110:1607-1616.
MITCI-IISON Kinetochores
TJ:
Modulation in Vitro.
J
GORUSKY GJ, SAME 91, Bo~lss GG: ics and Chromosome Motion Visualized Cells. J Cd Biol 1988, 106:1185-1192.
Microtubule in Living
17.
NICKIAS RR: The Motor for Poleward ment in Anaphase is in or Near the Riot 1989, 109:224%2255.
Chromosome Kinetochore.
18.
19.
assembly
and chromosome
motion
Wadsworth
27.
RIEDER CL DAVISON EA, JENSEN LCW, CASSIMERIS L, SALMON Oscillatory Movements of Monooriented Chromosomes their Position Relative to the Spindle Pole Result from Ejection Properties of the Aster and Half-Spindle. J Cell 1986, 103:581-591.
28.
MITCHISON TJ, KIRXXINER tubule Growth. Nafure
29.
HYhlAN Motor Nature
MW: 1984,
AA, MITCHISON TM: Two Activities with Opposite 1991, 35 I:20621 1.
Dynamic 312:237-242. Different Polarities
Instability
127
ED: and the Biol
of Micro-
Microtubule-based in Kinetochores.
SHELDEN E, WADSWORTH P: Microinjection of Biotin-Tubulin into Anaphase Cells Induces Transient Elongation of Kinetochore Microtubules and Reversal of Chromosome-to-pole Motion. J CeN Biol 1992, 116:1409-1420. Anaphase cells were injected with biotin-labeled tubulin to examine the relationship between chromosome motion and MT assembly. At high, but not low, concentrations of injected Nbulin, subunit incorpowtion wds detected at the kinetochore proximal ends of KMTs and was accompanied by a transient reversal of chromosome-to-pole motion. Thus, kinetochores retain the ability to mediate plus end dependent assembly of KMTs and plus end directed chromosome motion in anaphase cells. The results suggest that chromosome motion may be regulated by the concentration of tubulin.
30. ..
of Miin PtKl
of Microtubule Cell Riol 1990,
16.
spindle
DynamAnaphase
MoveJ Cdl
31.
WISE D, CASSl~lElUS L, Rlene~ CL. WADSWORIH P, S/\L\ION ED: Chromosome Fiber Dynamics and Congression Oscillations in Metaphase PtK2 Cells at 23 C. Cell Mofil Qtmkeleton 1991. 18:131-142.
WAU~ER RA, O’BRIEN ET, PRYER NK, SOBEIRO MF. VOTER Wq ERICKSON HP, SALCION ED: Dynamic Instability of Individual Microtubules Analyzed by Video Light Microscopy: Rate Constants and Transition Frequencies. J Cell Eiol 1988, 107~1437-1448.
32.
MITCHISON TJ: Polewards Spindle: Evidence from Cell Biol, 1989 109r637-652.
WADSWORTH P, SHE~DEN E. RUPP G. RIED~R CL: Biotin-Tubulin Incorporates into Kinetochore Fiber Microtubules During Early but not Late Anaphase. J Cell Bioll989, 109~2257-2265.
33.
GORBSKY GJ, BORISY GG: Microtubules Fiber Turnover in Metaphase but Biol 1989, 109:653462.
Microtubule Photoactivation
Flux of
in the Mitotic Fluorescence.
J
20. ..
not
of the Kinetochore in Anaphase. J CeiI
MITCHISON TJ, SAI~\ION ED: Poleward Kinetochore Fiber Movement Occurs During Both Metaphase and Anaphase-A in Newt Lung Cell Mitosis. J Cell Biol 1992, 119569-582. Local photoacdvadon of MT fluorescence was used to examine pole. wards motion or flux in metaphase and anaphase. Comparison of chromosome-to-pole motion and polewards Hux revealed that MT depolymerization at the kinekxhore accounted for 63%. and depolymerization at the pole 37 %, of the rate of chromosome motion. Depolymerization at the pole also occurred in metaphase. The results are significant in that they reveal thar flux occurs throughout mitosis and that more than one mechanism contributes to chromosome motion.
SAWIN KE, MITCHISON TJ: Poleward Microtubule Flux in Spin. dles Assembled in Vitro. J Cell Riot 1991, 112:941-954. Poleward flux of microtubules was examined in spindles assembled in Xejzoplrsextracts using photoactivation methods. Poleward flux was ob.se~ed in these spindles that lack kinetochore fibers, suggesting that flux is a general property of MTs. Further, flux was inhibited by AMP-PNP, but not vanadate. suggesting that kinesin-like motors may contribute.
21.
36.
VAI~I! Based
37.
GILL SR. SCHROER TA, SZIIAK I, S~ELIER ER, actin. A Conserved, Ubiquitously Expressed an Activation of Vesicle Motility Mediated Dynein. J Cell Biol 1991, 115:1639-1650.
38.
RIEDER CL, AIE~ANDIZR SP: Kinetochores are Transported Poleward Along a Single Astral Microtubule During Chromosome Attachment to the Spindle in Newt Lung Cells. J Cell Biol 1990, 1 lO:Sl-95.
39.
AIEXANDER SP, RIEDER CL Chromosome Motion During tatchment to the Vertebrate Spindle: Initial Saltatory-like Behavior of Chromosomes and a Quantitative Analysis Force Production by Nascent Kinetochore Fibers. J Cell 1990, 113:805-815.
lNOr$ S, SATO H: CeU of Molecules. The Nature their Role in Chromosome
Motility by of Mitotic Movement.
Labile Spindle .I Get1
Association Fibrcs and PlyGo/ 1966,
50~259-292. 22.
INOlle
S: CeU
Division
and
the
Mitotic
Spindle.
J CeN
34.
KOSHIAND DE, MIIXHISON TJ. KIKXXNEK MW: mosome Movement Driven by Microtubule tion in Vitro. Nature 1988, 331:499-5Oq.
Rio/
Polewards ChroDepolymeriza-
24.
COLIE M, LOLIHIIJJO VA, MCINTOS~I JR: Microtubule Depolymerization Promotes Particle and Chromosome Movement in Vitro. J Cell Biol 1991, 112:1165-1175. Chromosome motion ~3s examined in an in vitro system consisting of MTs nucleated from 7ii/r&)~f)zetzu cortices and isolated chromosomes. Chromosomes were observed to move to the attached MT ends when MT disassembly wds induced by dilution. Motion occurred when the level of ATP was very low and in the presence of orthovanadate, sug gesting that MT depolymerization provided the free energy for motion. ..
25.
HIU. TL KII~CHNER MW: Bioenergetics and Kinetics tubule and Actin Filament Assembly-Disassembly. Q/o/ 1982, 78:1-125.
26.
BAJER AS: Functional Autonamy of Evidence for Oscillatory Movement 1982, 93:33X!.
Monopolar in Mitosis.
of MicroIn! /&L,
Spindle J CX/
and Riot
in
TJ: Mitotic Spindle Vitro. J Cell Riol
Assembly by Two 1991. 112:925-940.
35.
1981. 91:1315-1475. 23.
SAWIN KE, MITCHISON Different Pathways
RB: Cytoplasmic Motility. Tren&
Dynein: Cell Biol
Advances in 1991, 1:25-29.
Microtubule
SHEETZ M: DynComponent of by Cytoplasmic
40.
PFARR CM, COLIC M, GRISSON PM, HAYS Ts, MCINTOSH JR: Cytoplasmic Dynein is Localized chores During Mitosis. Nafrrre 1990, 345:263-265.
41.
S’I’IXI~R ER, WORDE~MAN L, SCHROER TA, tion of Cytoplasmic Dynein to Mitotic tochores. N&Ire 1930, 3451266268.
42.
WORDELIA~ Subdomains CHO Mitotic
Atof Bid
PORTER ME, to Kineto-
SHEETZ MP: LocalizaSpindles and Kine-
L, STLXIER E. SHEE~Z M. MITCHISON Within the Kinetochore Domain Chromosomes. J Cell Biol 1991,
T: Chemical of Isolated 114:285-294.
128
Cytoplasm
and cell motility
43.
GOIU%Y GJ, SAMMAK PJ, BORING GG: Microtubule ics and Chromosome Motion Visualized in Living Cells. / Cell Biol 1988, 106:1185-1192.
44.
NICK~AS RB: The Motor for Poleward Chromosome ment in Anaphase is in or Near the Kinetochore. Bid 1988, 109~2245-2255.
DynamAnaphase
found to perform overlapping and essential iclentilied as a component of the spindle.
roles in mitosis.
CIN8 W;IS
56.
tiGuaurc R, PARIS J, COIJTTIIRIER A, ROCHI C, PHIUI’PB M: Cloning by Differential Screening of a Xenopus cDNA that Encodes a Kinesin-related Protein. r\!o/ Ce// Biol 1991, 11:3395-3398.
HYMAN M, MIDDLFTON K, CENI-OIA M, MITCHI~ON TJ, CARI~ON J: Microtubule-motor Activity of a Yeast Centromere-binding Protein Complex. Nature 1992, 359:533-536. A complex of proteins, CpF3, that binds to yeast centromeric DNA was found fo link CEN DNA to MTs in vitra Further, the complex contains minus end directed motor activity. While the identity of motor protein in the complex is not yet available, the ability of the complex to utilize ATP analogs suggests that it is kinesin-like.
57.
MBLLII~ PB, ROSE MD: KAR3, A Kinesin-related Gene quired for Yeast Nuclear Fusion. Ceil 1990, 60:102+1041
58.
ZHANG P, KNOWLES BA, GOLDSTEIN ISB, HA~IIY RS: A Kinesin-like Protein Required for Distributive Segregation in Dmsophila. Cell 1990, 62:1053-1062.
59.
BLOOM
46. .
60.
’
MoveJ Ce//
45. ..
VERDE F, B~RREZ J-M, ANIONS C, KNlseh7’1 E: Taxol-induced Asters in Mitotic Extracts of Xenopus Eggs: Requirement for Phosphorylated Factors and Cytoplasmic Dynein. J Ce// Bid 1991. 112:1177-1187. Tax01 wbs used to ‘induce aster assembly in mitotic Xetroplts extracts. A$er formation occurred by a reorganization of MTs in the extract. Aster formation and maintenance required dy&in activity and phosphotylated factors. 47.
HEPIER PK: Membranes in the Mitotic Apparatus. In Mitosis Molecules and Mecbankms Edited by Hyams JS and Brinkley BR New York:
Academic
Press;
1989:241-269.
48.
SAXION WM, HICKS J, f&X.DWilN ISB, GAFF EC: Kinesin Heavy Chain is Essential for Viability and Neuromuscular Functions in Dmsopbilia, but Mutants Show No Defects in Mitosis. Cell 1991, 64:10931102.
49.
WRIGHT BD, HENSON JH, WEDA~IAN KP, WIILY PJ, MORAND JN, ScHOI.IZY JM: Subcellular Localization and Sequence of Sea Urchin Kinesin Heavy Chain: Evidence for its Association with Membranes in the Mitotic Apparatus and Interphase Cytoplasm. J CL?//Biol 1991, 113~817-833.
50.
PFI~TER KK, WAGNER MC, STENOII%N ST, Blu~v ST AND BIXX)M GS: A Battery of Monoclonal Antibodies to Kinesin Heavy and Light Chains Stain Vesicle-like Structures, but not Microtubules in Cultured Cells. J Cell Rio/ 1989. 108:1453-1463.
51.
ENDOW Meiotic
SA, I~ENIKOFF S, SOIEH-NIEDZIELA L: Mediation of and Early Mitotic Chromosome Segregation in Dmsopbila by a Protein Related to Kinesin. Nature 1990, 345:81--83.
52.
ENOS AP, MORRIS RN: Mutation of a Gene Kinesin-like Protein Blocks Nuclear Division Cell 1990, 60:101+1027.
53.
that Encodes a in A nidulans
HAGAN I, YANAGIDA M: Kinesin-related Cut7 Protein ciates with Mitotic and Meiotic Spindles in Fission Nature 1992, 356:74-76.
AssoYeast.
54. ..
ROOF DM, MELUH PB, ROSE MD: Kinesin-related Proteins Required for Assembly of tbe Mitotic Spindle. J Cell Biol 1992, 118:95-108. The existence of multiple kinesin-related proteins in S. cerevisiae is reported. Double mutants for IWO of these genes, KIpl and C/N& are inviable and arrest with duplicated but unseparated spindle poles. lmmunocytochemistry reveals that KIPl is associated with the spindle. These results reveal that these kinesin-related proteins are functionally redundant. HOYT MA, HE L, LOO KK, .. cenwisiue Kinesin-related totic Spindle Assembly. Genetic screens for chromosome to identify kinesin-related genes. work are very similar to [%a*]: 55.
SAUNDERS WS: Two Sacchammyces Gene Products Required for MiJ Cell Biol 1992, 118:109-120. instability in S. cerervkiae were used The results and significance of this CfN8 and K/P1 gene products are
GS: Motor
Proteins
for
Cytoplasmic
Re-
Microtubules.
Curs Opin Cell Biol 1992, 4:66-73. SALIN~~R~ WS, HOYT MA: Kinesin-related Proteins required for Structural Integrity of the Mitotic Spindle. Cc// 1992, 70:451358. Experiments were perfomied to test the role of CINS/KlPl folknving spindle assembly. When hmction was eliminated, the previously sepa. rated poles were drawn together, suggesting that acti\iry is required to resist opposing forces. Loss of hmction of another kinesin-related gene, w, partially suppresscul the spindle collapse, further suggesting that opposing forces are generated hy kinesin-related pro’eins.
..
61. ..
SAU;IN KE, IIGLI~LIIC K, PHII.II~I~~ M, M~~CHISON TJ: Mitotic Spindle Organization by a Plus-end Directed Microtubule Motor. Nature 1992, 359:54Cbj43. The contribution of Eg5, a Xenoplts kinesin related protein, to spindle assembly in vifro was examined. Antibodies to Eg5, or immunodeple tion of Eg5. prevented bipolar spindle formation and disrupted fomied spindles. Eg5 was further demonstrated to be a slow, plus end directed motor irr vitm, 62. ..
YEN TJ. LI G, SCHAAR BT. S%IIAK I, C~~EIAND DW: CENP-E is a Putative Kinetochore Motor that Accumulates Just Before Mitosis. Nature 1992, 359:536-539. CENP-E, previously localized to kinetochores during prometaphase and metaphase and fo interzonal MTs during late anaphase. was identified as a member of the kinesin superfamily. Pulse-labeling experiments reveal thar CENP-E accumulates and is lost in a cell cycle dependent manner. NISI.• W C, LoMniLIX) VA, KLIHN,~~IA R. MCINTOSH JR: A Plusend Directed Motor Enzyme that Moves Antiparallel Microtubules in Vitro Localizes fo the Interzone of Mitotic Spindles. Nature 1992. 359543-547. Identification of the CHOI antigen as MKLP-I. a member of the kinesin superfamily. Demonstrates that MKLP-1 mediates the ATP-sensitive formation of antiparallel MT bundles and the sliding of antipardllel MTs 63. ..
in vilro. 64.
COLE DC. CANDE WZ. B*KIN
RJ, SKOLIFW DA, HOGAN CJ, SCHOIIY JM: Isolation of a Sea Urchin Kinesin-related Protein Using Peptide Antibodies. J Cell Sci 1992, 101:291-301. Polyclonal antibodies to synthetic peptides corresponding to conserved regions of kinesin were used to identify kinesin-related proteins in sea urchin eggs and embryos. The antibodies recognize a complex of three polypeptides that bind to MTs in an ATP-sensitive manner and bundle MTs in c~itro.These results demonstrate that this kinesin.related complex mediates MT-MT interactions in mirotic cells. .
65.
WAU~ER RA, SAL~ION ED, ENDOW SA: The Drosophila Claret Segregation Protein is a Minus End-directed Motor Molecule. Nature 1990, 347~780-782.
66.
MCDONALII like Ned Microtubule
HB, STEWART RJ, GOLDS~:IN LSD: Tbe KinesinProtein of Drosophila is a Minus End-directed Motor. Ce// 1990, 651159-l 165.
P Wadswonh, Department of Biology, Morrill Science Center, of Massachusetts, Amherst, Massachusetts 01003, USA
University