Cell Biology Program, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
In Xenopus egg extracts, spindles assembled around sperm nuclei contain a centrosome at each pole, while those assembled around chromatin beads do not. Poles can also form in the absence of chromatin, after addition of a microtubule stabilizing agent to extracts. Using this system, we have asked (a) how are spindle poles formed, and (b) how does the nucleation and organization of microtubules by centrosomes influence spindle assembly? We have found that poles are morphologically similar regardless of their origin. In all cases, microtubule organization into poles requires minus end-directed translocation of microtubules by cytoplasmic dynein, which tethers centrosomes to spindle poles. However, in the absence of pole formation, microtubules are still sorted into an antiparallel array around mitotic chromatin. Therefore, other activities in addition to dynein must contribute to the polarized orientation of microtubules in spindles. When centrosomes are present, they provide dominant sites for pole formation. Thus, in Xenopus egg extracts, centrosomes are not necessarily required for spindle assembly but can regulate the organization of microtubules into a bipolar array.
DURING cell division, the correct organization of microtubules in bipolar spindles is necessary to distribute chromosomes to the daughter cells. The
slow growing or minus ends of the microtubules are focused at each pole, while the plus ends interact with the chromosomes in the center of the spindle (Telzer and
Haimo, 1981 To begin to address these questions, we have used Xenopus egg extracts, which can be used to reconstitute different types of spindle assembly. Spindle assembly around
Xenopus sperm nuclei is directed by centrosomes (Sawin
and Mitchison, 1991 Preparation of Extracts, DMSO Asters, and Spindles
10,000-g cytoplasmic extracts of unfertilized Xenopus eggs arrested in
metaphase of meiosis II by CSF activity were prepared fresh as described
(Murray, 1991 Immunofluorescence and Video Experiments
For immunofluorescence of sperm DNA and chromatin bead spindles, 20-µl reactions were diluted with 1 ml 30% glycerol, 1% Triton X-100 in
BRB80 (80 mM Pipes, 2 mM MgCl2, 1 mM EGTA) and spun onto coverslips in modified corex tubes as described (Mitchison and Kirschner,
1984 Dynein Inhibition
The monoclonal IgM anti-dynein intermediate chain antibody (mAb
70.1) and control IgM anti-mouse IgG ascites were obtained from Sigma
Chemical Co. (St. Louis, MO) and dialyzed against 50 mM potassium
glutamate, 0.5 mM MgCl2, then concentrated to 20 mg/ml, flash frozen,
and stored in small aliquots at Preparation of EM Samples
Chromatin bead spindles in 200 µl of extract were magnetically retrieved
at 20°C. The extract was removed, and the spindles were gently resuspended in 100 µl of hooking solution containing 1.5 mg/ml calf brain tubulin, 0.5 M Pipes, pH 6.9, 1.5 mM MgCl2, 1 mM EGTA, 1 mM GTP, and
2.5% DMSO. After a 5-7-min incubation at 20°C, spindles were again
magnetically retrieved, and the hooking solution was removed. 1 ml of
25% glycerol, 0.2% glutaraldehyde in BRB80 was added, and then more
glutaraldehyde was added immediately afterward, bringing the final concentration to 2%. After 30 min at 20°C, the spindles were pelleted by centrifugation and washed several times with PBS. Samples were then dehydrated, imbedded in epon and sectioned, and observed with an electron
microscope (Carl Zeiss, Inc., Thornwood, NY). For data acquisition, hook handedness was assessed in 50-100 microtubules in each section, and
three sections were analyzed for each condition.
Pole Assembly Proceeds by a Common Mechanism with
or without Centrosomes
To compare the mechanism of spindle pole assembly in
the presence and absence of centrosomes, we examined
how spindle poles formed in Xenopus egg extracts under
three different conditions: around sperm nuclei that contain centrosomes (Sawin and Mitchison, 1991
While the visual similarity of these microtubule arrays
was striking, it did not indicate whether pole assembly occurred by a common mechanism. To characterize how
poles form, we took advantage of an assay developed to
follow microtubule movements during spindle assembly.
We have shown previously that stable polarity-marked microtubule "seeds," containing a brightly labeled minus end
and a dimly labeled plus end, act as markers for the movement of endogenous microtubules during spindle assembly
around chromatin beads (Heald et al., 1996
The similar behavior of stable microtubules added to
spindles and asters suggested that this movement was due
to a common mechanism. We have shown that seed movement on chromatin bead spindles is dependent on cytoplasmic dynein. Inhibition of dynein with vanadate or by
addition of a monoclonal antibody to the dynein intermediate chain (mAb 70.1) not only blocked seed translocation but caused dissolution of the poles (Heald et al., 1996
We wanted to understand how mAb 70.1 was disrupting
dynein activity. Microtubule gliding assays performed with
purified dynein revealed that mAb 70.1 did not block motility of the motor (data not shown). We therefore examined whether dynein localization was disrupted by addition of mAb 70.1 to extracts. A polyclonal antibody to the
dynein heavy chain was used for immunofluorescent analysis of spindles before and after mAb 70.1 addition (Fig. 5). In agreement with published reports, the dynein heavy
chain was localized to the poles of spindles, and faint punctate staining was also visible on chromosomes, probably
corresponding to kinetochores (Pfarr et al., 1990
Centrosomes Are Tethered to Spindle Poles by Dynein
The disruption of sperm DNA spindle poles by mAb 70.1 showed that centrosomes are not sufficient to maintain the
cohesion of poles in the absence of dynein activity. We
wanted to know what happened to the centrosomes after
dynein inhibition. It has been proposed that centrosomes
are tethered to spindle poles by the action of a dynein
complex that cross-links spindle microtubules to centrosomal microtubules (Karsenti, 1991 Sorting of Microtubules into an Antiparallel Array Does
Not Require Pole Formation
Our results thus far indicate that cytoplasmic dynein plays
a central role in spindle organization. We wondered to
what extent dynein function and pole assembly contributed to the polarized organization of microtubules in spindles, with microtubule minus ends at the poles and antiparallel microtubule interactions in the center of the
spindle. We have shown previously that in the presence of
mAb 70.1, a parallel array of microtubules formed around
chromatin beads with frayed, unfocused ends, and the
beads located in the center of the array (Heald et al., 1996
Centrosomes Regulate Bipolarity by Providing Focal
and Dominant Pole Assembly Sites
We have shown that pole formation occurs by a general
dynein-dependent mechanism in Xenopus egg extracts
whether or not centrosomes are present, creating morphologically indistinguishable structures (Fig. 1). These results
raise the fundamental question of the role of centrosomes
in spindle assembly, and more specifically in the establishment of spindle bipolarity. One potential explanation for
our results is that pole assembly mechanisms seem to be
equivalent because microtubule organizing centers, similar
to centrosomes, are created at poles during microtubule
self-organization. To address this issue, we examined the
fate of spindle poles after microtubule depolymerization.
If chromatin bead spindle pole formation creates stable
microtubule organizing structures, we reasoned that this
organization should persist after pole disassembly. To test
this, we cooled spindle reactions containing either sperm DNA spindles or chromatin bead spindles on ice to cause
complete microtubule depolymerization. The reactions
were then incubated at 20°C for various times and examined for microtubule regrowth (Fig. 7). In extracts containing sperm nuclei, microtubule asters were visible within 5 min after reheating. Two asters were often found near
condensed sperm chromatin, apparently growing from the
centrosomes that were at the spindle poles, no longer
closely associated with sperm chromatin. In contrast, chromatin bead reactions contained no asters or microtubules
5 min after reheating. Detectable microtubule growth did
not occur until ~15 min later, when microtubules began to
grow close to the chromatin beads. This nucleation phenotype corresponds to the first step in spindle assembly in
the absence of centrosomes (Heald et al., 1996
Although the self-assembly of spindle poles does occur
in some systems, centrosomes, when present, seem to determine the number of poles formed (Mazia et al., 1981 The Role of a Dynein Complex in Pole Formation
In this paper we show that in Xenopus egg extracts, mitotic
poles formed from DMSO-stabilized microtubules, around
chromatin beads or around sperm chromatin, are generated by the same mechanism; the minus end-directed
movement of microtubules across one another, driven by
dynein (Verde et al., 1991 Another protein known to be required for microtubule
organization into poles is NuMA (Kallajoki et al., 1991 Microtubule Sorting
In the process of spindle assembly around chromatin
beads, randomly nucleated microtubules are organized
into a bipolar array with microtubule minus ends at poles
and plus ends at chromatin (Heald et al., 1996 The Nature of a Spindle Pole
The precise nature of spindle poles and centrosomes and
the relationship between them has long been a source of
confusion and controversy in cell biology (see Mazia,
1984 A concept emerging from these studies is that the organization of microtubules into a spindle pole is a process
that is superimposed on the centrosomal aster (Karsenti,
1991 The Role of Centrosomes
Since centrosomes are not always required to form spindle
poles, what is their role? We and others have shown that a
single centrosome can create a dominant site for pole assembly, enforcing monopolarity (Mazia et al., 1981 Why are centrosomes required in some systems and not
in others? One distinguishing feature of early embryonic
systems such as Xenopus results from the presence of
stored components in eggs that may be limiting in somatic
cells. Therefore, centrosomes may be required in most systems because of the lack of stored microtubule nucleating
material in the cytoplasm, which is present only on centrosomes, preventing microtubule growth around chromosomes (discussed in Karsenti et al., 1996; McIntosh and Euteneuer, 1984
). Current
concepts of spindle assembly are based primarily on mitotic spindles of animal cells, which contain centrosomes.
Centrosomes are thought to be instrumental for organization of the spindle poles and for determining both microtubule polarity and the spindle axis. In the prevailing
model, termed "Search and Capture," dynamic microtubules growing from two focal points, the centrosomes, are
captured and stabilized by chromosomes, generating a bipolar array (Kirschner and Mitchison, 1986
). However,
while centrosomes are required for spindle assembly in
some systems (Sluder and Rieder, 1985
; Rieder and Alexander, 1990
; Zhang and Nicklas, 1995a
,b), in other systems
they appear to be dispensable (Steffen et al., 1986
; Heald
et al., 1996
). Furthermore, centrosomes are not present in
higher plant cells and in female meiosis of most animal
species (Bajer and Mole, 1982
; Gard, 1992
; Theurkauf and
Hawley, 1992
; Albertson and Thomson, 1993
; Lambert
and Lloyd, 1994
). In the absence of centrosomes, bipolar
spindle assembly seems to occur through the self-organization of microtubules around mitotic chromatin (McKim
and Hawley, 1995
; Heald et al., 1996
; Waters and Salmon,
1997
). The observation of apparently different spindle assembly pathways raises several questions: Do different
types of spindles share common mechanisms of organization? How do centrosomes influence spindle assembly? In
the absence of centrosomes, what aspects of microtubule
self-organization promote spindle bipolarity?
). Like other meiotic systems (Bastmeyer et al., 1986
; Steffen et al., 1986
), Xenopus extracts
also support spindle assembly around chromatin in the absence of centrosomes through the movement and sorting of randomly nucleated microtubules into a bipolar structure (Heald et al., 1996
). In this process, the microtubule-based motor cytoplasmic dynein forms spindle poles by
cross-linking and sliding microtubule minus ends together.
Increasing evidence suggests that the function of dynein in
spindle assembly depends on its interaction with other
proteins, including dynactin, a dynein-binding complex, and NuMA1 (nuclear protein that associates with the mitotic apparatus) (Merdes et al., 1996
; Echeverri et al.,
1996
; Gaglio et al., 1996
). In this paper, we demonstrate
that both in the presence and absence of centrosomes,
spindle pole assembly occurs by a common dynein-dependent mechanism. We show that when centrosomes are
present, they are tethered to spindle poles by dynein. In
the absence of dynein function, microtubules are still sorted
into an antiparallel array, indicating that other aspects of
microtubule self-assembly independent of pole formation
promote spindle bipolarity around mitotic chromatin. Since centrosomes are dispensable for pole formation in
this system, what is their function? We show here that if
only one centrosome is present, it acts as a dominant site
for microtubule nucleation and focal organization, resulting in a monopolar spindle. Therefore, although centrosomes are not required in this system, they can influence spindle pole formation and bipolarity.
Materials and Methods
). FITC-labeled tubulin prepared from calf brain tubulin
was added to 0.2 mg/ml (Hyman, 1991
). DNA beads and chromatin bead
spindles were prepared as described (Heald et al., 1996
). DMSO asters
were assembled by the addition of 5% DMSO and a 30-min incubation at
20°C (Sawin and Mitchison, 1994
). Mitotic spindles were assembled
around demembranated sperm nuclei by either the half spindle or the interphase to mitotic pathway as described (Sawin and Mitchison, 1991
).
). For DMSO aster reactions and for visualizing dissociating centrosomes, 15% glycerol was used instead of 30%. For samples to be processed for dynein heavy chain immunofluorescence, 4% formaldehyde
was included. After spinning, coverslips were fixed in methanol at
20°C
for 5 min and then blocked in 3% BSA for 10 min at room temperature.
Primary antibodies used were raised against Xenopus proteins and affinity
purified. Polyclonal anti-NuMA antibodies were provided by A. Merdes
(University of California, San Diego, CA). Polyclonal anti-
tubulin antibodies were raised to a COOH-terminal peptide by T. Ashford (European Molecular Biology Laboratory, Heidelberg, Germany), and anti-
dynein heavy chain antibodies raised to a conserved sequence in the motor domain (Vaisberg et al., 1993
) were provided by S. Reinsch (European Molecular Biology Laboratory). Rhodamine-conjugated secondary antibodies were used, and in some cases DNA was stained with propidium iodide. Rhodamine-labeled seeds were prepared and video microscopy was
performed as described (Heald et al., 1996
). Seed movement data was acquired using NIH Image. Seed distance from the center of DMSO asters
was measured at 5-s intervals.
80°C. For spindle pole disruption experiments, antibodies were diluted 1:10 in assembly reactions, which were
then diluted and spun after 5 or 10 min as described above. To block pole
assembly, mAb 70.1 was added 1:10 to extracts before spindle assembly.
Results
), around
chromatin beads in the absence of centrosomes (Heald et
al., 1996
), and in self-assembled mitotic asters, which form in the absence of centrosomes and chromatin but in the
presence of DMSO, a microtubule stabilizing agent (Sawin
and Mitchison, 1994
). We first compared the structure of
the three different types of poles by immunofluorescence
microscopy by examining the distribution of two different
proteins usually found associated with microtubule minus
ends in polarized arrays,
tubulin (Lajoie et al., 1994
;
Stearns and Kirschner, 1994
; Debec et al., 1995
; Li and
Joshi, 1995
) and NuMA (Maekawa et al., 1991
; Gaglio et al., 1995
; Merdes et al., 1996
). These proteins were localized similarly in all three cases (Fig. 1).
Tubulin was enriched at poles but was also present throughout the microtubules. NuMA was highly focused in the center of the
poles. Thus, by morphological criteria, centrosomal and
noncentrosomal poles appear to be quite similar.
Fig. 1.
Spindle pole antigens are similarly localized on poles whether or not centrosomes are present. Microtubules are green, antigens and DNA are red, and overlap is yellow. Tubulin (A-C) and NuMA (D-F) immunofluorescence of chromatin bead spindles (A
and D) and DMSO asters (B and E) with self-assembled poles and sperm DNA spindles (C and F) with a centrosome at each pole. Bar,
5 µm.
[View Larger Version of this Image (29K GIF file)]
). Seeds added
to extracts bind to and translocate along microtubules towards poles with their minus ends leading (Fig. 2 a). We
compared seed motility on the three types of polarized microtubule arrays (Fig. 2 b). Just as on chromatin bead spindles, microtubule seeds translocated poleward in DMSO
asters, forming bright foci at the poles. A complete analysis of seed movement on DMSO asters is shown in Fig. 3.
Poleward seed movement was saltatory, with an average
speed of 6 µm/min and a peak velocity of 30 µm/min. Polarity-marked seeds moved poleward with their brightly
labeled minus ends leading. Since both chromatin bead and DMSO aster pole structures are generated by microtubule self-organization, these results were not surprising.
Importantly, however, seeds also translocated poleward
on sperm DNA spindles containing poles derived from
centrosomes (Fig. 2 b). In all three cases, seed movement
was qualitatively and quantitatively similar (data not shown).
Therefore, minus end-directed microtubule movement is a general property of poles, whether or not centrosomes
are present.
Fig. 2.
Stable microtubule seeds translocate to and accumulate at mitotic poles in the presence and absence of centrosomes. Seeds are
red, microtubules are green, and overlap is yellow. (a) Diagram of seed experiment: polarity-marked microtubules polymerized from pure tubulin with a nonhydrolyzable GTP analogue, GMP-CPP, were added to extracts containing spindles. Seeds bound to spindle microtubules and moved poleward, where they accumulated. (b) Seeds accumulate at poles of chromatin bead spindles, DMSO asters, and sperm DNA spindles that contain centrosomes. Bar, 5 µm.
[View Larger Versions of these Images (15 + 25K GIF file)]
Fig. 3.
Video analysis of seed movement on DMSO asters. (a) Successive video frames at 20-s intervals showing that individual seeds move poleward. (b) Rates of seed movement over 5-s intervals and distances of individual seeds from the pole over time. (c) Polarity-marked microtubule seeds are oriented with their minus ends directed toward the center of the aster and its focus of microtubule minus ends. Bars: (a) 5 µm; (c) 8 µm.
[View Larger Versions of these Images (36 + 14 + 63K GIF file)]
;
and Fig. 4 A). We wanted to test whether dynein was also
involved in the organization of DMSO asters and in spindle poles containing centrosomes. The addition of mAb
70.1 to DMSO asters caused disruption of aster integrity
(Fig. 4 B), confirming a requirement for dynein in spontaneous aster assembly shown previously (Verde et al., 1991
;
Gaglio et al., 1996
). Furthermore, addition of mAb 70.1 also disrupted sperm DNA spindle poles containing centrosomes, causing them to splay outwards and become disorganized within 10 min (Fig. 4 C). In all cases, addition of
antibody prevented seed movement towards poles (data
not shown). When added before initiation of spindle assembly reactions, mAb 70.1 blocked pole formation, yielding similar structures with frayed, unfocused ends (Heald
et al., 1996
; see Fig. 8 a, D). Therefore, these results indicate that dynein-dependent translocation of microtubules
is required for pole formation and maintenance both in
the presence and absence of centrosomes.
Fig. 4.
Disruption of poles by antidynein antibodies. The monoclonal antibody (mAb 70.1) that recognizes the intermediate chain of cytoplasmic dynein was added to extracts containing chromatin bead spindles (A), DMSO asters (B), or sperm DNA spindles (C) with
centrosomes. Pole structures were disrupted within 10 min. Bar, 5 µm.
[View Larger Version of this Image (55K GIF file)]
Fig. 8.
Centrosomes are dominant sites of pole assembly. (a)
Sperm half spindle reactions in the presence of control antibodies
(A and B) or mAb 70.1 (C and D) were evaluated for microtubule polarity by NuMA immunofluorescence. (b) Quantification
of monopolar and bipolar structures under each condition. At
least 300 spindles were counted for each condition in two separate
experiments. Bar, 5 µm.
[View Larger Versions of these Images (38 + 14K GIF file)]
; Steuer et
al., 1990
). Upon addition of mAb 70.1, all spindle staining
with the heavy chain antibody disappeared within 5 min.
Therefore, mAb 70.1 seems to inhibit dynein by preventing localization and/or accumulation of the motor on spindle microtubules, perhaps by disruption of the interaction
of dynein with other proteins, such as the dynactin complex or NuMA.
Fig. 5.
Dynein tethers centrosomes to
spindle poles. (a) Cytoplasmic dynein is
eluted from spindles by addition of mAb
70.1. Immunofluorescent localization of
dynein heavy chain to spindle poles disappears within 5 min after mAb 70.1 addition. Microtubules are green, dynein
heavy chain is red, and overlap is yellow.
(b) Centrosomes are released from sperm
DNA spindle poles 3 min after addition of
mAb 70.1. Immunofluorescent localization of tubulin on sperm centriolar structures that are dissociating from spindle
poles. (c) Model of how dynein tethers
spindle microtubules to centrosomal microtubules and how this is disrupted by
mAb 70.1. Bars, 5 µm.
[View Larger Versions of these Images (25 + 8K GIF file)]
; Fig. 5 c). If this were
true, then dynein disruption should release centrosomes
from spindles. We therefore examined sperm DNA spindles after dynein inhibition and often noted small asters
near spindle poles (Figs. 4 and 5 a, arrows). These structures looked very similar to isolated centrosomes and were
not found upon mAb 70.1 addition to chromatin bead
spindles that lack centrosomes (not shown). To determine whether these structures were dissociated centrosomes, we
localized centrosomes shortly after mAb 70.1 addition by
immunofluorescent analysis of
tubulin (Fig. 5 b). Centriolar-like structures were often visible, tenuously linked to
spindle microtubules. These experiments indicate that
centrosomes are discrete entities independent of spindle
poles, tethered to the spindle by cytoplasmic dynein activity (Fig. 5 c).
;
Fig. 6 a). We wanted to know whether microtubules in
such bundles were randomly oriented or sorted into an antiparallel array around chromatin, with plus ends at the
chromatin and minus ends at spindle termini. To address
this question, we assessed microtubule polarity in spindles
assembled in the presence or absence of mAb 70.1 using two independent approaches. First, we examined the localization of NuMA, a protein normally associated with
the minus ends of microtubules in polarized arrays (Fig. 1)
(Maekawa et al., 1991
). We found that NuMA was still localized to the two frayed unfocused ends of the spindle
formed in the presence of mAb 70.1, albeit more diffusely
(Fig. 6 a). This result indicated that microtubule minus
ends were still sorted away from chromatin in the absence of pole formation. Second, we determined directly
whether microtubule polarity was uniform or not by the
hooking technique. In this technique, microtubules are incubated with pure tubulin under conditions that promote
addition of hooked protofilament appendages to the microtubule walls (Heidemann and McIntosh, 1980
). The polarity of individual microtubules can then be determined
by examination of hook handedness in serial sections by
electron microscopy (Euteneuer and McIntosh, 1981
; Euteneuer et al., 1982
). Here we analyzed single sections to
determine hook handedness in chromatin bead spindles
assembled in the presence of control antibodies or mAb 70.1 (Fig. 6). Under both conditions, sections that were cut
through beads, likely to correspond to the spindle centers,
contained microtubules with approximately equal proportions of right- and left-handed hooks, indicating that microtubules were of mixed polarity (Fig. 6, b-d). In control
spindles, sections through focused microtubule bundles,
corresponding to poles, contained hooks of which 90%
were the same handedness, indicating that microtubules were of almost uniform polarity (Fig. 6, b-d). Although
spindles assembled in the presence of mAb 70.1 did not
contain poles, microtubule bundles, likely to be close to
spindle termini, contained microtubules of >95% uniform
hook handedness. Therefore, both NuMA staining and
hook analysis show that microtubules are sorted into antiparallel arrays around chromatin beads. This sorting is independent of dynein activity and pole formation, indicating that other microtubule-based motors are contributing
to the antiparallel organization of microtubules in spindles.
Fig. 6.
Microtubules are sorted into antiparallel arrays around chromatin in the absence of pole formation. (a) Immunofluorescent localization of NuMA to the frayed ends of chromatin bead spindles that have formed in the absence of dynein activity. (b and c) Hooking analysis. (b) Quantification of hook handedness in control and poleless spindles. Percentage of right- and left-handed hooks in sections through spindle centers containing chromatin beads, and spindle ends containing microtubule poles (control), or bundles (+ mAb
70.1). (c) Low magnification micrographs (5-µm width) are shown to give an overall impression of microtubule organization seen in sections through spindle ends containing microtubule poles or bundles and through spindle centers containing beads. (d) Higher magnification micrographs (2-2.5-µm width) show hooks on cross sectioned individual microtubules. In the presence of control antibodies, a section through a pole contains right-handed (clockwise) hooks, while a section containing beads contains both right- and left-handed
hooks. In the presence of mAb 70.1, a section through a microtubule bundle likely to be at the spindle end is shown that contains almost
exclusively left-handed hooks. Note: Hook handedness does not give any information about the polarity of the microtubules in these
sections but indicates the degree to which microtubule polarity is uniform. 50-100 microtubules were evaluated in each section, and
three sections were evaluated for each condition. Bar, 5 µm.
[View Larger Versions of these Images (32 + 13 + 47 + 93K GIF file)]
). Thus,
poles assembled around chromatin beads do not become
permanent microtubule organizing centers, whereas centrosomes retain this property.
Fig. 7.
Centrosomes provide permanent microtubule organizing sites. Sperm DNA and chromatin bead spindles before and 5 or 20 min
after incubation on ice to depolymerize microtubules. No microtubules were visible on chromatin beads 5 min after cooling. Bar, 5 µm.
[View Larger Version of this Image (40K GIF file)]
;
Bajer, 1982
). This phenomenon has also been observed in
Xenopus egg extracts, in which monopolar spindles form
in the presence of a single centrosome, or when centrosome separation is blocked (Sawin and Mitchison, 1991
;
Boleti et al., 1996
). However, we have shown that in the
absence of centrosomes, predominantly bipolar spindles
formed around chromatin beads (Heald et al., 1996
).
These observations suggest that the presence of one centrosome, by providing a dominant focal microtubule nucleation site, could overrule the natural tendency of microtubules to self-organize into a bipolar array around
chromosomes. To test centrosome dominance directly, we
compared spindle assembly reactions in the presence and
absence of a single centrosome. Xenopus "half spindle" reactions mimic the single centrosome reaction because a
sperm nucleus containing a single centrosome directs assembly of a monopolar spindle when added directly to mitotic extracts (Fig. 8 a; Sawin and Mitchison, 1991
). To remove the centrosome, we added mAb 70.1, reasoning that
dynein inhibition would cause release of the centrosome
from the microtubule array, preventing its influence. We
then assessed the polarity of microtubule arrays formed by
immunofluorescent localization of NuMA (Fig. 8). In the
control reaction, as expected, more than 75% of the microtubule arrays formed after 45 min were monopolar,
with NuMA localized to the single spindle pole. Approximately 25% of the structures observed were bipolar, as a
result of half spindle fusion, as previously described
(Sawin and Mitchison, 1991
). In contrast, ~90% of the microtubule structures formed in the presence of mAb 70.1 were sorted into an antiparallel array around chromatin
with NuMA localized at each end. NuMA staining was no
longer focused because spindle poles did not form in the absence of dynein activity. However, these structures were
bipolar in the sense that microtubule minus ends were at
the two spindle termini. 10% of the structures had a monopolar distribution of NuMA with sperm chromatin asymmetrically located. These results indicate that bipolarity is
the favored self-assembly state of microtubules around
chromatin. However, the presence of a single centrosome
influences microtubule organization, leading to monopolarity. Centrosomes therefore create dominant sites for
pole formation.
Discussion
; Gaglio et al., 1996
; Heald et al.,
1996
). There is an increasing body of evidence supporting a general requirement for dynein in spindle assembly in
vivo (Pfarr et al., 1990
; Steuer et al., 1990
; Vaisberg et al.,
1993
). Both genetic and biochemical evidence suggest that
dynein function is dependent on its interaction with dynactin, a dynein-binding complex (for reviews see Schroer,
1994
; Schroer et al., 1996
). Here, we show that an antibody
directed against the dynein intermediate chain (mAb 70.1)
prevents the accumulation of dynein heavy chain on spindle microtubules. Because the dynein intermediate chain interacts with the dynactin subunit p150-glued (Karki and
Holzbaur, 1995
; Vaughan and Vallee, 1995
), mAb 70.1 may be dissociating dynein from spindles by disrupting its
interaction with dynactin. Interestingly, overexpression of
dynamitin (a component of dynactin) in somatic cells also
prevents proper targeting of dynein to spindles and disrupts the poles (Echeverri et al., 1996
). Both dynein and dynactin are also required for taxol aster formation in
HeLa cell extracts (Gaglio et al., 1996
), further supporting
the generality of this mechanism.
,
1992
, 1993
; Yang and Snyder, 1992
; Compton and Cleveland, 1993
; Compton and Luo, 1995
; Gaglio et al., 1995
).
Recently, experiments in Xenopus egg extracts have
shown that dynein and NuMA interact and that depletion
of NuMA results in a spindle pole defect very similar to
that of dynein inhibition (Merdes et al., 1996
). A model
consistent with these results is that NuMA is transported
to microtubule minus ends in a complex with dynein,
where NuMA could help cross-link microtubules and give
cohesion to the spindle pole (Merdes et al., 1996
). However, our results indicate that at least some NuMA was still
enriched at spindle ends in the absence of dynein activity (Fig. 6 a). Furthermore, we have found that inhibition of
NuMA blocks movement of microtubule seeds on spindle
arrays (Heald, R., and A. Merdes, unpublished data).
These data support the idea that, at least in Xenopus, a
complex of dynein and NuMA is essential for spindle pole
formation. Our results indicate that NuMA is targeted to
microtubule minus ends by another mechanism in addition to its association with dynein, perhaps in association with
another minus end-directed motor, as proposed (Gaglio et
al., 1996
), or NuMA could attain a polarized distribution
independently of motor activity (Maekawa et al., 1991
).
Taken together, the data emerging from several laboratories indicate that dynein functions in a multimeric complex
with NuMA and dynactin. However, the molecular mechanism by which these proteins interact to form spindle
poles is not yet understood.
). We have
referred to this process as microtubule sorting and proposed that microtubule-based motors could function as
sorting devices by recognizing microtubule polarity and
moving microtubules relative to one another and to chromatin. One possibility is that sorting occurs concomitantly
with spindle pole assembly, as dynein collects microtubule
minus ends into arrays of uniform polarity. Here we have
shown by two independent means that microtubule sorting
occurs in the absence of dynein activity and pole formation, resulting in an antiparallel array of microtubules
around chromatin. While NuMA staining indicated that
microtubule minus ends were enriched at the frayed spindle ends, hooking analysis revealed that microtubule bundles were of uniform polarity. Therefore, other motors besides dynein must contribute to microtubule sorting during
spindle assembly. One possibility is that plus end-directed motors associated with chromatin sort microtubules by
moving towards plus ends, thereby pushing microtubule
minus ends away from chromatin (Vernos et al., 1995
).
Alternatively, or concurrently, spindle tetrameric plus
end-directed motors of the BimC family (Walczak and
Mitchison, 1996
) or motors shown to promote antiparallel
microtubule sliding such as MKLP1 (Nislow et al., 1992
)
could also fulfill this role. Since polarity-marked seed motility appears to be driven predominantly by dynein, other
assays need to be developed to visualize the activities of
other motors in spindles.
). One reason for the confusion is that centrosomes
and mitotic poles are structurally similar, each containing
a focus of microtubule minus ends. However, several observations suggest that centrosomes and mitotic poles are
functionally different. First, several proteins, including NuMA (Merdes et al., 1996
) and the dynein/dynactin complex (Gaglio et al., 1996
; this work), are required for pole
formation but not for microtubule nucleation by centrosomes. Second, we have shown that after microtubule
depolymerization, centrosomes persist as microtubule organizing centers, while self-assembled poles do not (Fig.
7). Thus, focal nucleation and motor-dependent organization are two functionally different mechanisms for generating a focus of microtubule minus ends.
; Gaglio et al., 1996
). Several observations support a
dynamic association of spindle microtubules with centrosomal microtubules at poles. First, poleward microtubule flux requires that microtubule minus ends are free to
depolymerize (Mitchison and Sawin, 1990
). Second, the
centrosome and spindle pole appear to organize separate
populations of microtubules. These two populations are
easily recognized in spindles assembled around sperm nuclei in Xenopus egg extracts because there are two sources
of microtubules, chromatin and centrosomes, that can be
separated by dynein inhibition (Fig. 5 b). In other meiotic or embryonic systems, such as Lepidoptera and Drosophila, the centrosomes are physically located a significant distance away from the spindle poles (Wolf and Bastmeyer,
1991
). These two different microtubule populations seem
to exist in somatic systems as well. Serial sectioning of somatic spindles revealed that most spindle microtubules
ended a significant distance away from the centrosomes (Mastronarde et al., 1993
). In some centrosome-dependent systems, loss or removal of centrosomes does not
damage spindle integrity once the spindle is in metaphase
or in anaphase (Mitchison and Salmon, 1992
; Murray et
al., 1996
; Nicklas et al., 1989
). How are two populations of
microtubules created in such systems, in which chromatin-induced nucleation of microtubules does not occur? Spindle microtubules could be generated by release of microtubules from centrosomes. This has been shown to occur
in Xenopus egg extracts (Belmont et al., 1990
) and somatic
cells in interphase (McBeath and Fujiwara, 1990
). Thus,
centrosomes can provide a source of microtubules and not
themselves be poles.
; Bajer,
1982
; Sawin and Mitchison, 1991
; Fig. 8). In Xenopus egg
extracts, an explanation for this observation is that by
functioning as an efficient microtubule nucleator, a centrosome generates microtubules before they appear to
grow around chromatin (Fig. 7). Once microtubules begin
to grow around chromatin, they may be preferentially
shuttled by dynein towards the centrosome, which constitutes a preformed focal point of microtubule minus ends,
before they can be sorted into an antiparallel array. Therefore, centrosomes could be dominant for kinetic reasons,
suppressing aspects of microtubule self-organization and
thereby directing the sites of spindle pole formation. We propose that the critical function of centrosomes is to determine pole assembly sites. Centrosome positioning then
provides a mechanism for determining the orientation of
the cleavage plane, which is important in many cell types
and during development (White and Strome, 1996
).
). Therefore, the
difference between meiotic and mitotic spindle assembly
may reflect differences in microtubule nucleation rather
than different principles of spindle organization.
Received for publication 10 February 1997 and in revised form 28 May 1997.
1. Abbreviation used in this paper: NuMA, nuclear protein that associates with mitotic apparatus.We thank T. Ashford, A. Merdes, and S. Reinsch for providing antibodies, and S. Andersen, M. Glotzer, C. Gonzalez, and S. Reinsch for comments on the manuscript.
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