Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
The focusing of microtubules into mitotic spindle poles in vertebrate somatic cells has been assumed to be the consequence of their nucleation from centrosomes. Contrary to this simple view, in this article we show that an antibody recognizing the light intermediate chain of cytoplasmic dynein (70.1) disrupts both the focused organization of microtubule minus ends and the localization of the nuclear mitotic apparatus protein at spindle poles when injected into cultured cells during metaphase, despite the presence of centrosomes. Examination of the effects of this dynein-specific antibody both in vitro using a cell-free system for mitotic aster assembly and in vivo after injection into cultured cells reveals that in addition to its direct effect on cytoplasmic dynein this antibody reduces the efficiency with which dynactin associates with microtubules, indicating that the antibody perturbs the cooperative binding of dynein and dynactin to microtubules during spindle/aster assembly. These results indicate that microtubule minus ends are focused into spindle poles in vertebrate somatic cells through a mechanism that involves contributions from both centrosomes and structural and microtubule motor proteins. Furthermore, these findings, together with the recent observation that cytoplasmic dynein is required for the formation and maintenance of acentrosomal spindle poles in extracts prepared from Xenopus eggs (Heald, R., R. Tournebize, T. Blank, R. Sandaltzopoulos, P. Becker, A. Hyman, and E. Karsenti. 1996. Nature (Lond.). 382: 420-425) demonstrate that there is a common mechanism for focusing free microtubule minus ends in both centrosomal and acentrosomal spindles. We discuss these observations in the context of a search-capture-focus model for spindle assembly.
CHROMOSOME segregation during both mitosis and
meiosis is mediated by a complex microtubule-based structure called the spindle (McIntosh and
Koonce; 1989; Mitchison, 1989a One striking difference between spindles in vertebrate
somatic cells and some types of meiotic and plant cells is
that microtubules in vertebrate somatic cells are nucleated
from centrosomes, whereas plant cells and some meiotic
cells lack bonafide centrosomes. This single structural difference has spurred two different hypotheses regarding
the mechanism by which microtubule minus ends are focused at spindle poles (for discussions see Wilson, 1925 The dynein-specific monoclonal antibody 70.1 (Steuer et
al., 1991 Cell Culture
The human HeLa cell line and the monkey CV-1 cell line were both maintained in DME containing 10% fetal calf serum, 2 mM glutamine, 100 IU/ml
penicillin, and 0.1 µg/ml streptomycin. Cells were grown at 37°C in a humidified incubator with a 5% CO2 atmosphere.
Immunological Techniques
The control (mAb 154; Compton et al., 1991 Indirect immunofluorescence microscopy was performed on cultured
cells by immersion in microtubule stabilization buffer (MTSB: 4 M glycerol, 100 mM PIPES, pH 6.9, 1 mM EGTA, and 5 mM MgCl2) for 1 min at
room temperature, extraction in MTSB plus 0.5% Triton X-100 for 2 min,
followed by MTSB for 2 min. Cells were then fixed in Proteins from the mitotic extracts were solubilized directly with SDS-PAGE sample buffer. The proteins were then separated by size using
SDS-PAGE and transferred to PVDF (polyvinylidene difluoride) membrane (Millipore Corp., Bedford, MA). The membranes were blocked in
TBS containing 5% nonfat milk for 30 min at room temperature and the
primary antibody incubated for 6 h at room temperature in TBS containing 1% nonfat milk. Nonbound primary antibody was removed by washing five times for 3 min each in TBS, and the bound antibody was detected
using either horseradish peroxidase-conjugated protein A or horseradish
peroxidase-conjugated goat anti-mouse (Bio Rad, Hercules, CA). The
nonbound secondary reagent was removed by washing five times for 3 min
each in TBS and the signal detected using enhanced chemiluminescence
(Amersham Corp., Arlington Heights, IL).
Microinjection
CV-1 cells growing on photo-etched Mitotic Extracts
Mitotic extracts from HeLa cells were prepared according to Gaglio et al.
(1995) Immunodepletions from the extract before aster assembly was carried
out using 20-50 µg of either an anti-Eg5 affinity-purified rabbit polyclonal
IgG or the monoclonal antibody 70.1, which is an IgM specific for the
IC74 intermediate chain of cytoplasmic dynein. Each antibody was adsorbed onto ~25 µl of either protein A- or protein G-conjugated agarose
(Boehringer Mannheim, Indianapolis, IN). The 70.1 monoclonal antibody
against cytoplasmic dynein intermediate chain was coupled to protein
G-conjugated agarose using goat anti-murine IgM specific antibody (Vector Lab. Burlingame, CA). The antibody-coupled agarose was washed in
KHM buffer and then packed by centrifugation to remove the excess
fluid. Efficient depletion of each target protein was routinely achieved by
sequential depletion reactions in which the total quantity of packed agarose did not exceed 15 µl per 100 µl of extract. First, half of the antibody-coupled agarose was resuspended with the mitotic extract and incubated with agitation for 1 h at 4°C. After this incubation the agarose was removed from the extract by sedimentation at 15,000 g for 10 s and saved.
Next, the extract was recovered and used to resuspend the other half of
the antibody-coupled agarose and another incubation performed with agitation for 1 h at 4°C. After this incubation the agarose was removed by
sedimentation at 15,000 g for 10 s and pooled with the agarose pellet from
the initial depletion reaction. In all cases, immunoblot analysis indicates
that this depletion protocol results in nearly 100% efficient depletion of
the target protein as described previously (Gaglio et al., 1996 The Dynein-specific Antibody 70.1 Perturbs Mitotic
Spindle Assembly In Vivo
Heald et al. (1996)
To determine if the dynein-specific antibody is capable
of disrupting the assembly of the vertebrate mitotic spindle, we microinjected CV-1 cells during interphase with
the dynein-specific monoclonal antibody (70.1) and followed the fate of each cell as it entered mitosis. 60% (n = 20) of cells that entered mitosis after microinjection with
the dynein-specific antibody were significantly delayed in their completion of mitosis (>2 h). In contrast, 96% (n = 25) of cells that entered mitosis after microinjection with a
control antibody completed mitosis normally within 1 h.
The control antibody recognizes CENP-E. We specifically
chose it as a control because it is a monoclonal IgM that
recognizes a known spindle component, and we have previously determined that it does not perturb the progression of mitosis when injected into cultured cells (Compton et al., 1991
The data presented in Fig. 2 suggest that the 70.1 antibody prevents mitotic spindle formation but does not directly demonstrate whether cytoplasmic dynein plays a
specific role in maintaining the focused organization of microtubule minus ends at the mitotic spindle pole in the
presence of centrosomes. To address this point, we microinjected CV-1 cells during metaphase (i.e., with pre-assembled spindles) with either the control antibody or the dynein-specific antibody. 8 out of 8 metaphase cells injected
with the dynein-specific antibody were blocked in mitosis
for at least 2 h, whereas 8 out of 10 metaphase cells
injected with a control antibody completed mitosis normally within 1 h. This indicates that the injection of the dynein-specific antibody into metaphase cells with pre- assembled spindles delays the completion of mitosis. To
explore the effects of this antibody we examined the morphology of the mitotic spindle in cells at various times after antibody microinjection. 95% (n = 21) of metaphase
cells injected with the control antibody had typical bipolar
mitotic spindles with NuMA concentrated in the characteristic crescent-like shape near the centrosomes (Fig. 3
A). In contrast, 91% (n = 43) of metaphase cells injected
with the dynein-specific antibody had abnormal mitotic
spindles (Fig. 3, B-D). As rapidly as 5 min after injection
with the dynein-specific antibody, the mitotic spindles
were barrel shaped and the poles were unusually broad as
judged by the organization of the microtubules and the
distribution of NuMA (Fig. 3 B). If the injected cells were
examined between 15 and 30 min after injection, the spindles lacked a typical fusiform organization. The microtubule minus ends appeared splayed, and NuMA was dislocated from the centrosomal region of the spindle and
localized on the microtubules at the splayed ends of the
spindle (Fig. 3, C and D). At later time points, NuMA was
localized along the length of many of the microtubules (Fig. 3 D, arrows). Importantly, the centrosomes in these
injected cells stained positively for
To determine if the 70.1 antibody perturbs the association of cytoplasmic dynein with the mitotic spindle, we microinjected metaphase cells with the 70.1 antibody and
stained the cells with the dynein-specific monoclonal antibody 74.1 (Dillman and Pfister, 1994
The Dynein-specific Antibody 70.1 Perturbs Mitotic
Aster Assembly In Vitro
We next tested if mitotic asters that form through a centrosome-independent mechanism in somatic cell mitotic
extracts (Gaglio et al., 1995
The Dynein-specific Antibody 70.1 Reduces
the Efficiency with Which Dynactin Associates
with Microtubules
To determine if the presence of these antibodies in the mitotic extract altered the efficiency with which proteins
known to be involved in spindle pole and aster assembly
associated with microtubules, we performed immunoblot
analysis on the soluble and insoluble fractions derived
from the mitotic extract containing the control and dynein-specific antibodies (Fig. 5 D). The efficiency with
which tubulin (data not shown), NuMA, cytoplasmic dynein, or Eg5 was converted from the soluble fraction to the
insoluble fraction during the reaction was not significantly
altered by the presence of the dynein-specific antibody,
despite the fact that this antibody disrupted the formation
of mitotic asters. This was true whether the antibody was
added before (Fig. 5 D, PRE) or after (Fig. 5 D, POST)
the formation of mitotic asters. In contrast, we consistently
observed a reduction in the efficiency with which dynactin
associated with the microtubules in the presence of the dynein-specific antibody. Typically, 15-30% of dynactin associates with the mitotic asters in the insoluble fraction
(Gaglio et al., 1996 The alteration in the efficiency with which dynactin associates with microtubules in the presence of the dynein-specific antibody suggests that addition of this dynein-specific
antibody to the extract may have functional consequences
that are different from inactivation of cytoplasmic dynein
alone. To determine if the reduction of dynactin association with microtubules induced by the addition of the dynein-specific antibody is functionally involved in the disruption of mitotic aster formation, we compared the effects
of the addition of the dynein-specific antibody to the depletion of cytoplasmic dynein (Fig. 6). In this experiment, it was necessary to either deplete cytoplasmic dynein or
add the 70.1 antibody to extracts depleted of the plus end-directed kinesin-related protein Eg5. The formation of mitotic asters in this system requires cytoplasmic dynein, and
asters fail to organize in the absence of cytoplasmic dynein
alone due to the imbalance in forces generated by microtubule motors during aster assembly. Mitotic asters will form in the absence of cytoplasmic dynein, however, if the
forces generated by microtubule motors are partially
equilibrated by the simultaneous depletion of the plus
end-directed motor Eg5 (Gaglio et al., 1996
Finally, to determine if the association of dynactin with
the mitotic spindle was also altered in vivo by the presence
of the 70.1 antibody, we microinjected this dynein-specific
antibody into metaphase cells and performed immunofluorescence microscopy with a dynactin-specific antibody
(45A, which recognizes the Arp1 subunit; Fig. 7). In
metaphase cells that have been microinjected with the
control antibody, dynactin is localized in a crescent-like pattern concentrated at the polar regions of the spindle as
well as throughout the cytoplasm, consistent with previously published reports (Fig. 7 A; Gill et al., 1991
The data presented here indicate that microtubule minus
ends located at mitotic spindle poles in vertebrate somatic
cells are organized in a complex manner requiring contributions from both the centrosomes and noncentrosomal
protein components. Centrosomes are essential to nucleate microtubules in somatic cells (McIntosh, 1983 Based on these data, we propose that mitotic spindle
formation in somatic cells proceeds through a search-capture-focus mechanism (Fig. 8). This model expands on the
search-capture model (Kirschner and Mitchison, 1986
This search-capture-focus model for spindle assembly
accounts for both our observations of spindle pole formation in somatic cells (i.e., centrosomal spindles) as well as
spindle pole formation in systems such as plants and some
meiotic cells that assemble spindles in the absence of centrosomes. In acentrosomal meiotic systems, microtubules
appear to be nucleated from free According to this search-capture-focus model for spindle assembly it may be possible to separate the microtubule nucleating activity associated with centrosomes in somatic cells from the focusing activity exerted by the
noncentrosomal structural and motor proteins. In fact, this
possibility has already been confirmed under a variety of
different experimental conditions. First, there are several
reports in the literature that centrosomes have detached from the body of the spindle while the microtubule minus
ends remain focused in a pole (Rieder and Hard, 1990 Finally, prevailing evidence indicates that the mechanism for focusing free microtubule minus ends into spindle
poles in both centrosomal and acentrosomal spindles is
driven by a common group of noncentrosomal accessory
proteins including NuMA, cytoplasmic dynein, dynactin,
Eg5, and a minus end-directed kinesin-related protein
(Verde et al., 1991 In the end, we speculate that free microtubule minus
ends are necessary for proper spindle function, because
they are necessary for the mechanism of poleward microtubule flux, which exerts force through the spindle (Waters et al., 1996; Rieder, 1991
). The spindle is assembled in a spatially and temporally regulated
manner during the cell cycle, and its assembly and function
are intimately associated with microtubule dynamics (Inoué and Salmon, 1995
; Hyman and Karsenti, 1996
; Nicklas, 1997
). The organization of microtubules into spindles
is governed largely by the interaction of microtubules and
microtubule ends with accessory proteins that regulate microtubule dynamics. These accessory proteins are located on the chromosomes (kinetochores), derived from the cytosol (some motor proteins), and/or found at the microtubule minus ends (centrosomes and peri-centrosomal region
in somatic cells). The result of these complex interactions
is a typical fusiform microtubule array in both mitotic and
meiotic cells with microtubule plus ends attached to the
chromosomes and minus ends focused into spindle poles.
;
Schrader, 1953
; Rieder et al., 1993
; Waters and Salmon,
1997
). In acentrosomal spindles, microtubules associate
with chromatin and are drawn into two focused poles
through the action of minus end-directed microtubule motors (Bastmeyer et al., 1986
; Steffen et al., 1986
; Theurkauf
and Hawley, 1992
; McKim and Hawley, 1995
; Vernos and Karsenti, 1995
; Heald et al., 1996
; Matthies et al., 1996
;
Merdes et al., 1996
). In contrast, in mitotic spindles in vertebrate cells the predominant view holds that microtubule
minus ends are focused at the poles as a consequence of
their nucleation from the centrosomes (Kirschner and
Mitchison, 1986
; Hayden et al., 1990
; Holy and Leibler,
1994
; Rieder and Salmon, 1995
). It is currently unclear,
however, if mitotic spindles containing centrosomes, like
acentrosomal spindles, also use minus end-directed motor activity to promote the focusing of microtubule minus
ends at spindle poles despite the presence of centrosomes.
) was recently shown to perturb the function of cytoplasmic dynein during spindle assembly in meiotic extracts prepared from Xenopus eggs (Heald et al., 1996
). It
blocked the formation of spindle poles as well as induced
the disorganization of the polar regions of preassembled
spindles, suggesting that dynein function was important to
establish and maintain these spindle poles. Spindles assembled under those conditions, however, do not contain
centrosomes, and the spindle poles are focused through an
acentrosomal mechanism (Lohka and Maller, 1985
; Sawin
and Mitchison, 1991
; Heald et al., 1996
; Merdes et al.,
1996
). Thus, in this article we have used the 70.1 antibody
to investigate whether the organization of microtubules at
the polar ends of the mitotic spindle also relies on the action of cytoplasmic dynein despite the inherent focusing
activity of centrosomes. We report that perturbation of
cytoplasmic dynein function with the 70.1 antibody in somatic cells leads to the disruption of mitotic spindle poles
and the separation of the centrosomes from the body of
the spindle. Furthermore, the 70.1 antibody prevents the
assembly of mitotic asters when added to a cell-free mitotic extract, and in both cases, reduces the efficiency with
which dynactin associates with microtubules. These data
indicate that microtubule minus ends are focused at mitotic
spindle poles through contributions from both centrosomes
and accessory proteins, including the minus end-directed
motor cytoplasmic dynein and dynactin, and suggest that
there are common aspects to the mechanism by which free
microtubule minus ends are focused into poles in centrosomal and acentrosomal spindles. These results are discussed in the context of a search-capture-focus model for
mitotic spindle assembly.
Materials and Methods
) and dynein-specific (mAb
70.1; Steuer et al., 1991
) IgMs were purified from ascites fluid by mannose-binding protein affinity chromatography (Pierce, Rockford, IL). The
purified antibodies were dialyzed into 0.1 M Tris, pH 7.4, and concentrated using centricon-30 concentrators (Amicon, Beverly, MA) to 8-16
mg/ml. The remaining antibodies used in this study were a rabbit anti-
nuclear mitotic apparatus (NuMA)1 (Gaglio et al., 1995
), mouse anti-tubulin (DM1A; Blose et al., 1984
), rabbit anti-Eg5 stalk-tail (Sawin et al.,
1992
), mouse anti-Arp1 (45A; Schafer et al., 1994
), mouse anti-p150 dynactin (150B; Gaglio et al., 1996
), and mouse anti-dynein (74.1; Dillman and Pfister, 1994
).
20°C methanol for
10 min. Indirect immunofluorescence microscopy on mitotic asters assembled in the cell-free mitotic extract was performed by dilution of 5 µl of
the extract into 25 µl of KHM buffer (78 mM KCl, 50 mM Hepes, pH 7.0, 4 mM MgCl2, 2 mM EGTA, 1 mM DTT; Burke and Gerace, 1986
). The
diluted sample was then spotted onto a poly-L-lysine coated glass coverslip and fixed by immersion in
20°C methanol. Both the fixed cells and
mitotic asters were rehydrated in TBS (10 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 1% albumin, and all antibody incubations and washes
were performed in TBS plus 1% albumin. Each primary antibody was incubated on the coverslip for 30 min except for the 45A antibody against
dynactin and the 74.1 antibody against cytoplasmic dynein, which were incubated on the coverslip for 2 h. After primary antibody treatment, the
coverslips were washed for 5 min in TBS plus 1% albumin, and the bound
antibodies were detected using either fluorescein- or Texas red-conjugated species- and antibody isotype-specific secondary antibodies at dilutions of 1:500 (Vector Labs, Burlingame, CA). DNA was detected using
DAPI (4
,6-diamidino-2-phenylindole) at 0.4 µg/ml (Sigma Chemical Co.,
St. Louis, MO). After a final wash the coverslips were mounted in FITC-guard mounting medium (Testog, Inc., Chicago, IL) and observed on a
Nikon Optiphot microscope equipped for epifluorescence (Nikon, Inc.,
Meliville, NY).
-numeric glass cover slips (Bellco
Glass Co., Vineland, NJ) were microinjected following the procedures of
Compton and Cleveland (1993)
and Capecchi (1980)
. Interphase cells
were microinjected in the cytoplasm with either the control antibody or
the dynein-specific antibody and followed by phase contrast microscopy
as they progressed into mitosis. Metaphase cells were selected for injection by phase contrast microscopy by virtue of a clearly identifiable bipolar mitotic spindle. Injected cells were followed for up to 4 h after injection unless otherwise stated in the text and were processed for immunofluorescence microscopy as described above.
. HeLa cells were synchronized in the cell cycle by double block
with 2 mM thymidine. After release from thymidine block the cells were
allowed to grow for 6 h, and then nocodazole was added to a final concentration of 40 ng/ml. The mitotic cells that accumulated over the next 4 h
were collected by mitotic shake off and incubated for 30 min at 37°C with
20 µg/ml cytochalasin B. The cells were then collected by centrifugation at
1,500 rpm and washed twice with cold PBS containing 20 µg/ml cytochalasin B. Cells were washed one last time in cold KHM buffer containing 20 µg/ml cytochalasin B and finally Dounce homogenized (tight pestle) at a
concentration of ~3 × 107 cells/ml in KHM buffer containing 20 µg/ml cytochalasin B, 20 µg/ml phenylmethylsulfonyl fluoride, and 1 µg/ml each of
chymostatin, leupeptin, antipain, and pepstatin. The crude cell extract was then subjected to sedimentation at 100,000 g for 15 min at 4°C. The supernatant was recovered and supplemented with 2.5 mM ATP (prepared as
Mg2+ salts in KHM buffer) and 10 µM taxol, and the mitotic asters were
stimulated to assemble by incubation at 30°C for 30-60 min. After incubation, samples were processed for indirect immunofluorescence microscopy
as described above, and the remainder of the extract containing the assembled mitotic asters was subjected to sedimentation at 10,000 g for 15 min at 4°C. The supernatant and pellet fractions were both recovered and
solubilized in SDS-PAGE sample buffer for immunoblot analysis.
). The depleted extract was recovered and microtubule polymerization induced by
the addition of taxol and ATP and incubation at 30°C. Each depletion experiment was performed at least two times and in all cases the efficiency
of mitotic aster formation (as determined by counting the average number
of asters per microscope field) was not significantly different from the values determined previously (Gaglio et al., 1996
).
Results
recently showed that an antibody
raised against the 74-kD subunit of cytoplasmic dynein purified from chicken embryo fibroblast cells (mAb 70.1;
Steuer et al., 1991
) perturbed the formation and maintenance of the polar ends of spindles assembled in meiotic
extracts prepared from Xenopus eggs. To determine the
specificity of this antibody in primate somatic cells, we performed immunoblot analysis of total cell protein from
the primate cell lines HeLa and CV-1. Fig. 1 shows that
this antibody reacts specifically with a single polypeptide
of apparent molecular mass of 74 kD. This band most
likely represents the light intermediate chain of cytoplasmic dynein derived from these two cell lines, because this
immunoreactive polypeptide has the same apparent molecular weight as the original chicken protein, and we have previously shown that this antibody specifically immunoprecipitates cytoplasmic dynein from HeLa cell mitotic extracts (Gaglio et al., 1996
). In some cases where we performed immunoblot analysis with this antibody on enriched
cellular fractions, we observed three reactive species that
ranged in molecular mass from 70-76 kD (see below). This
result is consistent with both the expression of multiple
isoforms and the complex post-translational regulation of
this subunit of cytoplasmic dynein (Paschal et al., 1992
;
Niclas et al., 1996
; Pfister et al., 1996
). Thus, mAb 70.1 is
specific for the light intermediate chain of cytoplasmic dynein in these primate cell types in accordance with its specificity for this subunit of cytoplasmic dynein in avian cells
(Steuer et al., 1991
).
Fig. 1.
mAb 70.1 specifically recognizes
the light intermediate chain of cytoplasmic
dynein in primate cells. Immunoblot analysis of total cell protein from 100,000 HeLa or CV-1 cells (~50 µg protein) using
the 70.1 monoclonal antibody. The migration position of myosin (200), -galactosidase (116), phosphorylase B (97), and
BSA (66) are indicated in kD.
[View Larger Version of this Image (16K GIF file)]
). Examination of the mitotic spindles in cells
injected with the control antibody by immunofluorescence
microscopy showed a typical fusiform microtubule array
with NuMA concentrated in a characteristic crescent-like
position at the polar ends of the spindle (Fig. 2 A). In contrast, cells that entered mitosis in the presence of the dynein-specific antibody have disorganized mitotic spindles
that lack well organized poles with NuMA localized along
the length of many of the microtubules (Fig. 2 B). These results suggest that this antibody perturbs an essential
function of cytoplasmic dynein during the formation of a
normal bipolar spindle in somatic cells. This conclusion is
consistent with the results previously reported by Heald et
al. (1996)
using this antibody to perturb acentrosomal
spindle formation in extracts prepared from Xenopus eggs
as well as evidence supporting a role for cytoplasmic dynein during spindle assembly in somatic cells (Pfarr et al.,
1991
; Steuer et al., 1991
; Vaisberg et al., 1993
; Echeverri et
al., 1996
; Gaglio et al., 1996
).
Fig. 2.
The dynein-specific 70.1 antibody blocks the formation of the mitotic
spindle. Monkey CV-1 cells were monitored as they progressed through mitosis
after microinjection with either a control
antibody (A) or the dynein-specific 70.1 monoclonal antibody (B). The mitotic cells were fixed and processed for immunofluorescence microscopy using the
DNA-specific dye DAPI, and antibodies
specific for tubulin and NuMA as indicated. Bar, 10 µm.
[View Larger Version of this Image (82K GIF file)]
-tubulin (data not
shown) and were nucleating microtubules normally forming small astral microtubule arrays, but were separated
from the body of the spindle (Fig. 3, C and D, arrowheads). Thus, despite the presence of functional centrosomes, the minus ends of the microtubules became unfocused and were displaced from the centrosomes after the
injection of this dynein-specific antibody into metaphase
cells.
Fig. 3.
The dynein-specific 70.1 antibody disrupts preassembled mitotic spindles despite the presence of
functional centrosomes. Monkey CV-1
cells in metaphase with bipolar mitotic spindles were selected by phase
contrast microscopy and microinjected with either a control antibody
(A) or the dynein-specific 70.1 monoclonal antibody (B-D). 5 min (B) or
15-30 min (A, C, and D) after microinjection, the cells were fixed and
processed for immunofluorescence
microscopy using the DNA-specific
dye DAPI and antibodies specific for
tubulin and NuMA as indicated. Arrowheads in C and D indicate centrosomes and arrows in D indicate
NuMA. Bar, 10 µm.
[View Larger Version of this Image (97K GIF file)]
). Cytoplasmic dynein
is localized on the mitotic spindle and throughout the cytosol of mitotic cells after injection with the control antibody
(Fig. 4 A) consistent with previously published reports
(Pfarr et al., 1991
; Steuer et al., 1991
). In contrast, the signal intensity for cytoplasmic dynein on mitotic spindles of
cells injected with the dynein-specific 70.1 antibody is significantly reduced (Fig. 4 B). The intensity of staining of
the cytosolic fraction of cytoplasmic dynein is equivalent
between these two samples, indicating that the staining efficiency for each sample was similar (Fig. 4, A and B). The
possibility that the 70.1 antibody sterically hinders the
binding of the 74.1 antibody to cytoplasmic dynein is unlikely, because the 74.1 antibody decorates the cytosolic fraction of cytoplasmic dynein to the same extent in the
presence of either the 70.1 or control antibodies (Fig. 4, A
and B). Also, preincubation of fixed cells with the 70.1 antibody does not reduce the signal observed using the 74.1 antibody in immunofluorescence microscopy (data not
shown). These results indicate that the dynein-specific 70.1 antibody disrupts the interaction of cytoplasmic dynein
with the mitotic spindle in somatic cells.
Fig. 4.
The dynein-specific 70.1 antibody causes a reduction in
the efficiency with which cytoplasmic dynein associates with the
mitotic spindle in vivo. Monkey CV-1 cells in metaphase with bipolar mitotic spindles were selected by phase contrast microscopy
and microinjected with either a control antibody (A) or the dynein-specific 70.1 monoclonal antibody (B). The cells were then
fixed and processed for immunofluorescence microscopy using
the DNA-specific dye DAPI and the 74.1 antibody, which is specific for the light intermediate chain of cytoplasmic dynein as indicated. Bar, 10 µm.
[View Larger Version of this Image (108K GIF file)]
, 1996
) are perturbed by the addition of this dynein-specific antibody. Addition of the
control antibody to the extract either before mitotic aster
assembly (Fig. 5 A) or after mitotic aster assembly (data
not shown) had no observable effect on the organization of microtubules within the mitotic asters or the localization of NuMA at the central core of each aster. Addition
of the dynein-specific antibody, however, prevented the
assembly of mitotic asters if added to the extract before induction of aster assembly and perturbed the organization
of preassembled mitotic asters if it was added to the extract after the induction of aster assembly (Fig. 5, B and
C). In both cases, NuMA was associated with the microtubules but was distributed along the length of many of the
microtubules instead of concentrated at any one position.
This indicates that addition of this antibody before mitotic
aster formation prevented the accumulation of NuMA in
any one position, while addition of this antibody to pre-
assembled mitotic asters caused the displacement of
NuMA from the region near the microtubule minus ends.
Thus, perturbation of cytoplasmic dynein function by the
addition of this antibody is sufficient to not only prevent
the assembly of mitotic asters in this system but also to disrupt the organization of pre-assembled mitotic asters in a
manner very similar to the disruption of the polar regions
of mitotic spindles in cultured cells (see above) and in
acentrosomal spindles assembled in extracts prepared
from Xenopus eggs (Heald et al., 1996
).
Fig. 5.
The dynein-specific 70.1 antibody disrupts both the formation and maintenance of mitotic asters assembled in a cell-free
mitotic extract. The control antibody (A) and the dynein-specific
70.1 antibody (B and C) were added to a HeLa cell mitotic extract either before (A and B) or after (C) the induction of mitotic
aster assembly by the addition of taxol and incubation at 30°C.
After incubation, a portion of the sample was fixed and processed
for immunofluorescence microscopy (A-C) using antibodies specific for tubulin and NuMA as indicated. The remainder of the
sample, in which either the control antibody (154) or the dynein-specific antibody (70.1) were added before (PRE) or after
(POST) mitotic aster assembly, was separated into 10,000-g soluble (S) and insoluble (P) fractions. These fractions were subjected to immunoblot analysis using antibodies specific for
NuMA, Eg5, cytoplasmic dynein, and dynactin as indicated (D).
Bar, 10 µm.
[View Larger Version of this Image (61K GIF file)]
). Fig. 5 D shows that 15% of dynactin
is associated with the mitotic asters in the insoluble pellet
in the presence of the control antibody, but <5% of dynactin is found in the insoluble fraction in the presence of
the dynein-specific antibody (Fig. 5 D; percentages determined by densitometry). The alteration in the efficiency
with which dynactin associates with the microtubules in
the presence of the dynein-specific antibody was not dependent on when the antibody was added to the extract,
because the same result was obtained if the dynein-specific
antibody was added before mitotic aster assembly or if the
antibody was added to pre-assembled mitotic asters. This
effect on dynactin, while indirect because the antibody is
directed against cytoplasmic dynein (Fig. 1), is specific because the efficiency with which both NuMA and Eg5 associated with microtubules in the presence of this antibody was not appreciably altered. Thus, these data suggest that
in addition to its direct effects on cytoplasmic dynein, this
dynein-specific antibody perturbs the efficiency with which
dynactin associates with microtubules in this system.
). Thus, if the
depletion of cytoplasmic dynein is functionally equivalent
to the addition of the dynein-specific 70.1 antibody, then
mitotic asters should be observed after either the simultaneous depletion of cytoplasmic dynein and Eg5 or addition of the 70.1 antibody to an Eg5-depleted extract. Fig. 6
shows that in the absence of Eg5 alone, microtubules organize into astral arrays that are larger than typical mitotic
asters, they lack a well formed central core, and NuMA is
diffusely localized at the center (Fig. 6 B; Gaglio et al.,
1996
). In the absence of both Eg5 and cytoplasmic dynein,
mitotic asters form that resemble those formed in the
absence of Eg5 alone, although they are somewhat less
well organized (Fig. 6 C; Gaglio et al., 1996
). Thus, if the
70.1 antibody only affects the function of cytoplasmic dynein, then addition of the 70.1 antibody to an Eg5-depleted extract should also yield asters. Contrary to this
prediction, mitotic asters did not form when the 70.1 antibody was added to an Eg5-depleted extract, while addition
of the control antibody had no observable effect on the
formation of astral microtubule arrays (Fig. 6, D and E).
Thus, in this cell-free system the depletion of cytoplasmic dynein is not functionally equivalent to the addition of this dynein-specific antibody. The most likely explanation for
this difference is that the dynein-specific antibody both directly affects cytoplasmic dynein and indirectly affects dynactin such that the presence of the 70.1 antibody is analogous to the simultaneous disruption of dynein and dynactin.
While we can not rule out the possibility that this antibody
has additional deleterious effects beyond the perturbation
of cytoplasmic dynein and dynactin, this interpretation is
consistent with our previous results showing that the depletion of dynactin is more deleterious to mitotic aster formation than the depletion of cytoplasmic dynein (Gaglio et al., 1996
).
Fig. 6.
The addition of mAb 70.1 to the cell-free mitotic aster
assembly system is more deleterious to mitotic aster assembly
than the depletion of cytoplasmic dynein. The cell-free HeLa mitotic extract was depleted using either a preimmune antibody (A)
or an Eg5-specific antibody (B-E). The Eg5-depleted samples
were further treated by either the depletion of cytoplasmic dynein (C) or the addition of the dynein-specific (D) or control (E) antibodies. After the induction of mitotic aster assembly under these conditions, the samples were fixed and processed for immunofluorescence microscopy using antibodies specific for tubulin
and NuMA as indicated. Bar, 10 µm.
[View Larger Version of this Image (51K GIF file)]
). In contrast, the staining intensity for dynactin at the spindle
poles is significantly reduced after the microinjection of
the dynein-specific antibody (Fig. 7 B). The staining intensity for dynactin at centrosomes of adjacent, uninjected
cells is equivalent, indicating that the staining efficiency was similar for the two samples (Fig. 7, A and B, arrowheads). Thus, while this result is difficult to quantitate, it
indicates that the dynein-specific antibody is affecting the
efficiency with which dynactin associates with the mitotic
spindle in vivo, consistent with our observations using the
cell-free system for mitotic aster assembly. The disruption
of both cytoplasmic dynein and dynactin by the dynein-specific 70.1 antibody suggests that cytoplasmic dynein
and dynactin associate with microtubules in a cooperative
manner, which is consistent with the following: the current
models for the interaction of dynein and dynactin with microtubules (Allan 1996
; Schroer et al., 1996
); the original functional characterization of dynactin (Schroer and
Sheetz, 1991
); the fact that both dynein and dynactin have
microtubule binding domains (Waterman-Storer et al.,
1995
); our previous data showing that the efficiency with
which cytoplasmic dynein associates with microtubules in
a cell-free mitotic extract can be modulated by manipulating the quantity of dynactin in the extract (Gaglio et al.,
1996
); and the reduction in the association of cytoplasmic dynein with mitotic structures following the disruption of
dynactin (Echeverri et al., 1996
). This interpretation offers
the most likely explanation for the discrepancy between
the present data and the results of Vaisberg et al. (1993)
,
who previously investigated the role of cytoplasmic dynein
in the assembly of the mitotic spindle by microinjection of
an antibody directed against the cytoplasmic dynein heavy
chain. Their antibody, while inhibitory to dynein-mediated
motility in vitro, is not known to inhibit dynein-mediated
motility in vivo and would not be predicted to disrupt the
interaction of dynein with dynactin, which is mediated by
the 74-kD light intermediate chain of dynein (Karki and
Holzbaur, 1995
; Vaughan and Vallee, 1995
). The 70.1 antibody, on the other hand, has effects on both cytoplasmic
dynein and dynactin, implying that cytoplasmic dynein and
dynactin act as a functionally relevant unit that may have
structural activities in addition to minus end-directed motor activity.
Fig. 7.
The dynein-specific 70.1 antibody causes a reduction in the efficiency with which dynactin associates with the mitotic spindle in vivo. Monkey CV-1 cells in metaphase with bipolar mitotic spindles were selected by phase contrast microscopy and microinjected
with either a control antibody (A) or the dynein-specific 70.1 monoclonal antibody (B). The cells were then fixed and processed for immunofluorescence microscopy using the DNA-specific dye DAPI and the 45A antibody, which is specific for the Arp1 subunit of dynactin as indicated. The arrowheads indicate centrosomal staining for dynactin in adjacent uninjected cells, which verifies that these two
samples were stained equivalently. Bar, 20 µm.
[View Larger Version of this Image (72K GIF file)]
Discussion
; Mazia,
1984
; Maniotis and Schliwa, 1991
; Zhang and Nickals, 1995a,b). Contrary to the traditional view that the formation of a spindle pole in somatic cells is a consequence of
this nucleation event, we demonstrate here that the noncentrosomal proteins cytoplasmic dynein and dynactin are
also required for both the formation and maintenance of
the organization of microtubule minus ends at the mitotic
spindle pole. Disruption of their activities using the dynein-specific 70.1 monoclonal antibody leads to the splaying of microtubule minus ends and disruption of the connection between the centrosome and the body of the
mitotic spindle. This result underscores the concept that
the mitotic spindle pole is not synonymous with a mitotic
aster nucleated from a centrosome, but that the spindle
pole is a specialized entity of noncentrosomal components that is superimposed onto the centrosomally nucleated microtubule aster.
)
and begins with the nucleation of microtubule asters from
centrosomes. The plus ends of these microtubules "search" the cytoplasm by rapidly converting between growing and
shrinking states and are "captured" and stabilized by kinetochores. At some point during the search and capture
process microtubules are released (or severed) from the
centrosome, and these microtubules become "focused" by
structural and motor proteins into a spindle pole with their
free minus ends near the centrosome (Fig. 8). The centrosome remains tethered to this newly focused array
through a lateral interaction between microtubules within
this array and astral microtubules that continue to emanate from the centrosome (Fig. 8).
Fig. 8.
The search-capture-focus model for mitotic spindle assembly. Microtubules in somatic cells are nucleated from centrosomes that form symmetrical mitotic asters. These microtubules are relatively unstable (dashed lines) and "search" the
cytoplasm by continuously converting between growing and
shrinking states (arrows). Occasionally a microtubule plus end
will contact a kinetochore and be "captured" and stabilized (solid
lines). At some point during the search and capture events, some
of the microtubules will release from the centrosome, resulting in
free microtubule minus ends. These free microtubule minus ends
are "focused" at the spindle pole by noncentrosomal proteins, including cytoplasmic dynein, dynactin, NuMA, Eg5, and a minus
end-directed kinesin-related protein. The centrosome is tethered
to this focused group of microtubules by the lateral interaction of
microtubules within this array and astral microtubules that emanate from the centrosome.
[View Larger Version of this Image (14K GIF file)]
-tubulin ring complexes
(Zheng et al., 1996). These short microtubules associate
randomly with chromatin in disorganized arrays and are then organized into parallel bundles (Steffen et al., 1986
;
Theurkauf and Hawley, 1992
; Heald et al., 1996
). In somatic cells, microtubules are nucleated from
-tubulin ring
complexes that are associated with centrosomes (Moritz et
al., 1996), which establishes an oriented microtubule array
(mitotic asters) with microtubule plus ends that search the
cytoplasm and are captured by kinetochores. Despite these differences in the initial search and capture stages of spindle formation, both systems appear to use a common
mechanism to "focus" the free microtubule minus ends
into spindle poles. The parallel bundles of microtubules in
acentrosomal spindles are focused at their minus ends
(Theurkauf and Hawley, 1992
; Matthies et al., 1996
; Merdes et al., 1996
; Heald et al., 1996
), while in centrosomal
spindles free microtubule minus ends are focused onto the
astral microtubule array. This process of focusing free microtubule minus ends is much more pronounced in acentrosomal meiotic systems, because unlike mitotic systems
in which microtubules are inherently focused from centrosomes, microtubules in acentrosomal meiotic systems
have no organization before the focusing activity exerted
by the noncentrosomal components.
;
Mitchison and Salmon, 1992
; Murray et al., 1996
). These
poles are functional because they support chromosome
segregation, and in one case it was observed that the microtubule turnover associated with poleward microtubule
flux continued to converge towards this pole (Mitchison and Salmon, 1992
). Second, centrosomes can be mechanically detached from the body of the mitotic spindle at
metaphase, and the minus ends of the mitotic spindle remain focused; and in some cases the chromosomes still migrate toward that focused collection of microtubule minus
ends despite the removal of the centrosome (Hiramoto and Nakano, 1988
; Nicklas, 1989
; Nicklas et al., 1989
).
Third, in rare cases where microtubules associate with
chromosomes in the absence of centrosomes and/or centrioles in cultured cells (Brenner et al., 1977
; Keyer et al.,
1984
; Debec et al., 1995
), or if the requirement for centrosomes in microtubule nucleation is bypassed by the addition of taxol both in vivo during mitosis (DeBrabander
et al., 1981
) and in vitro in mitotic extracts (Gaglio et al.,
1995
, 1996
), then microtubules are still organized into astral and polar arrays. The focusing of microtubule minus
ends under these conditions is likely the manifestation of
the microtubule minus end focusing activity exerted by the
noncentrosomal components. Fourth, electron microscopy
has shown that many microtubule minus ends within both
mitotic and centrosome-containing meiotic spindles are
not associated with centrosomes (Wolf and Bastmeyer,
1991
; Mastronarde et al., 1993
). Fifth, microtubule release
from centrosomes has been documented in cell-free extracts (Belmont et al., 1990
) as well as under nonmitotic
circumstances in living cells (Keating et al., 1997
). Finally,
free microtubule minus ends within the mitotic spindle are
necessary for tubulin subunit loss during poleward microtubule flux (Mitchison, 1989b
). Taken together, these results clearly discriminate between the processes of microtubule nucleation and minus end focusing during mitosis
in somatic cells, and demonstrate that noncentrosomal
structural and motor proteins will focus microtubule minus ends independently of centrosomes in somatic cells
through a mechanism that is probably related to spindle
pole formation in acentrosomal systems.
; Sawin et al., 1993; Endow et al., 1994
;
Gaglio et al., 1995
, 1996
; Blangy et al., 1996; Heald et al.,
1996
; Matthies et al., 1996
; Merdes et al., 1996
; Walczak et
al., 1997
). Experimental data indicate that all of these proteins participate in the organization of spindle poles in
both centrosomal and acentrosomal systems. Despite the
complex nature of this process and the involvement of numerous components, recent evidence suggests that a trimolecular complex composed of NuMA, cytoplasmic dynein, and dynactin may be crucial for focusing free
microtubule minus ends at spindle poles. These three
proteins form a stable complex in extracts prepared from
Xenopus eggs, and this complex of proteins is essential for
the organization of spindle poles in that system (Merdes et
al., 1996
). While no evidence exists for a stable complex between these proteins in extracts prepared from somatic
cells, we show that NuMA fails to concentrate near microtubule minus ends in the absence of dynein and/or dynactin under both in vitro and in vivo conditions, consistent
with a dynein/dynactin-dependent movement of NuMA to
microtubule minus ends (Figs. 3 and 5, and Gaglio et al.,
1996
). Indeed, in a striking group of experiments, the perturbation of either cytoplasmic dynein (mAb 70.1 microinjection; present work), dynactin (dynamitin over expression; Echeverri et al., 1996
; Gaglio et al., 1996
), or NuMA
(antibody microinjection; Gaglio et al., 1995
) in cultured
cells all produced a similar effect on the spindle pole organization characterized by splaying of microtubule minus
ends and detachment of centrosomes. Given that the interaction of NuMA with cytoplasmic dynein and dynactin is
mitosis-specific in somatic cells (NuMA is nuclear during
interphase), it is possible that NuMA confers a unique mitosis-specific property to the minus end-directed motor activity of cytoplasmic dynein and dynactin, which contributes to the essential function of this trimolecular complex
during spindle formation.
) and (depending on the cell type) contributes to poleward chromosome movement (Salmon, 1992;
Wilson et al., 1994
; Mitchison and Salmon, 1992
; Zhai et al., 1995
). Free microtubule minus ends are inherently produced in acentrosomal spindles, whereas in centrosomal
spindles they must be generated by microtubule release
from centrosomes. In both cases, a common pole-forming
activity focuses the free microtubule ends. In somatic cells,
these free microtubule minus ends that are obligatory to
poleward microtubule flux remain attached to the astral
microtubules emanating from the centrosome, thus allowing the centrosome-associated astral array to convey positional cues derived from the cell cortex to the body of the
spindle.
Received for publication 19 June 1997 and in revised form 14 July 1997.
1. Abbreviation used in this paper: NuMA, nuclear mitotic apparatus.This work was supported by grants from the National Institutes of Health (GM51542) and American Cancer Society (JFRA-635).
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