* Ludwig Institute for Cancer Research; and Division of Cellular and Molecular Medicine, University of California at San Diego,
La Jolla, California 92093-0660
A central event during cell division is the transformation of an interphase network of microtubules into
a bipolar spindle. For most animal cells the centrosomes, a pair of centrioles surrounded by electron-dense pericentriolar material, represent the microtubule organizing centers from which interphase microtubules
are nucleated, with the microtubule minus ends at the pole
and the rapidly growing, free plus ends extending away.
At, or just before, the time of nuclear envelope fragmentation, the duplicated centrosomes separate from each other
using microtubule-dependent motors that push against the
astral microtubules nucleated by each centrosome. Microtubules penetrate the nucleus, and in a mechanism called
"search and capture" (Kirschner and Mitchison, 1986
Attractive as this paradigm is, several examples of meiotic spindles, as well as early embryonic mitotic spindles in
animals, have been found without centrosomes, displaying
a spindle morphology that is more reminiscent of a barrel
shape and lacking astral microtubules. Also, many plant
cells are devoid of morphologically recognizable centrosomes. Several proposals to explain this discrepancy have been offered, perhaps the most prominent of which
was that of Mazia (1984) Spindle Poles without Centrosomes
How do spindles form in the absence of preexisiting microtubule organizing sites? Live observations of meiotic
spindle formation in Drosophila oocytes (Matthies et al.,
1996
Dynein-dependent Spindle Pole Assembly with or
without Centrosomes
The concept of centrosome-free spindle pole formation
depending on the action of microtubule motors was directly demonstrated during spindle assembly in extracts
from metaphase-arrested frog eggs (Heald et al., 1996 A Complex of Cytoplasmic Dynein, Dynactin, and
NuMA Tethers Centrosomes to Spindle Microtubules
Through what mechanism can dynein provide stability to
spindle poles with or without centrosomes? In work using
Xenopus egg extracts, Heald et al. (1997) Cytoplasmic dynein-dependent microtubule tethering at
poles requires at least two microtubule binding sites that
in principle could be achieved by the dimeric dynein heavy
chain head domains. However, other efforts have clearly
demonstrated that dynein does not act by itself. Rather,
dynein-associated proteins are needed, including its motility-activating complex dynactin (Gaglio et al., 1996 Several lines of evidence demonstrate that NuMA plays
a critical role in microtubule tethering at poles. Immunodepletion of the NuMA/cytoplasmic dynein/dynactin
complex from frog egg extracts does not affect assembly of
a bi-oriented array of microtubules with centrally oriented
chromosomes but does completely block the aggregation
of the microtubule minus ends into focused spindle poles,
despite the presence of centrosomes (Merdes et al., 1996 The simplest view is that microtubule tethering into
poles is mediated by a large complex containing NuMA,
dynein, and dynactin, using the motor activity of dynein to
power the complex toward the microtuble minus ends and
the distinct microtubule binding sites on NuMA (Merdes
et al., 1996 Why Is Microtubule Tethering to Spindle Poles Needed?
From the viewpoint of the centrosome as the nucleator of
spindle microtubules, why is a NuMA/dynactin/cytoplasmic dynein complex necessary for pole assembly? The
most obvious and direct answer emerged initially from serial sectioning of a mammalian mitotic spindle. Unlike
many simplified text book views, this revealed that up to
75% of the interpolar microtubules do not connect directly to the centrosome but end within a distance of While Heald et al. (1997) Centrosomes Versus No Centrosomes
With many conserved components between centrosome-containing and centrosome-free spindles, a key difference
may simply be the high abundance of these components in
eggs and early embryos that may enable these systems to
form spindles by self organization of microtubules through
recruitment of any necessary factors from the large cytoplasmic pool. In somatic cells, such supplies are more limited, and spatial constraints within the cell, as well as the
requirement of a specific orientation of the cell in a tissue,
may favor spindle formation from preexisting, centrosomal microtubule organizing centers. As demonstrated in
the paper by Heald et al. (1997))
some attach to kinetochores, specialized regions that assemble onto the surface of centromeres (Fig. 1 A). As a result, most mitotic animal cells have spindles with two
clearly defined spindle poles at which the microtubules
(kinetochore attached, pole-to-pole, or astral) converge in
a focal area around each centrosome (Fig. 1 B). This, and
the proven ability of centrosomes to nucleate microtubules efficiently in vitro (Mitchison and Kirschner, 1984
),
have fueled the general view that centrosomal microtubule organizing centers are essential features of spindle assembly and organization.
Fig. 1.
Spindle formation in centrosome-containing cells. (a)
Microtubules are nucleated from the duplicated centrosomes
with their growing plus ends pointing away from the centrosomes. Microtubules that penetrate the perforated nuclear envelope in prometaphase are captured by the kinetochores of the
chromosomes. Multivalent plus end-directed motors of the bimC
family may be involved in the separation of the two centrosomes
and the establishment of a symmetric spindle axis (big arrows).
(b) In the mature spindle, microtubule minus ends disconnect
from the centrosomes and are anchored to the body of the spindle by complexes of NuMA/dynein/dynactin. (The chromosomes
are indicated in blue.)
[View Larger Version of this Image (36K GIF file)]
, who suggested the existence of
flexible centrosomal material, aligned on a ribbon-like
structure that can fold or extend in cell-type specific ways
and act in microtubule organization in all cells. Two candidate proteins for such microtubule organizing material are
tubulin and pericentrin, both centrosomal components in "conventional" spindles.
Tubulin has been found in
ring-like structures (Zheng et al., 1995
) that may be
aligned on a cage-like lattice, most likely provided by pericentrin (Dictenberg, J., W. Carrington, F.S. Fay, and S.F.
Doxsey. 1995. Mol. Biol. Cell. 6:40a), and both proteins
are found at poles in mouse oocytes and early embryos, although no centrosomes can be detected (Gueth-Hallonet et al., 1993
). Surprisingly, however, there are mitotic and
meiotic cells (e.g., in Drosophila; Matthies et al., 1996
;
Wilson et al., 1997
) that appear to have neither centrosomes nor detectable amounts of
tubulin; furthermore, pericentrin is dispensable for centrosome-independent formation of microtubule asters and half spindles in
vitro (Kallajoki et al., 1992
; Gaglio et al., 1996
).
) have revealed that the spindles form by an "inside-out" mechanism in which microtubules reorganize around
the mass of chromatin (Fig. 2 A). This process may involve
the action of chromatin-bound, plus end-directed microtubule motors, including such candidates as the chromatin-associated, kinesin-like proteins chromokinesin (Wang
and Adler, 1995
), its frog homologue Xklp1 (Vernos et al.,
1995
), and Drosophila Nod (Afshar et al., 1995
). With the
microtubule minus ends oriented away from the chromatin in these developing spindles, the organization of the
microtubules into bipolar spindles may then be achieved by the action of multivalent, minus end-directed microtubule motor complexes that can tether parallel-oriented microtubules into bundles and stabilize converging microtubules into poles (Fig. 2 B). As Matthies et al. (1996)
showed, in Drosophila oocytes this process is clearly dependent on the presence of the minus end-directed motor
Ncd, although there seem to be other motor proteins with
redundant functions involved.
Fig. 2.
Spindle formation in centrosome-free cells. (a) Spindle
formation is driven by chromatin-associated, plus end-directed
microtubule motors, orienting chromatin-attached microtubules
with their minus ends outward (arrows). Multivalent plus end-directed microtubule motors of the bimC family can interconnect
antiparallel microtubules and establish a bipolar organization of
the spindle by moving the microtubule ends apart. (b) During
spindle pole formation, complexes composed of NuMA, dynein,
and dynactin induce convergent arrays of microtubules at the
spindle poles and provide stability to the spindle by tethering the
microtubule minus ends.
[View Larger Version of this Image (34K GIF file)]
).
Using DNA-coated beads as chromosomal substitutes, microtubules were nucleated and organized into a bipolar
spindle apparatus without specialized centromere sequences on the DNA and without centrosomes at the
poles. Addition of an antibody to the intermediate chain
subunit of the microtubule motor cytoplasmic dynein
blocked the organization of microtubule arrays into focused poles without affecting the assembly of a bi-oriented
array of microtubules emerging from the centrally localized, bead-bound DNA. The establishment of bipolarity
without centrosomes thus involves two independent mechanisms. The first is sorting of microtubules into a bipolar
axial array, which may be achieved by plus end-directed,
multimeric motors that can promote anti-parallel microtubule sliding and axial alignment. Candidates for such an
activity are the tetrameric motors of the BimC kinesin family, such as Eg5 and KRP130 (Kashina et al., 1996
).
The second is bundling of these oriented microtubules into
poles, involving the minus end-directed, microtubule motor cytoplasmic dynein. Consistent with this, dynein has
been implicated by a variety of in vitro studies in frog and
mammalian mitotic extracts in which dynein-dependent,
centrosome-free spindle pole formation was mimicked by
the induction of microtubule asters in the presence of the
drug taxol (Verde et al., 1991
; Gaglio et al., 1996
), as well
as by immunolocalization of dynein on spindle poles in dividing cells (Pfarr et al., 1990
; Steuer et al., 1990
).
showed that addition of an antibody against cytoplasmic dynein intermediate chain blocks the translocation of fluorescently tagged
spindle microtubules along each other. Since organization
of microtubules into poles is also blocked by this same dynein antibody, the evidence suggests that minus end-directed
microtubule gliding is a prerequisite to organize microtubules into convergent polar arrays. Furthermore, Heald et
al. (1997)
demonstrated that dynein acts as a microtubule
tethering factor at the spindle poles, irrespective of the
presence or absence of centrosomes.
) and
NuMA, a 240-kD protein with a ~1,500-amino acid-long helical domain, separating globular head and tail regions.
NuMA is nuclear during interphase but localizes to the
spindle poles in mitosis as well as to centrosome-free spindles in meiosis (Tang, T.K., C.J.C. Tang, and H.M. Hu.
1995. Mol. Biol. Cell. 6:422a; Navara, C.S., C. Simerly,
D.A. Compton, and G. Shatten. 1996. Mol. Biol. Cell. 7:
208a). The NuMA tail binds to microtubules in vitro, and
NuMA in frog egg extracts is associated in a nearly stoichiometric complex with cytoplasmic dynein and dynactin
(Merdes et al., 1996
).
). As there is a many-fold excess of cytoplasmic dynein and
dynactin over NuMA, disruption of pole formation must
reflect a necessity for NuMA not diminution of cytoplasmic dynein or dynactin. This phenotype is almost indistinguishable from the effect of inhibitory anti-dynein antibodies added to a similar spindle formation assay (Heald
et al., 1996
, 1997
). Similarly, immunodepletion of NuMA from mammalian mitotic extracts completely blocks taxol-induced microtubule aster formation (Gaglio et al., 1995
,
1996
), as does addition of a monoclonal antibody to the
dynein intermediate chain. The latter, reported on pages
1055-1066 of this issue, apparently leads to the disconnection of dynein from dynactin (Gaglio et al., 1997
), even
though this antibody does not affect the motility of purified dynein itself (Heald et al., 1997
). Further, depletion of
either cytoplasmic dynein or dynactin in the taxol-induced aster formation assay yielded only randomly oriented microtubules with NuMA scattered all over the microtubule
length. Direct support for an involvement of dynactin in
spindle formation came from overexpression of p50/dynamitin, one of the nine known components of the dynactin
complex. This caused disruption of the complex and resulted in aberrant spindle morphology with irregular poles (Echeverri et al., 1996).
) and the associated p150 dynactin component
(Karki and Holzbaur, 1995
; Vaughan and Vallee, 1995
) to
provide the needed crosslinking. The displacement of
NuMA upon dynein or dynactin depletion or upon microinjection of anti-dynein antibody into cells (Gaglio et al.,
1997
) supports the idea that NuMA is one of the specific
cargos of the dynein motor during cell division.
1
µm thereof (Mastronarde et al., 1993
). Hence, most spindle microtubules cannot be directly attached to the pole.
Moreover, removal of the centrosome by micromanipulation does not grossly affect the integrity of the spindle
(Nicklas, 1989
; Nicklas et al., 1989
). A plausible model for
what keeps these microtubules in place invokes the NuMA
complex, which is distributed in a broad, crescent-shaped
area between the centrosome and the spindle microtubule
bundles, rather than focused directly at the centrosome.
NuMA thus is likely to be one of the connecting molecules
that anchor the large number of free microtubule minus ends to the microtubules still directly nucleated by the centrosome. Furthermore, as Heald et al. (1997)
and Gaglio et
al. (1997)
now demonstrate, cytoplasmic dynein plays an
essential role in linking centrosomes to spindles. Addition
of one dynein intermediate chain antibody to spindles
formed in vitro (Heald et al., 1997
), as well as microinjection of that same antibody into cultured cells (Gaglio et
al., 1997
), leads to the disconnection of the centrosome from the rest of the spindle. Similar effects were previously observed upon microinjection of anti-NuMA antibodies
(Gaglio et al., 1995
) or overexpression of p50/dynamitin of
the dynactin complex (Echeverri et al., 1996). Thus, in
both centrosome-free and centrosome-containing spindles, NuMA, dynein, and dynactin are involved in stabilizing the spindle poles.
do demonstrate that there is
dynein-dependent poleward flow of labeled microtubules
added in vitro and provide a plausible explanation for pole
formation by dynein-driven poleward microtubule movement in noncentrosomal meiotic spindles, it seems less
likely that this reflects the in vivo mechanism of pole formation in centrosome-containing cells, because the dominant microtubule nucleation site is located at the centrosome and not at the kinetochore (Mitchison et al., 1986
;
Geuens et al., 1989
). Thus, early in mitosis, the majority of
the microtubules should be centrosome bound, but as the
mitotic cycle proceeds, some of these disconnect from
their nucleation centers. The newly freed (possibly uncapped) minus ends may be essential for a mechanism called "poleward microtubule flux," seen in metaphase
and anaphase of mitosis. Poleward flux involves translocation of microtubules towards the spindle poles, while the
microtubule plus ends at the spindle equator elongate and
the minus ends at the poles shorten simultaneously. This
flux is likely to be powered by microtubule motors such as
dynein bound to the moving microtubules or alternatively, plus end-directed microtubule motor proteins such as Eg5
or Xklp2 (Sawin et al., 1992
; Boleti et al., 1996
) bound to
an immobilized spindle pole matrix, comprised, at least in
part, of NuMA. Further evidence consistent with this is
the ability of NuMA to form a filamentous meshwork
when overexpressed in the cytoplasm (Saredi et al., 1996
),
as well as unusually long spindle-like structures assembled
in the absence of NuMA (Merdes et al., 1996
), suggesting the loss of part of the "flux" machinery necessary for the
shortening of microtubule minus ends.
, the potential of centrosomes to organize microtubules provides a kinetic advantage to the cell, and when present, centrosomes are
dominant over self assembly of microtubules around chromatin. In both cases of spindle assembly, however, the underlying principles of pole organization relying on NuMA/ cytoplasmic dynein/dynactin-dependent microtubule tethering remain largely similar.
Received for publication 20 July 1997 and in revised form 31 July 1997.
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