1 Department of Biology, Indiana University, Bloomington, IN 47405, USA
2 Howard Hughes Medical Institute, Indiana University, Bloomington, IN 47405,
USA
* Present address: Cecil H. and Ida Green Center for Reproductive Biology
Sciences and Dept of Pharmacology, University of Texas Southwestern Medical
Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9051, USA
Author for correspondence (e-mail:
kaufman{at}bio.indiana.edu)
Accepted 27 August 2002
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Summary |
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Key words: Drosophila, Centrosomes, Centrosomin (Cnn), PCM, Flares
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Introduction |
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In the absence of centrosomes, the bipolar spindle assembles by an
alternate, anastral, pathway where the microtubules are nucleated on the
chromosomes and subsequently organized into the bipolar spindle with the minus
ends of the microtubules focused at the poles
(Hyman, 2000;
Compton, 2000
). This process
involves the coordinate activities of motor proteins
(Wittmann et al., 2001
;
Walczak, 2000
;
Heald, 2000
;
Sharp et al., 2000
;
Compton, 2000
). Since
centrioles are degraded during oogenesis in many species, this alternate
spindle assembly pathway appears to act exclusively during female meiosis
(Theurkauf and Hawley, 1992
;
Rieder et al., 1993
;
McKim and Hawley, 1995
;
Megraw and Kaufman, 2000
;
Compton, 2000
).
Found throughout the animal kingdom, the centrosome consists of over 100
proteins (Kalt and Schliwa,
1993; Kellogg et al.,
1994
). At the heart of the centrosome lies a pair of centrioles.
These are surrounded by the pericentriolar material (PCM), which has a
filamentous structure (Dictenberg et al.,
1998
; Schnackenberg et al.,
1998
; Schnackenberg and
Palazzo, 1999
). The centromatrix, a substructure of the PCM, is a
filamentous basal component of the centrosome that remains after many PCM
proteins are removed by high salt
(Schnackenberg et al., 1998
;
Schnackenberg and Palazzo,
1999
; Moritz et al.,
1998
; Dictenberg et al.,
1998
; Palazzo et al.,
2000
). Additionally,
-tubulin ring complexes (
TURCs)
are bound to the PCM and are the sites of microtubule nucleation at the
centrosome (Gunawardane et al.,
2000
).
Cnn is a core centrosome constituent, and a major protein component of
purified Drosophila centrosomes
(Lange et al., 2000), but it
is not part of the centromatrix (Moritz et
al., 1998
). In the hierarchical assembly of the centrosome
(Palazzo et al., 2000
), Cnn
lies between the centromatrix and the incorporation of other PCM components
including CP60, CP190 and
-tubulin
(Megraw et al., 1999
;
Megraw et al., 2001
). Changes
in the centrosome composition with the cell cycle are reflected in the
discovery that Cnn is required for
-tubulin accumulation at mitotic,
but not interphase, centrosomes (Megraw et
al., 2001
).
Currently, the role of the centrosome in cell division is being
re-evaluated (Rieder et al.,
2001; Marshall,
2001
; Raff, 2001
;
Hyman, 2000
;
de Saint Phalle and Sullivan,
1998
; Bonaccorsi et al.,
1998
; Megraw et al.,
1999
; Vaizel-Ohayon and
Schejter, 1999
; Bonaccorsi et
al., 2000
; Khodjakov et al.,
2000
; Megraw et al.,
2001
; Khodjakov and Rieder,
2001
; Hinchcliffe et al.,
2001
; Piel et al.,
2001
; Giansanti et al.,
2001
). In the absence of centrosomes, or in cells where functional
centrosomes are not assembled, such as in asterless or
centrosomin (cnn) mutants, the bipolar spindle apparatus is
assembled by the anastral pathway described above
(de Saint Phalle and Sullivan,
1998
; Bonaccorsi et al.,
1998
; Megraw et al.,
1999
; Vaizel-Ohayon and
Schejter, 1999
; Bonaccorsi et
al., 2000
; Khodjakov et al.,
2000
; Megraw et al.,
2001
; Khodjakov and Rieder,
2001
; Hinchcliffe et al.,
2001
). When centrosomes are completely removed from cultured cells
by laser ablation (Khodjakov et al.,
2000
; Khodjakov and Rieder,
2001
) or by microsurgery
(Hinchcliffe et al., 2001
),
the cells enter mitosis and divide, but then arrest at G1. However, a
Drosophila cell line that lacks centrosomes can be perpetually
maintained, although a delay at cytokinesis is observed
(Debec and Abbadie, 1989
;
Piel et al., 2001
). These
results demonstrate that bipolar spindle assembly can occur efficiently
without centrosomes in vivo and in vitro. Furthermore, much of
Drosophila development can occur without fully functional mitotic
centrosomes (Megraw et al.,
2001
). These data also indicate that although the centrosome plays
an essential role in the cell cycle, it is not required at mitosis but for
progression through G1 (Khodjakov and
Rieder, 2001
; Hinchcliffe et
al., 2001
; Piel et al.,
2001
). The mother centriole may mediate a checkpoint at G1 by
migrating to the midbody and signaling the completion of cytokinesis
(Piel et al., 2001
). Whatever
its total functional repertoire, it is evident that the centrosome has
functions that change with the cell cycle.
In Drosophila cnn mutants, the mitotic centrosome is not fully
functional, but the interphase centrosome may be normal
(Megraw et al., 2001).
cnn mutant animals develop into adults, with their mitotic divisions
occurring by the anastral pathway for bipolar spindle assembly
(Megraw et al., 2001
).
cnn mutant females produce no offspring because the eggs they lay
arrest in early embryogenesis (Megraw et
al., 1999
; Vaizel-Ohayon and
Schejter, 1999
) due, at least in part, to the fusion of nuclei at
telophase (T.L.M., S.K., F.R.T. and T.C.K., unpublished).
In the early Drosophila embryo, where the first 13 cleavage
divisions occur synchronously in a syncytium, the centrosome has an
indispensable role. Here, spindle assembly and nuclear divisions can occur in
the absence of centrosomes, but the actin cytoskeleton does not organize
properly and cellularization of the blastoderm does not occur
(de Saint Phalle and Sullivan,
1998; Megraw et al.,
1999
; Vaizel-Ohayon and
Schejter, 1999
). Although the centrosome has long been viewed as
an MTOC, it also organizes the actin cytoskeleton and appears to function in
this capacity independently of microtubules
(Stevenson et al., 2001
).
Fluorophore-conjugated and GFP-fused centrosomal proteins have been used to
understand the assembly and dynamics of the centrosome in several studies
(Oegema et al., 1995;
Khodjakov and Rieder, 1999
;
Kubo et al., 1999
;
Young et al., 2000
;
Piel et al., 2000
;
White et al., 2000
;
Strome et al., 2001
;
Piel et al., 2001
).
Pericentrin is pre-assembled into particles with
-tubulin prior to
import/assembly into the centrosome
(Dictenberg et al., 1998
).
GFP-Pericentrin revealed the presence of Pericentrin particles in the cell
that move in a microtubule- and dynein-dependent manner into the centrosome
(Young et al., 2000
).
GFP-
-tubulin was used to show that
-tubulin dynamically
exchanged at the centrosome and is accumulated at the centrosome in higher
levels at mitosis in a microtubule-independent manner
(Khodjakov and Rieder, 1999
).
Using GFP-centrin, the dynamics of the centrioles and their genesis were
examined (Piel et al., 2000
;
White et al., 2000
). With
GFP-Centrin it was shown that the mother and daughter centrioles have a far
less static relationship with one another than was previously thought,
especially following telophase, and the movements of mother and daughter
centrioles depend upon the microtubule and actin cytoskeletons
(Piel et al., 2000
;
Piel et al., 2001
). GFP-PCM-1
was used to show the dynamics of satellites, which are subcentrosomal
structures of the PCM. In this study, it was shown that satellites are very
dynamic and move in a microtubule-dependent manner back-and-forth from the
centrosome (Kubo et al.,
1999
). Taken together, live imaging of centrosomes and its
components has revealed some surprisingly dynamic features of this organelle.
Here, we show that during the syncytial cleavage divisions of the early
Drosophila embryo, the centrosome has a dynamic structure, from which
PCM material is ejected. Ejected particles of PCM material move upon
microtubules, have properties in common with vertebrate satellites and may
have a relationship with actin cytoskeleton organization.
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Materials and Methods |
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Drosophila stocks
Transgenic lines were recovered following microinjection of plasmids
directly into early embryos collected over a 30-40 minute period as described
(Robertson et al., 1988). We
injected plasmids at a concentration of 1 mg/ml in water directly through the
chorion and vitelline membrane of early embryos of the genotype:
w*;TMS, P{ry+t7.2=Delta2-3}99B/TM6B, Tb1.
Embryos were collected, rinsed with water, arranged onto coverslips in a
parallel register with a spacing of approximately two embryos' width and then
blotted dry with a paintbrush. After drying briefly, the embryos stick to the
coverslip. Embryos were injected at their posterior end. We recovered
transgenic lines of the pUASpGFP-Cnn plasmid with inserts on chromosomes I, II
and III (w*; P{w+mC=UASp-GFP-Cnn1}). Two stocks were
used in this study, lines 26-1 and 29-3. To express pUASpGFP-Cnn and
GFP-
-tubulin in the embryo a stock that expresses Gal4 in the ovary
from the nanos promoter was used
(P{GAL4::VP16-nos.UTR}MVD1)
(Van Doren et al., 1998
). A
fly stock expressing a GFP-Histone 2A variant (w1118;
P{w+mC=His2AvT:Avic\GFP-S65T}62A)
(Clarkson and Saint, 1999
) was
a gift from Rob Saint. The GFP-
-tubulin
(P{w+,UASp-GFPS65C-al-tub84B}) stock, an insertion on chromosome
III, (Grieder et al., 2000
)
was a gift from Allan Spradling. The cnn allele used in these studies
was the null cnnhk21
(Megraw et al., 1999
). All
mutant animals analyzed were hemizygous
cnnhk21/Df(2R)cnn.
Immunostaining
Embryos were fixed using either MeOH or formaldehyde fixation according to
Theurkauf (Theurkauf, 1994).
Fixed embryos were incubated with Guinea pig antibodies raised against Cnn at
a 1:1000 dilution, anti-
-tubulin monoclonal antibody DM1A (Sigma) at
1:500, and the fluorescent DNA dye TOTO-3 (Molecular Probes) at 1:400. Rabbit
antibodies to D-TACC, a gift from Bill Theurkauf, were used at a 1:500
dilution. Fluorescent secondary antibodies (Jackson ImmunoResearch Labs) were
used at a 1:200 dilution. In order to detect Cnn colocalization with membrane
bearing particles or Golgi, embryos were stained for Cnn and Discontinuous
actin hexagon (Dah), or Lava lamp (Lva) or beta-Coatomer protein (ß-Cop).
Rabbit Dah (1:120) (Zhang et al.,
1996
) and rabbit Lva (1:3000)
(Sisson et al., 2000
)
antibodies were generously provided by Bill Sullivan and John Sisson. Rabbit
anti-ß-Cop (1:200) (Ripoche et al.,
1994
) was a gift from Vivek Malhotra. Embryos were incubated with
primary antibodies and RNase A (50 µg/ml) overnight at room temperature
with rocking in 300 µl PBT [phosphate-buffered saline
(Theurkauf, 1994
) with 0.1%
Triton X-100]. Embryos were then rinsed three times with PBT, and then washed
three times for 20 minutes each with PBT. Secondary antibodies were diluted in
PBT and incubated with embryos with rocking for 90 minutes. Embryos were
rinsed and washed as above, then mounted on glass slides in PBS containing 90%
glycerol. Images were captured with a Leica TCS NT scanning confocal
microscope and saved as a maximal projection through several optical sections
totaling approximately 10 µm, then imported into Adobe Photoshop 5.0. For
deconvolution microscopy, images were captured on a Nikon TE 300 inverted
microscope and processed with Applied Precision deconvolution software. For
Fig. 4, images were captured
using an Olympus FV500 Confocal microscope equipped with an IX81 inverted
microscope.
|
|
Microinjection and live cell imaging
Embryos were collected for 1-2 hours, dechorionated by rolling on
double-stick tape, placed onto a coverslip that was coated with double-stick
tape extract and, for injections, dehydrated mildly
(Schubiger and Edgar, 1994).
The coverslip was mounted onto an upright microscope using a coverslip holder
with a gas permeable Teflon membrane
(Kiehart et al., 1994
). For
both microinjection and viewing of live material the embryos were mounted in
Halocarbon Oil 700. Colchicine (Sigma) and Cytochalasin-D (Sigma) were
dissolved in water and injected at a concentration of 0.1 mg/ml.
Rhodamine-tubulin (Molecular Probes, Inc) was injected at a concentration of
10 mg/ml. Rhodamine-actin (Cytoskeleton, Inc) was injected at a concentration
of 1 mg/ml. Embryos were treated with 10 µM Paclitaxel (Taxol, Sigma) in
Grace's Insect Medium (Invitrogen) while being permeated with high grade
octane (Sigma) for 30 seconds as described
(Raff et al., 1993
). Taxol
mock control embryos were similarly treated, except their medium contained
only 0.4% DMSO and no Taxol. Time-lapse images were collected every3 seconds
with a Leica TCS NT scanning confocal microscope using a 63x/1.2NA water
immersion lens. Images were processed for animation using NIH Image software
(http://rsb.info.nih.gov/nih-image/)
and, for the two-color images, Final Cut Pro software.
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Results |
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|
Cnn has a dynamic relationship with the centrosome
To examine Cnn and centrosome dynamics in living embryos, we constructed a
green fluorescent protein (GFP)-Cnn fusion construct that contains Gal4 UAS
transcription control elements 5' of the ORF and ovary permissive
control elements 5' and 3' of the ORF
(Rorth, 1998). This construct,
contained in a P element transformation vector, was integrated into the fly
genome. A nanos-Gal4 `driver' construct, in which nanos
transcription regulatory elements control the expression of the yeast Gal4 ORF
(Van Doren et al., 1998
), was
used to express GFP-Cnn during oogenesis. Embryos from mothers containing both
transgenes express GFP-Cnn in laid eggs. When this combination of ovarian Gal4
and GFP-Cnn constructs are present in a cnn mutant female, she
produces viable embryos, thereby rescuing the maternal effect lethal phenotype
associated with the cnn mutation
(Megraw et al., 1999
).
In the early embryo, GFP-Cnn is localized to the centrosome and more weakly to the mitotic spindle (Fig. 2). When live GFP-Cnn embryos were viewed by time-lapse imaging, the dynamics of centrosomes during the early embryonic cleavage cycles were apparent (Fig. 2; Movie 1, available at jcs.biologists.org/supplemental). Changes in Cnn intensity at the centrosome with the cell cycle are also apparent. The GFP-Cnn signal increases at the centrosome as the cleavage cycles progress from interphase into mitosis, beginning at prophase.
|
Closer inspection revealed that the centrosome and the punctate Cnn-containing particles seen by immunostaining (Fig. 1B) share an intimate and dynamic relationship (Fig. 3; Movie 2, available at jcs.biologists.org/supplemental). The particles emerge from the centrosome and move back and forth radially. We will refer to this process as `flaring' and will refer to the moving particles of PCM material as `flare particles'. Flare particles move in a saltatory fashion. They jump 0.2 to 1.0 µm in less than 3 seconds and then may continue in the same or in the reverse direction. The rate of movement of the flare particles could not be precisely measured because each step occurs more quickly than the time it takes to capture each individual image. From these observations, however, it is clear that flare movements are in the range of 0.06-0.3 µm/second.
|
The number of flares associated with centrosomes varies with the cell cycle. We compared the average number of flare particles associated with centrosomes at different stages of the cleavage cycle, and these data are presented in Table 1. The intensity of flares is highest at cleavage telophase/interphase centrosomes and lowest at mitotic centrosomes, especially during metaphase and anaphase. In addition, Cnn appears to be associated with metaphase/anaphase centrosomes more tightly, giving the centrosome a more rounded appearance, with fewer of the projections that spawn flare particles seen on centrosomes at other phases of the cleavage cycle (see Fig. 1B). Furthermore, at telophase when the centrosome is dividing, projections of Cnn material transiently span the divide between the separating centrosomes.
To observe the dynamics of both Cnn and microtubules in living embryos, fluorescent-rhodamine-labeled tubulin protein was injected into GFP-Cnn embryos (Fig. 4). In a recorded time-lapse series the formation of astral microtubules around centrosomes and the dynamics of the spindle through the cleavage cycle can be seen (Movie 3, available at jcs.biologists.org/supplemental).
Flares contain a subset of known centrosomal proteins
We examined the colocalization of several centrosomal proteins with Cnn in
wild-type syncytial stage embryos. We found that the core centrosomal protein
D-TACC colocalized with Cnn at centrosomes and flare particles
(Fig. 5). However, not all
Cnn-containing particles contained a detectable signal of D-TACC, and
conversely some D-TACC particles did not have a detectable amount of Cnn.
Immunostaining against the centrosomal proteins -tubulin and CP190
showed no signal at flare particles when co-stained with anti-Cnn antibodies
(data not shown).
|
Flares are dependent on microtubules for their activity
To investigate the involvement of microtubules in flare activity, we
injected the microtubule-destabilizing drug colchicine into GFP-Cnn embryos.
The inhibition of microtubule polymerization caused the cleavage cycle to
arrest and the normal movement of flare particles to cease
(Fig. 6A). Specifically, the
centrosomes in colchicine-treated embryos oscillate and appear to attempt to
release flares but fail to do so (see Fig.
6A; Movie 4, available at
jcs.biologists.org/supplemental).
Thus although flare particles are clearly seen in colchicine-treated embryos,
their normal movement is disrupted and is therefore dependent on microtubules.
Although we injected rhodamine-labeled tubulin into GFP-Cnn embryos to observe
the microtubules and Cnn simultaneously
(Fig. 4), we were unable to
resolve the astral microtubules that the flare particles are apparently moving
upon using our imaging conditions. To see the microtubules more clearly, we
immunostained for both Cnn and -tubulin in fixed embryos and
deconvoluted the images. From this, we observed that flare particles appear
associated laterally and with the plus (+) ends of astral microtubules
(Fig. 6B).
|
Flare movement was also inhibited by the drug Taxol
(Fig. 7). Treatment of embryos
with Taxol stabilized the microtubules and their cleavage was arrested
(Schiff et al., 1979;
Wani et al., 1971
;
Callaini and Riparbelli, 1997
).
Taxol caused the spindle to arrest in metaphase, which could be readily seen
in GFP-tubulin embryos (compare Fig.
7A to Fig. 7B;
Movie 5A-D, available at
jcs.biologist.org/supplemental).
In GFP-Cnn embryos we found that Taxol inhibited flare movement significantly
(compare Movie 5C with Movie 7D), although some small movements can still be
detected in these embryos (Fig.
7D; Movie 5, available at
jcs.biologists.org/supplemental).
In control animals that were permeabilized with octane but not exposed to
Taxol, cleavage defects were observed. The nuclei did not reassemble properly
following cleavage and often collided with each other
(Fig. 7A; Movie 5, available at
jcs.biologists.org/supplemental)
(data not shown). Nonetheless, flaring was not inhibited under these control
conditions (Fig. 7C; Movie 5,
available at
jcs.biologists.org/supplemental).
|
We investigated the relationship between the actin cytoskeleton and flares using GFP-Cnn and Rhodaminelabeled actin together in living embryos. At anaphase B and telophase, actin assembles densely along the outer edge of the centrosome on the face opposite to the spindle (Fig. 8, 297 seconds). These dense patches of actin juxtapose at division, preventing neighboring centrosomes from colliding. The characteristic formation of actin caps over the centrosomes in interphase and the assembly of actin cages at mitosis around the mitotic spindle occurred (Fig. 8; Movie 6, available at jcs.biologists.org.supplemental). At mitosis, flare particles moved to the boundary of the actin cage but did not go beyond it.
|
To determine whether there was any dependence of flares on the actin
cytoskeleton, actin polymerization was inhibited by the injection of
cytochalasin-D into GFP-Cnn embryos. Cytochalasin-D did not inhibit flare
activity. However, flare particles appeared to move farther from the
centrosome than they did in untreated embryos
(Fig. 9; Movie 7, available at
jcs.biologists.org/supplemental).
Flare particles also jumped to neighboring centrosomes in the
cytochalasin-treated embryos (Fig.
9; Movie 7, available at
jsc.biologists.org/supplemental),
which rarely occurred in untreated embryos. Notably, multiple (more than two)
centrosomes appear with each nucleus in the cytochalasin-treated embryos. This
is due to the fusion of nuclei at division
(Zalokar and Erk, 1976;
Callaini et al., 1992
;
Sullivan et al., 1993
). Thus,
the nuclei become larger as the chromosomes and centrosomes become hyperploid
at each division.
|
![]() |
Discussion |
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Cnn is localized at the periphery of the centrosome, with the center
appearing devoid of the protein. This was observed by both immunostaining
(Fig. 1A) and by live imaging
with GFP-Cnn. Imaging gives the appearance of a `doughnut-like' structure, but
it is more likely that the structure being viewed is actually a sphere devoid
of Cnn at its center. With GFP-Cnn, as with immunostaining, the hole is
visible only at low gain, where flare particles are difficult to detect
(images of GFP-Cnn at low gain are not shown). The center of the centrosome is
where the centrioles lie, and these structures may preclude the accumulation
of Cnn. A `hollow sphere' structure has also been observed in C.
elegans centrosomes during mitosis when viewed by live imaging of
GFP-- or GFP-ß-tubulin (Strome
et al., 2001
). Additionally, EM studies on interphase cleavage
stage Drosophila centrosomes reported `clouds' of PCM material
(Callaini and Riparbelli, 1990
)
and structures with `spider-like' extensions of electron dense PCM material
(Debec et al., 1999
), that are
similar to those in Fig. 1C.
Moreover, similar projections of PCM material from centrosomes like those seen
here with anti-Cnn antibodies (Fig.
1B, Fig. 5 and
Fig. 6B), with GFP-Cnn
(Fig. 4,
Fig. 7C and
Fig. 9) and by EM
(Fig. 1C) were also observed on
mouse centrosomes with anti-Pericentrin antibodies
(Mogensen et al., 1997
).
The localization of GFP-Cnn to the centrosome and to particles (flares) was
consistent with immunostaining seen previously
(Megraw et al., 1999;
Li and Kaufman, 1996
).
However, live imaging revealed a strikingly dynamic relationship between Cnn
and the centrosome. Live imaging revealed that flare particles emerged from
centrosomes and moved back and forth in a microtubule-dependent manner to the
boundary of the actin cage. Flares appear to be extrusions of Cnn-containing
PCM material that contain only a subset of known PCM components (see below).
Flares are probably not unique to cleavage stage centrosomes and may exist in
other cells in addition to the early embryo. We have observed particles of Cnn
near centrosomes in neuroblasts and spermatocytes by immunostaining (data not
shown). Live imaging will be required to demonstrate whether these particles
have the dynamic flaring relationship with the centrosome that we observed in
the cleavage-stage embryo.
Flare activity oscillates with the cleavage division cycle. Specifically there are few flare particles associated with metaphase centrosomes, and this persists through mitosis until telophase, when flare activity increases again (see Table 1). When flare numbers are high, especially at interphase, the centrosomes have a more tentacled appearance when viewed with GFP-Cnn. Flare particles are born from these extrusions that emanate from the PCM. At metaphase Cnn has a relatively more intense and `tight' association with the centrosome, producing a rounded appearance for the centrosome as compared with interphase.
Flares appear to be associated primarily with astral microtubules. Spindle
microtubules had few flare particles associated with them. These observations
are consistent with the changes in astral microtubule length that occur during
the cleavage cycle (Karr and Alberts,
1986). Thus, flare numbers per centrosome approximate to the
changes in astral microtubule length during the cleavage cycle. That is, in
metaphase, when astral microtubules are short, flaring is less intense,
whereas in interphase, when astral microtubules are long, flares are more
numerous. It should be noted however that flare numbers do not absolutely
mirror astral microtubule length changes. Asters grow longer during anaphase
(Karr and Alberts, 1986
),
which precedes the observed increase in flaring that occurs at the ensuing
telophase.
It is unclear what, if any, relationship flares have with Cnn assembly into centrosomes. To examine assembly, we attempted to use FRAP (fluorescence recovery after photobleaching) analysis, but were unable to photobleach centrosomes sufficiently without causing damage to the nuclei. These attempts at photobleaching caused the associated nucleus to arrest at interphase and fall away from the cortex toward the center of the embryo.
Flares are dependent upon microtubules for their movements. Colchicine
injection stops all movement of flares, but does not affect the localization
of Cnn to the centrosome or to the immobilized flare particles. The movie of
GFP-Cnn with colchicine (Fig.
6A; Movie 4, available at
jcs.biologists.org/supplemental)
shows that the centrosome forms projections that appear to attempt budding of
new flare particles, but these fail to exit the PCM, probably because the
astral microtubules have been destabilized. Cnn may not bind flare particles
directly to microtubules, since little microtubule-binding activity is
detected with Cnn in vitro (Li and
Kaufman, 1996). However, if Cnn is associated with Msps (see
below), as D-TACC is (Lee et al.,
2001
; Cullen and Ohkura,
2001
), then Cnn may be bound to microtubules indirectly through
the microtubule-binding activity of Msps.
Flare particles contain a subset of centrosomal proteins. While we were
unable to detect -tubulin or CP190 at flare particles, D-TACC protein
does colocalize with Cnn at centrosomes and flare particles. In a recent
report, D-TACC-GFP was examined in the early embryo where it localized to
centrosomes and to punctate particles among astral microtubules at interphase
(Gergely et al., 2000
).
Additionally, D-TACC was found to be in a complex with Msps
(Lee et al., 2001
;
Cullen and Ohkura, 2001
), and
the two proteins interact directly with one another
(Lee et al., 2001
). When
D-TACC-GFP and Msps-GFP fusion proteins were both expressed in
Drosophila embryos, both were localized in particles that move to and
fro from the centrosome (Lee et al.,
2001
). It is therefore likely that Cnn, D-TACC and Msps are all
components of flares. However, colocalization will need to be examined to
verify this for Msps.
We favor a model that posits that flare particles are passive passengers on
dynamic microtubules, as opposed to a model where flare particles are
transported by microtubule-based motor proteins. Taxol stabilizes microtubules
but does not inhibit motor proteins as taxol is used routinely in vitro to
stabilize microtubules for motor movement assays
(Saxton, 1994). Thus since
Taxol inhibited the activity of flares, flare movement is probably attributed
to the dynamic instability inherent to microtubules and not to
microtubule-based motor proteins. This suggests that flare particles are
mostly static passengers upon dynamic microtubules. Thus, flare particles
emerge from the centrosome, are associated with astral microtubules and move
back and forth upon them as they grow and shrink. There may be a minor
component of motor protein contribution to flare movement since flare
particles moved to a small degree when microtubules were stabilized with
taxol.
Consistent with the above model is the apparent rate at which flare
particles move. We estimate that flares move at a speed of 4-20 µm/minute,
a rate faster than that of spindle microtubule flux, estimated at 0.3-2
µm/minute (Mitchison, 1989;
Mitchison and Salmon, 1992
;
Zhai et al., 1995
;
Desai et al., 1998
), and more
similar to the rate of interphase microtubule treadmilling measured at 12
µm/minute (Rodionov et al.,
1999
). Our estimate of flare velocity is also consistent with the
rates of plus-end dynamics of centrosomal microtubules
(Rodionov et al., 1999
). Thus,
the movement of flare particles on astral microtubules is probably a direct
consequence of dynamic instability at the plus ends of these
centrosome-anchored microtubules.
Injection of cytochalasin to disrupt the actin cytoskeleton does not affect
flare movement but does cause flare particles to transfer to neighboring
centrosomes. Such transfers rarely occur in embryos not treated with the drug.
So actin is required to limit the extent of flare particle movement. During
the cortical cleavage divisions, actin is organized into caps at interphase
and furrows at metaphase (Warn et al.,
1984; Karr and Alberts,
1986
; Kellogg et al.,
1988
). Centrosomes orchestrate these cytoskeletal rearrangements
(Raff and Glover, 1989
) but
can do so independently of microtubules during cleavage in Drosophila
(Stevenson et al., 2001
). Live
observations from GFP-Cnn embryos injected with Rhodamine actin showed that
flares transit to the edge of the actin cage at mitosis. From this
observation, it is tempting to speculate that flares act with the centrosome
to organize the actin cage boundary. If flares, which clearly are microtubule
dependent, participate in promoting actin organization by the centrosome
during cleavage, their role in this process may not be absolutely
required.
During the cortical cleavage divisions, membrane-bearing particles are
recruited to the cleavage furrows for deposition of membrane, actin and
membrane proteins such as Discontinuous actin hexagon (Dah)
(Zhang et al., 2000;
Rothwell et al., 1999
).
Membrane-bearing particle recruitment requires the activity of Nuclear fallout
(Nuf), a protein found at the centrosome
(Rothwell et al., 1999
). Flare
particles are probably not these membrane-bearing particles since Dah and Cnn
did not colocalize when we immunostained for the two proteins (data not
shown). In addition, immunostaining for Golgi bodies using anti-ß-Cop
(ß-coatomer protein) (Ripoche et al.,
1994
) or anti-Lva (Lava lamp)
(Sisson et al., 2000
)
antibodies also showed no colocalization to Cnn flare particles (data not
shown).
To our knowledge, the ejection of PCM particles from centrosomes has not
been described previously. The appearance of `ejected asters' from frog oocyte
spindle poles in vitro (Murray et al.,
1996) may be related to the flaring phenomenon. However, unlike
ejected asters, flare particles do not appear to emanate microtubules.
Additionally, particles that contain Pericentrin, a centrosomal protein that
is required for microtubule nucleation at centrosomes
(Doxsey et al., 1994
), were
reported recently (Young et al.,
2000
). Pericentrin particles also contain
-tubulin and move
toward centrosomes where they dock and facilitate proper centrosome assembly
(Young et al., 2000
). However,
Cnn-containing flare particles behave differently, because they emerge from
the centrosome and move bi-directionally, both retreating and advancing toward
the centrosome. In addition, although Pericentrin particles are carried by the
minus-end-directed motor cytoplasmic dynein
(Young et al., 2000
), movement
of Cnn-containing flare particles appears to be largely dependent upon
intrinsic microtubule dynamics.
In vertebrate cells, early electron micrograph studies of centrosomes
revealed the presence of electron-dense spherical granules approximately
70-100 nm in diameter that localized around centrosomes and within the PCM and
have been called satellites or centriolar satellites
(Rattner, 1992;
Kalt and Schliwa, 1993
;
Kubo et al., 1999
). PCM-1, a
component of the centrosome PCM that localizes to the centrosome during
interphase, but dissociates from it at mitosis, is required for mouse zygotes
to pass through interphase (Balczon et al.,
1994
; Balczon et al.,
2002
). PCM-1 is a component of satellites, and live imaging of
GFP-PCM-1 particles showed that satellites move bi-directionally in a
microtubule- but not actin-dependent manner
(Kubo et al., 1999
). Thus the
behavior of satellites in vertebrate cells is similar to that of the flares we
describe here in Drosophila embryos except that the reported rate of
satellite movement is 0.7-0.8 µm/second
(Kubo et al., 1999
), which is
approximately four-fold faster than the maximum rates we measured for flare
movement. Since TACC, and probably Msps, appear to be components of flares
it will be interesting to investigate whether the homologs of these
proteins in vertebrates are localized at satellites.
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Acknowledgments |
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