* Department of Biochemistry and Biophysics, University of California, San Francisco, California 94143-0448; Department of
Embryology, Carnegie Institute of Washington, Baltimore, Maryland 21210; § National Academy of Sciences, Washington, DC
20418; and
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
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Abstract |
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Extracting isolated Drosophila centrosomes
with 2 M KI generates salt-resistant scaffolds that lack
the centrosomal proteins CP190, CP60, centrosomin,
and -tubulin. To clarify the role of these proteins in
microtubule nucleation by centrosomes and to identify
additional centrosome components required for nucleation, we have developed an in vitro complementation
assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a
soluble fraction of a Drosophila embryo extract. The
CP60 and CP190 can be removed from this extract
without effect, whereas removing the
-tubulin destroys the complementing activity. Consistent with
these results, we find no evidence that these three proteins form a complex together. Instead,
-tubulin is
found in two distinct protein complexes of 240,000 and
~3,000,000 D. The larger complex, which is analogous
to the Xenopus
-tubulin ring complex (
TuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison.
1995. Nature. 378:578-583), is necessary but not sufficient for complementation. An additional factor found
in the extract is required. These results provide the first
evidence that the
TuRC is required for microtubule nucleation at the centrosome.
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Introduction |
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IN animal cells, centrosome-nucleated microtubule arrays are essential for a wide variety of cellular processes including cell division and chromosome segregation, directed cell movement and interphase cytoplasmic
organization (for reviews see Mazia, 1987; Vorobjev and
Nadezhdina, 1987
; Kellogg et al., 1994
). EM studies have
shown that centrosomes consist of a pair of centriolar cylinders surrounded by an electron-dense cloud of pericentriolar material (PCM),1 and that the PCM originates the
many microtubules that are nucleated by the centrosome
(Rieder and Borisy, 1982
; Vorobjev and Chentsov, 1982
;
Keryer et al., 1984
).
The molecular characterization of the centrosome and
its ability to nucleate microtubules is still in its early stages.
The centrosome may contain as many as 100 different proteins (for review see Kalt and Schliwa, 1993; Kellogg et al.,
1994
), but it is not known how many of these are actual components of the PCM, with direct or indirect roles in microtubule nucleation. The identification of centrosomal components is further confounded by the fact that the centrosome,
as the focus of the cell's microtubule array, is also a hub for
intracellular trafficking. This makes it difficult to distinguish
actual components of the PCM from molecules recruited by
the microtubule array. To simplify this problem, we define
the "core" centrosome as the structure that remains when all
of its microtubules have been depolymerized.
The discovery of one such core centrosomal protein,
-tubulin, has led to a breakthrough in our understanding
of microtubule nucleation by centrosomes.
-Tubulin is a
highly conserved member of the tubulin family shown to
be involved in microtubule nucleation (Oakley et al., 1990
;
Stearns et al., 1991
; Zheng et al., 1991
; Joshi et al., 1992
;
Felix et al., 1994
; Stearns and Kirschner, 1994
). Recently, a
-tubulin-containing ring complex (
TuRC), capable of
nucleating microtubules in vitro, was purified from Xenopus eggs (Zheng et al., 1995
). EM tomography on centrosomes isolated from Drosophila revealed the presence
of rings containing
-tubulin in the PCM in both the presence and absence of nucleated microtubules. The
-tubulin rings are found at the microtubule minus ends in centrosome-nucleated microtubule asters (Moritz et al., 1995b
).
Ring structures are also visible in the PCM of centrosomes
of the surf clam, Spisula (Vogel et al., 1997
). These results
suggest that the
TuRC is a highly conserved structure responsible for the microtubule-nucleating capacity of the
PCM (Moritz et al., 1995b
; Zheng et al., 1995
).
Although these studies indicate that the TuRC is likely
to be essential for microtubule nucleation by centrosomes,
many important questions remain. These include: What is
the structural organization of the PCM and how is it assembled? How is the
TuRC anchored within the PCM?
Is the attachment of the
TuRC to the centrosome matrix
important for its activity? Do other centrosomal proteins contribute to microtubule nucleation?
Other core centrosomal proteins that may have direct or
indirect roles in microtubule nucleation include pericentrin, CP190, CP60, and centrosomin (CNN). Pericentrin is
a human autoimmune antigen that has also been identified
in mouse and Xenopus. It is thought to be a structural
component of the PCM that may play an essential role in
its organization (Doxsey et al., 1994). CP60 and CP190 are
two core centrosomal proteins of unknown function identified in Drosophila. CP190 is a novel, zinc-finger-containing protein identified by microtubule affinity chromatography (Kellogg et al., 1989
; Whitfield et al., 1995
). Native
CP190 localizes primarily to nuclei during interphase, becoming prominent at centrosomes upon nuclear envelope
breakdown at the onset of mitosis (Frasch et al., 1986
;
Whitfield et al., 1988
; Oegema et al., 1997
). CP60, a novel
protein identified by immunoaffinity chromatography by virtue of its ability to interact with CP190 (Kellogg and Alberts, 1992
), also localizes to both nuclei and centrosomes
in a cell cycle-dependent manner, but with slightly different timing (Kellogg et al., 1995
; Oegema et al., 1997
). Previous work from our laboratory suggested that CP190,
CP60, and
-tubulin are components of a soluble protein
complex present in embryo extracts (Raff et al., 1993
), but
further studies show that this is not the case (see below).
In Drosophila, the centrosome core also contains at least one developmentally important component, called CNN,
which is the target of a homeotic gene and is essential for
proper centrosome function (Li and Kaufman, 1996
).
To examine the role of the TuRC in microtubule nucleation at centrosomes and to test the potential contributions to nucleation by other known centrosomal components, we sought to develop an in vitro complementation
assay for aster formation using isolated Drosophila centrosomes (Moritz et al., 1995a
; Moritz and Alberts, 1998
).
Previous work has shown that the microtubule-nucleating activity of mammalian centrosomes can be destroyed by
salt or urea treatments, and that the activity can be restored by injecting the treated centrosomes into Xenopus
eggs, or by mixing them with egg extract. This suggests
that factors in the egg cytoplasm can associate with the
damaged centrosomes, restoring their ability to nucleate
microtubules (Klotz et al., 1990
; Buendia et al., 1992
).
With this information in mind, we developed an assay in which microtubule nucleation by Drosophila centrosomes
is reconstituted from two components, inactive salt-stripped
centrosome scaffolds and the high speed supernatant of a
Drosophila embryo extract.
In this paper, we characterize both the salt-stripped scaffolds and the soluble components in the extract that are
necessary for nucleation. In particular, we test for a role in
nucleation for CP190, CP60 and the Drosophila TuRC.
Our assay also allows us to begin to address what components, if any, are required for attachment of the
TuRC to
the salt-stripped scaffolds.
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Materials and Methods |
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Buffers
BRB80: 80 mM K-Pipes, pH 6.8, 1 mM MgCl2, 1 mM Na3EGTA (prepare
as a 5× stock, dilute to 1× for use). Hepes buffer: 50 mM K-Hepes, pH
7.6, 1 mM MgCl2, 1 mM Na3EGTA. Hepes block: Hepes buffer + 100 mM KCl, 10 mg/ml BSA (fraction V; Sigma Chemical Co., St. Louis, MO),
and 1 mM -mercaptoethanol. Embryo extract buffer for making complementing extract: Hepes buffer + 100 mM KCl, 10% glycerol, 1:100 protease inhibitor stock, and 1 mM PMSF. Protease inhibitor stock: 10 mM
benzamidine-HCl, 0.1 mg/ml phenanthroline, 1 mg/ml aprotinin, 1 mg/ml
leupeptin, 1 mg/ml pepstatin A in ethanol. GTP stock: 0.5 M GTP (Sigma
Chemical Co.) in 1× BRB80. Tubulin dilution buffer (TDB): 1× BRB80,
10% glycerol, 1 mM GTP. TDB wash: TDB + 10 mg/ml BSA (fraction V;
Sigma Chemical Co.). Extract buffer for characterizations of CP60,
CP190,
-tubulin protein complexes: 50 mM K-Hepes, pH 7.6, 75 mM
KCl, 1 mM Na3EGTA, 1 mM EDTA, 0.05% NP-40, 1:50 protease inhibitor stock, 2 mM PMSF. Gradient buffer: Hepes buffer + 1 mM
-mercaptoethanol, 1:200 protease inhibitor stock. Column buffer: Hepes buffer + 2% wt/vol glycerol, 1 mM
-mercaptoethanol, 1:200 protease inhibitor
stock. PBS: 5.4 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM
KCl, adjusted to pH 7.2. PBST: PBS + 0.1% Tween-20. Sample buffer: 63 mM Tris-HCl, pH 6.8, 3% SDS, 5%
-mercaptoethanol, 10% glycerol.
TBS: 10 mM Tris-Cl, pH 8, 150 mM NaCl, 0.05% Tween-20.
Centrosome Isolation
Drosophila centrosomes were isolated on sucrose gradients from 0-3.5-h-old embryos and tested for activity as previously described (Moritz et al.,
1995a; Moritz and Alberts, 1998
).
Tubulin
Tubulin was purified from bovine brain (Mitchison and Kirschner, 1984)
and labeled with N-hydroxy-succininimidyl-rhodamine (Hyman et al.,
1991
) as described previously.
Acid-washed, Poly-lysine-coated Coverslips
Acid-washed, 12-mm round glass coverslips were prepared in large batches by incubating the coverslips in a large glass beaker with 1 N HCl at 65°C for 4 h to overnight with occasional swirling. The coverslips were rinsed extensively in ddH2O, until the pH was neutral, and then incubated in 0.1% wt/vol poly-L-lysine for 20 min. The coverslips were dried by laying them out on a large piece of filter paper, or in a drying oven.
On-Glass Complementation Assay
To destroy the microtubule-nucleating activity of centrosomes, an equal
volume of 4 M KI in 1× BRB80 was mixed with the centrosomes, and the
mixture was incubated on ice for 10 min. 20 µl of this mixture was then applied to a 12-mm round acid-washed, poly-lysine-coated glass coverslip,
which was placed on a piece of Parafilm inside a humidified Petri dish
kept in a 30°C water bath. The centrosomes were allowed to bind to the
coverslip for 5 min, and then washed briefly by pipetting on and aspirating
off three times with 60 µl Hepes block. (For controls in which the centrosomes were omitted, the coverslips were washed in the same way with
Hepes block, and then the sample was applied and treated in the same
way as coverslips with centrosomes.) The final wash was allowed to incubate on the coverslip for 5 min. Depending on the experiment, the dish
containing the coverslips was either kept at 30°C, or transferred to a 0°C ice bath. The final wash was then replaced with 10-60 µl of the sample to
be tested. The centrosomes were incubated with the sample for 10 min (at
0° or 30°C), and then the sample was washed away briefly three times with
60 µl TDB wash. A 25-µl mixture of unlabeled and rhodamine-labeled tubulin (usually in a 7:1 ratio) diluted to 2 mg/ml in TDB was then incubated
on the coverslips for 10 min at 30°C. Any resulting microtubules or asters
were fixed by a 3-min incubation with 60 µl 1% glutaraldehyde in BRB80
(EM grade; Ted Pella, Inc., Redding, CA), followed by a 3-min incubation with 20°C methanol. The coverslips were then inverted and mounted on
slides on drops of mounting medium (80% glycerol in PBS + 1 mg/ml
para-phenylenediamine). The slides were viewed on a Nikon Microphot-FXA, 100× objective (1.4 NA), and either photographed using Kodak
Ektachrome 400 Elite or Ektachrome P1600 film, or on a Nikon Optiphot-2, 60× or 100× objective (1.4 NA) using a cooled CCD camera
(Princeton Scientific Instruments, Inc., Monmouth Junction, NJ). Micrographs were processed using Photoshop (Adobe Systems, Inc., Mountain
View, CA). For quantitation, asters were counted in 50 randomly selected,
100× microscope fields.
For immunofluorescence, unlabeled tubulin was used during the microtubule regrowth step, and the samples on coverslips were rehydrated after
methanol fixation by washing in TBS. Residual glutaraldehyde from the
fixation step was reduced by incubation with 0.1% sodium borohydride in
TBS for 7 min. The samples were washed and blocked in TBS + 3% BSA
for 5 min, and incubated simultaneously for 1 h with rabbit anti--tubulin
and DM1
(mouse anti-
-tubulin, T2096; Sigma Chemical Co.), each diluted 1:1,000. After washing, the coverslips were incubated for 1 h with a
mixture of fluorescein-labeled goat anti-rabbit (1:500) and Texas red-
labeled donkey anti-mouse (1:50), washed, and then mounted for viewing
under the fluorescence microscope. Images were obtained on a Nikon Optiphot-2 (60× objective, 1.4 NA) using a cooled CCD camera (Princeton
Scientific Instruments, Inc.). WinView software (Princeton Scientific Instruments, Inc.) was used to quantitate fluorescence intensity (see Fig. 3 h).
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Drosophila Embryo Extracts
Drosophila embryos between 0- and 2-h old (for preparation of complementing extract), or 0- and 4.5-h old (for characterization of protein complexes) were harvested, dechorionated, and then washed as described previously (Moritz and Alberts, 1998). The embryos were dried by blotting
with paper towels, weighed, and then resuspended in 1 vol of extract
buffer. The embryos were immediately homogenized by five passes of a
motor-driven Teflon pestle in a glass Dounce homogenizer. The extract
could be frozen in liquid nitrogen at this point and stored at
80°C. To
prepare high speed supernatant for complementation tests and their associated immunodepletions, the crude extract was centrifuged for 20 min at
~228,000 g (TL100; Beckman Instruments, Inc., Fullerton, CA), the supernatant was transferred to a new tube, and then centrifuged again in the
same way. For complementation tests, nocodazole (100 µM final concentration) was added to the extract before centrifugation. To prepare the supernatant for characterizations of protein complexes, the crude extract
was centrifuged for 10 min at 30,000 rpm in a Beckman TLA 100.3 rotor,
transferred to a new tube and centrifuged again at 100,000 rpm for 8 minutes in the same rotor.
Antibodies
The rabbit antibodies to CP60 and to amino acids 385-508 of CP190 have
been previously described (Kellogg et al., 1995; Oegema et al., 1995
). The
rabbit antibody to amino acids 705-789 of CP190 was prepared according
to Oegema et al. (1995)
. One of the rabbit anti-
-tubulin antibodies used
was raised against the full-length maternal form of Drosophila
-tubulin
(these sequence data are available from GenBank/EMBL/DDBJ under
accession number P42271) expressed in baculovirus. The second antibody
recognizing
-tubulin was raised against the COOH-terminal peptide
QIDYPQWSPAVEASKAG of the maternal form of Drosophila
-tubulin. The production and purification of these antibodies will be described elsewhere.
Immunoprecipitations
To prepare the antibodies used for immunoprecipitation, 20-30 µg of antibody was coupled to 50 µl of packed Affiprep protein A beads (Bio-Rad Laboratories, Hercules, CA). The beads were first mixed by gentle rotation with antibody in PBST for 0.5-1 h at room temperature, and then washed three times with PBST, followed by three washes and resuspension in 0.2 M sodium borate, pH 9.0. To covalently attach the antibodies to the beads, dimethyl pimelimidate was added to 20 mM and the beads were incubated while rotating the tube gently for 0.5-1 h at room temperature. To inactivate residual cross-linker, the beads were washed into 0.2 M ethanolamine, pH 8.0, and rotated at room temperature for 2 h to overnight before use. The beads were then pre-eluted three times with 100 mM glycine, pH 2.3, before washing into extract buffer. To begin the immunoprecipitation, 50 µl of packed beads were rotated with 300 µl of concentrated embryo extract for 1 h at 4°C. The beads were pelleted and the supernatants sampled. The beads were washed four times with column buffer (or with extract buffer) plus 75 mM KCl, 0.05% NP-40 or 0.05% Triton X-100, and 1:200 protease inhibitor stock, and then once with the same buffer without detergent. In some cases (for immunoprecipitations to characterize protein complexes), proteins were eluted three times sequentially with 150 µl of 100 mM glycine, pH 2.3. The elutions were pooled and neutralized by addition of 200 µl 0.5 M K-Hepes, pH 7.6. For gel analysis, 20 µg of porcine insulin was sometimes added as carrier and the samples were precipitated with TCA.
In experiments to test the ability of immunodepleted extracts to complement salt-stripped centrosomes, the beads were pelleted, washed as above, and then boiled in sample buffer for SDS-PAGE and Western analysis. Samples of the supernatants were kept for this purpose as well, and the remainder of the supernatant was used in the on-glass complementation assay.
Sucrose Gradient Sedimentation and Gel-filtration Chromatography
Sucrose gradients were poured as step gradients (five 950-µl steps) that were allowed to diffuse into continuous gradients overnight at 4°C before use. The gradients were formed from 5-20% or 5-40% sucrose (Ultra-pure; ICN Biomedicals, Costa Mesa, CA) in gradient buffer plus 75 mM, 100 mM, or 500 mM KCl, as indicated for each experiment. A 50-75-µl aliquot of sample was loaded onto each gradient, and the gradients were centrifuged at 4°C at 50,000 rpm in a Beckman SW55 rotor for 4 to 8 h, as indicated. The gradients were fractionated from the top by hand into 16 300-µl fractions. Protein standards (0.5 mg/ml each) were loaded in an equivalent volume and were run in parallel over identical gradients for each experiment.
Gel-filtration chromatography was carried out on a Superose-6 column
by FPLC (Pharmacia Biotech Sevrage, Uppsala, Sweden) in column buffer
plus 75 mM, 100 mM, or 500 mM KCl, as indicated. The column was calibrated with standards of known Stokes radii as indicated in the legend to
Fig. 5. The size and shape (Stokes radii) of protein complexes were estimated according to (Siegel and Monty, 1966). The axial ratios of the
equivalent prolate ellipsoids of revolution, {a/b}p, were estimated according to (Laue et al., 1992
), using the method of Kuntz (1971)
to estimate
the degree of hydration from amino acid sequence.
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Sucrose Gradient Quantitation
Standards were precipitated by the addition of TCA to 10%, resuspended
in sample buffer, separated by 8.5 or 11% PAGE, and then stained with
Coomassie blue. Gels were scanned into the computer using a UMAX scanner (Fremont, CA), and the program NIH Image was used to quantitate
band intensities. The peak fraction was assigned for each standard using Kaleidagraph (Synergy Software, Reading, PA). Standard curves of peak fraction versus sedimentation coefficient were then used to convert fraction
number to S value (essentially S20,w) for each sucrose gradient to allow direct comparison of protein complexes sedimented in 75 mM and 500 mM KCl. This use of standards to correct to S20,w from different buffers is valid
as long as the partial specific volumes are the same for the standard proteins
and the protein complexes being studied (Martin and Ames, 1961).
Quantitative Immunoblotting
For immunoblots, samples were precipitated by the addition of TCA to
10% and resuspended in Sample buffer before separation by SDS-PAGE
on 10 or 11% gels. Proteins were then transferred to nitrocellulose (pore
size 0.1 µm) in the presence of 25% methanol, 0.15 M glycine, 0.02% SDS.
The blots were incubated for 20 min in block (TBS + 0.1% Tween-20, 3%
nonfat dry milk, 10% glycerol). A chemiluminescent substrate system (SuperSignal CL-HRP; Pierce Chemical Co., Rockford, IL) was used to detect the HRP-conjugated secondary antibodies. The developed film was
scanned into the computer using a UMAX scanner and NIH Image was
used to quantitate band intensities. Serial dilutions of CP190, CP60, and
-tubulin were blotted simultaneously with all experimental fractions, allowing us to determine the relative concentrations of CP190, CP60, and
-tubulin in each fraction, as shown in Fig. 6.
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Results |
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In Vitro Complementation of Salt-stripped Centrosomes
To study the role of the TuRC in microtubule nucleation
at centrosomes and to test for potential contributions to
nucleation by other centrosomal components, we developed an in vitro assay in which the nucleating activity of
salt-stripped Drosophila centrosomes is restored by incubation with embryo extract (Fig. 1). Initially, several different salts, including NaCl, KCl, and KI, as well as urea at
various concentrations were tested for their ability to inactivate Drosophila centrosomes. Whereas all of the salts and
urea were destructive to some extent (data not shown), we found that treatment with 2 M KI consistently destroyed
the microtubule-nucleating activity of the centrosomes.
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Using this complementation assay, we determined that a 228,000 g supernatant of an extract made from 0-2-h-old embryos was able to complement the KI-stripped centrosomes (Fig. 2). When extract was incubated on the coverslip in the absence of centrosomes, many microtubules, but virtually no asters, formed (Fig. 2, b and c). When KI-stripped centrosomes were incubated with buffer instead of extract, very few microtubules and no asters formed (Fig. 2 d). When KI-stripped centrosomes were incubated with the complementing extract before the tubulin incubation, asters that look very similar to those that formed on buffer-treated centrosomes were produced (compare Fig. 2, a and e). Complementation occurred when the KI-treated centrosomes were incubated with extract containing nocodazole at either 0° or 30°C, indicating that aster regrowth was not merely due to the elongation of microtubules initiated during incubation with the extract (Fig 2, e and f).
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To be confident that centrosomes were indeed present
even when few or no asters could be found, the number of
centrosomes on the coverslips was independently verified
by immunofluorescent staining of -tubulin in the centrioles (and
-tubulin where possible). The number of centrosomes (whether intact or salt-stripped) bound to individual coverslips was found to be quite consistent (Fig. 2 g).
An initial characterization of the complementing extract showed that its activity could be destroyed by heating to 60°C or boiling, and that ATP was not required for complementation. In addition, when the extract was prepared under conditions that promote microtubule polymerization and the microtubules were removed by centrifugation, the extract did not complement. This suggests that at least one component required for the complementation binds to microtubules (data not shown).
The Centrosomal Proteins CP60, CP190, CNN, and
-Tubulin Are Removed from Centrosomes by 2 M KI
Since it was possible to complement the salt-stripped centrosomes with soluble factors present in embryo extract, yet
these same factors were not capable of inducing aster formation in the absence of the KI-treated centrosomes (Fig.
2), it appeared that a remnant structure must persist after
treatment with 2 M KI. This remnant might be a scaffolding
to which the soluble factors necessary for restoring microtubule nucleation attach. Therefore, we characterized what
remains at the centrosomes after salt treatment, and how
the known centrosomal proteins behave under such conditions. We began by determining whether the salt treatment
removed the known centrosomal proteins CP60, CP190,
CNN, and -tubulin (Kellogg et al., 1989
; Oakley and Oakley, 1989
; Kellogg and Alberts, 1992
; Whitfield et al., 1995
;
Li and Kaufman, 1996
). Centrosomes were incubated with
2 M KI, as described above for the complementation assay,
and then pelleted. The centrosomal protein profile becomes simpler after KI treatment (Fig. 3 a). Immunoblotting of the
pellets and supernatants of buffer- and KI-treated centrosomes shows that virtually all detectable CP60, CP190,
CNN, and
-tubulin are removed by the salt (Fig. 3 b). Electron microscopic examination of the KI-stripped centrosomes shows that the centrioles are destroyed to varying
degrees by the salt, but that there is little obvious structural
abnormality in the PCM (data not shown).
-Tubulin, but Not CP60 or CP190, Are
Required for Restoring Microtubule-nucleating Activity
to KI-treated Centrosomes
The fact that treatment of centrosomes with KI both led to
a loss of microtubule-nucleating activity and extracted
CP60, CP190, CNN, and -tubulin, suggested that one or
more of these proteins might be needed for this activity.
Therefore, we tested the effect of immunodepleting CP60,
CP190, and
-tubulin on the ability of the extract to complement salt-stripped centrosomes (we were not able to
deplete CNN to a sufficient extent for this assay). Each protein was quantitatively depleted (Fig. 3 i), and the resulting extracts were tested in the in vitro assay (Fig. 1).
Only the depletion of
-tubulin had an effect on the ability
of the extract to complement KI-inactivated centrosomes,
and this activity was consistently destroyed by the removal
of
-tubulin (Table I; see Fig. 8 c).
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|
We used immunofluorescence to further examine the effect of the KI treatment and extract complementation on
the presence of -tubulin at centrosomes. In these experiments, staining centrosomes with antibodies against
-tubulin and
-tubulin (which recognize PCM and centrioles, respectively) confirmed that
-tubulin is removed from
centrosomes by treatment with KI (Fig. 3, c and d; c' and
d'). We also found that
-tubulin reassociates with centrosomes during incubation with extract (Fig. 3, e and f; e'
and f'). To rule out the possibility that spurious asters form
in the absence of centrosomes resulting from clustering of
-tubulin into foci, we also stained coverslips that were incubated with extract followed by tubulin in the absence of
centrosomes, and found no foci of
-tubulin staining (Fig.
3, g and g'). These data were quantitated by measuring the
fluorescence intensity of
- and
-tubulin staining at intact
(buffer-treated) and KI-treated centrosomes (Fig. 3 h).
Cumulatively, these results suggest that -tubulin is required for aster formation, whereas CP60 and CP190 are
not. We had initially expected that CP60 and CP190 might
also be at least indirectly required since our laboratory
had previously found evidence for the existence of a protein complex containing these three proteins (Raff et al.,
1993
). These results led us to re-evaluate the interactions
of CP60, CP190, and
-tubulin. In addition, we were interested in characterizing further the
-tubulin component required for complementation.
Drosophila -Tubulin Is in a Protein Complex That Is
Similar to the Xenopus
TuRC
We used immunoprecipitations, gel filtration, and sucrose
gradient sedimentation to investigate centrosomal protein
complexes containing -tubulin, CP60, and/or CP190, which
might restore microtubule-nucleating activity to salt-stripped centrosomes. CP60, CP190, or
-tubulin were
each immunoprecipitated from concentrated embryo extracts and analyzed by SDS-PAGE (Fig. 4 a) and immunoblotting (Fig. 4, b and c). Antibodies recognizing
-tubulin
(Zheng, Y., unpublished observations) immunoprecipitated
-tubulin and a group of associated proteins that are
components of the Drosophila
TuRC. The protein profile of this complex is very similar to the that of the Xenopus
TuRC (Fig. 4 a, right two lanes; Oegema, K., and Y. Zheng, manuscript in preparation) (Zheng et al., 1995
).
CP190 antibodies brought down CP190 and a large fraction
of CP60 (Fig. 4 a, middle two lanes). Antibodies to CP60
immunoprecipitated CP60 and a small fraction of the
CP190 found in extracts (Fig. 4 a, second lane from left).
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Immunoblots of the supernatants and pellets from the immunoprecipitations confirmed the identity of the Coomassie-stained bands. Analysis of the immunoprecipitation supernatants showed that each protein was depleted by its corresponding antibody (Fig. 4 b). In addition, CP60 is largely depleted in the supernatants of extracts treated with antibodies against CP190 (Fig. 4 b).
Immunoprecipitation pellets from concentrated extract
(+) or from buffer controls () are shown in Fig. 4 c. Neither CP190 nor CP60 was detected in the pellets from immunoprecipitations performed with the antibodies to
-tubulin, nor was any
-tubulin present in the pellets of
immunoprecipitations performed with antibodies against
CP60 or CP190. The majority of the CP60 in extracts coimmunoprecipitated with CP190, however, and only a small
fraction of the CP190 coimmunoprecipitated with CP60.
These results suggest that, although CP60 and CP190 associate in these extracts, neither CP190 nor CP60 are in a cytoplasmic complex with
-tubulin.
In Embryo Extracts, -Tubulin Is Found in Two
Distinct Complexes, Neither of Which Contain CP190
or CP60
To characterize potential protein complexes involving
-tubulin, CP60, and CP190 further, we analyzed the behavior of these proteins by gel-filtration chromatography
and by sucrose gradient sedimentation. The same concentrated embryo extract was simultaneously fractionated by
both techniques in identical buffers containing either 75 or
500 mM KCl (Fig. 5). To facilitate the comparison between
complexes containing
-tubulin and those containing CP190 and CP60, we used a quantitative blotting technique that
allowed us to determine the relative concentrations of
CP190, CP60 and
-tubulin in each fraction (Fig. 6). Sucrose gradients run in different salt concentrations cannot
be directly compared because of differences in buffer density and viscosity that affect sedimentation rates. Therefore, standard curves of peak fraction versus S value, generated by loading proteins of known S value on identical
sucrose gradients run in parallel with each experimental
gradient, were used to convert fraction number to S value
for each gradient.
In buffer containing 75 mM KCl, most of the -tubulin is
found in two complexes that can be separated by both sucrose gradient sedimentation and by gel filtration (Figs. 5,
a and b; and 6, a and b, left panels). The large
-tubulin
complex can be converted to the small
-tubulin complex
by raising the KCl concentration to 500 mM (compare
-tubulin migration in Fig. 5 a, top and bottom; b, top and
bottom; Fig. 6, a and b, compare left and right). In addition,
the gel-filtration peak corresponding to the small
-tubulin complex, which appears heterogeneous in 75 mM KCl,
becomes much more homogeneous in 500 mM KCl. These
results suggest that the small
-tubulin complex is a subunit of the large
-tubulin complex.
The sedimentation coefficients of the large and small
-tubulin complexes are 36.9 S and 8.5 S, respectively.
(The sedimentation coefficient of the large
-tubulin complex was determined on a separate sucrose gradient using
the 30 S ribosome particle as a standard, in addition to the
standards mentioned in the legend to Fig. 5 [data not
shown]). The Stokes radii of the small and large
-tubulin
complexes, estimated from our gel-filtration results, are
6.9 nm and ~20 nm, respectively (the large
-tubulin complex fractionates close to the void volume of the Superose-6
column, preventing a more precise determination). From
these sedimentation coefficients and Stokes radii, the
masses of the small and large
-tubulin complexes were estimated to be 240,000 and ~3,000,000 D, respectively (Siegel and Monty, 1966
).
On sucrose gradients, CP60 comigrates with the small
-tubulin complex in both high and low salt (compare
CP60 with
-tubulin in Figs. 5 a, and 6 a, left panel). However, under the same buffer conditions, CP60 can be separated easily from the small
-tubulin complex by gel filtration (compare CP60 and
-tubulin peaks in Figs. 5 b and 6 b). By gel filtration in 75 mM KCl (Figs. 5 b, and 6 b, left
panel), CP60 elutes with the large
-tubulin complex, but
under identical conditions, CP60 and the
-tubulin large
complex are easily separated on sucrose gradients (Figs. 5
a, and 6 a, left panel). Similarly, CP190 can be separated
from the small
-tubulin complex by gel filtration (Figs. 5
b, and 6 b), and from the large
-tubulin complex on sucrose gradients (Figs. 5 a, and 6 a, left panel). These experiments indicate that neither CP60 nor CP190 are among
the components of either the large or small
-tubulin complexes. Interestingly, even in high salt conditions where
CP190 and CP60 do not associate (data not shown), both
proteins are found in large complexes. The properties of
these centrosomal protein complexes are summarized in
Table II.
|
The -Tubulin Ring Complex Is Necessary,
but Not Sufficient for Complementation of
Salt-stripped Centrosomes
Since our immunodepletion studies demonstrated a requirement for soluble -tubulin in the complementation
assay, we used the assay to determine if either the small or
large
-tubulin-containing complexes could complement
the centrosome scaffolds. For this test we fractionated embryo extract on sucrose gradients or by gel filtration and
then assayed the fractions for complementing activity. The
sucrose gradient fractions containing the large complex
complemented the salt-stripped centrosomes, whereas
fractions containing the small complex did not (Fig. 7 a).
Gel filtration fractions containing the large complex also
gave good complementation; in addition, we observed
some less robust complementation by the gel filtration
fractions containing the small complex (Fig. 7 b).
|
These results suggested that the -tubulin large complex
is not only essential, but possibly sufficient for complementation. To determine if this was the case, we used an
antibody that recognizes the COOH-terminal 17 amino
acids of the Drosophila maternal form of
-tubulin to purify the large complex (we will refer to this subsequently as
the Drosophila
TuRC) in a manner similar to that described for the purification of the Xenopus
TuRC (Zheng,
Y., manuscript in preparation; Zheng et al., 1995
), see Fig.
4 a for an immunoprecipitation showing the protein composition of this complex). The purified Drosophila
TuRC
has a ring structure very similar to that of the Xenopus
complex (Zheng, Y., manuscript in preparation). To our
surprise, we found that the immunoaffinity-purified
TuRC
was not able to complement.
We reasoned that an additional factor in the extract that
was removed upon immunoaffinity purification of the
TuRC was required to allow the
TuRC to complement.
To test this idea, equal volumes of pure
TuRC and extract that had been immunodepleted using an anti-
-tubulin antibody were mixed, and then tested for complementation of salt-stripped centrosomes in the on-glass assay
(Fig. 8). Asters formed when KI-treated centrosomes were
incubated with the partially purified large
-tubulin complex from a Superose-6 column (Fig. 8 a). Some free microtubules, but no asters formed when the centrosomes were omitted (Fig. 8 b). If KI-stripped centrosomes were
incubated with a mixture of pure
TuRC and
-tubulin-
depleted extract, asters formed (Fig. 8 e). However, centrosomes incubated with pure
TuRC or
-tubulin-depleted
extract alone were not able to produce asters (Fig. 8, c and
d). In the absence of centrosomes, the mixture of pure
TuRC and
-tubulin-depleted extract formed microtubules, but no asters (Fig. 8 f).
We concluded that the immunoisolated TuRC requires
an additional factor found in extracts to complement salt-stripped centrosomes. To determine whether the order of
addition of these two components is important, we incubated salt-stripped centrosomes sequentially with
-tubulin-depleted extract followed by a washing step, and then
by pure
TuRC, or vice versa. The centrosomes only regained their ability to nucleate microtubule asters when
they were first incubated with the
-tubulin-depleted extract, followed by pure
TuRC, or when they were incubated with a mixture of the two. The complementation
worked best when the two components were added simultaneously (data not shown). Initial characterization by sucrose gradient sedimentation and gel filtration of this additional required factor indicates that it has an estimated
molecular weight of 220,000 D (data not shown).
![]() |
Discussion |
---|
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---|
To begin to understand microtubule nucleation in the context of the centrosome, we developed an in vitro complementation assay in which centrosome aster formation is reconstituted from salt-stripped centrosome scaffolds and a soluble fraction provided by a Drosophila embryo extract. This assay opens several new avenues for studying the structure and composition of the centrosome matrix. Extraction of centrosomes with the strongly chaotropic salt, KI, removes all of the known Drosophila components of core centrosomes, reducing the centrosomes to a simpler structure that appears to contain a scaffolding on which microtubule-nucleating sites may reassemble. The proteins left in this salt-resistant structure are unknown, but it should be possible to identify them by peptide sequencing.
Electron microscopy of the salt-stripped centrosome
scaffolds did not reveal any striking modifications of the
PCM, suggesting that CP60, CP190, CNN, and -tubulin
are distributed throughout the PCM in intact centrosomes
rather than being confined to particular regions. This is
consistent with our previous EM observation that
-tubulin is found at all levels of the PCM (Moritz et al., 1995b
).
We have also characterized the soluble components
contributed by the embryo extract that are required for
centrosome aster reconstitution. By immunodepleting the
Drosophila centrosomal proteins CP60, CP190, and -tubulin from the complementing extract, we found that
-tubulin is absolutely required for aster formation in our assay.
This requirement is similar to the requirement for
-tubulin in the aster assembly assay in Xenopus extracts (Felix et al., 1994
; Stearns and Kirschner, 1994
). Previous work
from our laboratory suggested that CP60, CP190, and
-tubulin are in a protein complex together (Raff et al.,
1993
), yet we found that CP60 and CP190 were not necessary for complementation.
We therefore characterized the -tubulin-containing
complexes found in Drosophila embryo extracts to determine if we could identify a specific complex required for
complementation and to re-evaluate the association of
-tubulin with CP190 and CP60. Protein complexes were
immunoprecipitated or fractionated by sucrose-gradient sedimentation and gel-filtration chromatography at high
and low salt concentrations. We found that in low salt,
most of the
-tubulin is found in two distinct complexes of
240,000 and ~3,000,000 D, which can be separated by either fractionation technique. The larger
-tubulin complex
can be converted into the smaller complex by 500 mM
KCl, suggesting that the small
-tubulin complex is a subunit of the larger one. The large
-tubulin complex seen in
these extracts has been purified to near homogeneity and
is the Drosophila analogue of the
-tubulin ring complex
(
TuRC) isolated from Xenopus egg extracts (Zheng, Y.,
and K. Oegema, manuscript in preparation; Zheng et al.,
1995
).
These experiments also demonstrated that although
CP60, CP190, and -tubulin are all found in large protein
complexes neither CP60 nor CP190 are among the components of the
-tubulin complexes. How can we reconcile
these data with previous results favoring a cytoplasmic
complex containing CP190, CP60, and
-tubulin? In the
previous study (Raff et al., 1993
) the complexes were not examined by gel-filtration chromatography, experiments
that turned out to be crucial in the current work to distinguish the CP60 complex from the
-tubulin small complex.
However, in the previous study,
-tubulin was detected by
Western blotting in the elutions from immunoaffinity columns constructed from antibodies to CP60 or CP190, although it was a very minor component of these elutions, since a Coomassie blue-staining
-tubulin band was never
detected (Raff et al., 1993
). In contrast, in this study we
were not able to immunoprecipitate
-tubulin with antibodies to CP60 or CP190, nor could we immunoprecipitate
CP60 or CP190 with antibodies to
-tubulin. One possible
explanation for these disparate results is that although
-tubulin is not found in a soluble complex with CP60 or
CP190 in extracts, these proteins may assemble with each other to form a higher order complex on immunoaffinity
columns and in vivo at the centrosome under conditions
where the concentrations of these centrosomal proteins
are higher than they are in extracts.
To explore the role of the large and small -tubulin-
containing complexes in aster formation, we tested embryo extracts fractionated on sucrose gradients or by gel
filtration for complementing activity in our assay. Although a direct role for the small complex is unclear, both
sucrose gradient and gel filtration fractions containing the
large complex (the Drosophila
TuRC) can complement
salt-stripped centrosomes (Figs. 7 and 8). This result was
surprising because treatment with KI extracts all of the
known components of the pericentriolar material in
Drosophila (CP190, CP60, CNN, and
-tubulin) and appears to substantially simplify the protein composition of
the isolated centrosomes. The fact that both sucrose gradient and gel filtration fractions containing the
TuRC can
complement suggests that the connection between the
TuRC present in the extract and the salt-resistant scaffold is likely to be direct.
In contrast to the TuRC in the sucrose gradient and gel
filtration fractions, purified Drosophila
TuRC is unable
to complement, suggesting that a component or modification of the
TuRC that is required for complementation is
lost during immunoaffinity purification. The possibilities
for what this component or modification is doing include:
(a) providing a physical link between the centrosome and
the
TuRC, or (b) modifying the scaffolds or the
TuRC
in some way so that the
TuRC can bind to, or become activated at the centrosome. The fact that the purified
Drosophila
TuRC can nucleate microtubules in solution
(Zheng, Y., and C. Wiese, unpublished observation) and
when bound to the coverslips in our assay (Fig. 8, d and f),
suggests that this component or modification probably is
not necessary for nucleating activity but may instead be required for attachment of the
TuRC to the salt-stripped
centrosome scaffolds. The order of addition experiment
further supports the idea that
-tubulin-depleted extract
may supply an attachment factor required to link the purified
TuRC to the salt-stripped scaffolds.
We have found that this putative attachment factor has
an estimated molecular weight of ~220,000 D. Pericentrin
is one possible candidate for the factor, since it is a large
coiled-coil, core centrosome structural protein that may
interact with -tubulin (Doxsey et al., 1994
; Dictenberg et
al., 1998
). However, testing this possibility awaits the
development of pericentrin reagents that work well in
Drosophila.
In summary, we have developed an assay in which microtubule nucleation by Drosophila centrosomes is reconstituted from inactive salt-stripped centrosome scaffolds
and soluble components derived from a Drosophila embryo extract. We conclude that the TuRC is required for
microtubule nucleation at centrosomes and that the connection between the
TuRC and the simplified scaffolds is
likely to be directly mediated by a factor found in extracts
that is normally loosely associated with the
TuRC. We
believe that our approach of attempting to simplify centrosome structure and function by focusing on smaller centrosomal protein complexes in conjunction with an in vitro
complementation assay will be valuable in understanding this important but complicated organelle.
![]() |
Footnotes |
---|
Received for publication 2 March 1998 and in revised form 30 June 1998.
Address all correspondence to Michelle Moritz, Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143-0448. Tel.: (415) 476-4581. Fax: (415) 476-0806.We thank M. Welch, C. Walczak (University of California, San Francisco, CA), and C. Wiese (Carnegie Institute of Washington, Baltimore, MD) for helpful suggestions on the manuscript, and all members of the Mitchison lab (East and West) for a lively environment in which to study centrosomes and microtubules. Many thanks to Dr. T. Kaufman (Howard Hughes Medical Institute, Indiana University, Bloomington, IN) for antibodies against centrosomin, and to A. Desai (Harvard Medical School, Boston, MA) for help with centriole immunofluorescence.
This work was supported by the Herbert W. Boyer Fund (to M. Moritz) and the National Institutes of Health (No. GM23928 to B.M. Alberts).
![]() |
Abbreviations used in this paper |
---|
CNN, centrosomin;
PCM, pericentriolar
material;
TDB, tubulin dilution buffer;
TuRC,
-tubulin ring complex.
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A ![]() |