* The Department of Physiology and Cell Biology, University of Kansas, Lawrence, Kansas 66045, and the Marine Biological
Laboratory, Woods Hole, Massachusetts 02543; Department of Biological Sciences, Stanford University, Palo Alto, California
94305; and § Wadsworth Center for Laboratories and Research, Albany, New York 12201-0509
Centrosome-dependent microtubule nucleation involves the interaction of tubulin subunits with
pericentriolar material. To study the biochemical and
structural basis of centrosome-dependent microtubule
nucleation, centrosomes capable of organizing microtubules into astral arrays were isolated from parthenogenetically activated Spisula solidissima oocytes. Intermediate voltage electron microscopy tomography revealed
that each centrosome was composed of a single centriole surrounded by pericentriolar material that was
studded with ring-shaped structures ~25 nm in diameter and <25 nm in length. A number of proteins copurified with centrosomes including: (a) proteins that contained M-phase-specific phosphoepitopes (MPM-2), (b)
-,
-, and
-tubulins, (c) actin, and (d) three low molecular weight proteins of <20 kD.
-Tubulin was not
an MPM-2 phosphoprotein and was the most abundant
form of tubulin in centrosomes. Relatively little
- or
-tubulin copurified with centrosomes, and the ratio of
- to
-tubulin in centrosomes was not 1:1 as expected,
but rather 1:4.6, suggesting that centrosomes contain
-tubulin that is not dimerized with
-tubulin.
In most organisms, microtubules (Mts)1 are not randomly distributed in cells but exist as highly ordered
arrays that are essential for various functions (for review see Brinkley, 1985 Early electron microscopy studies revealed that in most
animal cells, centrosomes are composed of one or two centrioles surrounded by an amorphous cloud of electrondense material, the pericentriolar material (PCM) (Porter,
1966 Microtubule nucleation must involve the interaction of
tubulin subunits ( To address these and other questions, we have used the
unique properties of Spisula solidissima (surf clam) oocytes to develop an in vitro system for the study of centrosome function. These oocytes can be obtained in 100-g
quantities, thus facilitating preparative biochemistry. Furthermore, since they are arrested at prophase of meiosis I
(Rebhun, 1959 All reagents were from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted.
Lysate Preparation
Adult Spisula were collected by the Marine Resources Department of the
Marine Biological Laboratory (Woods Hole, MA) and maintained in
flow-through sea water tanks at ~13°C. Oocytes were dissected from ripe
ovaries, passed through cheese cloth, and washed in sea water by three cycles of suspension/sedimentation. Oocytes were activated by treatment
with KCl for 4 min, and lysates were prepared as previously described
(Palazzo et al., 1988 Tubulin Preparation
Sea urchin (Strongylocentrotus purpuratus) Mts were prepared by three
cycles of polymerization/depolymerization as previously described (Suprenant and Marsh, 1987 Three-cycled tubulin was also prepared from Spisula oocyte lysates
using a modification of the procedure described in Suprenant (1989) When necessary, Spisula tubulin was purified from three-cycled tubulin
by polymerizing in the presence of Na-glutamate (Simon et al., 1992 Centrosome Isolation
Centrosomes were isolated from lysates by sucrose density-gradient centrifugation using a modification of the procedures described by Mitchison
and Kirschner (1984, 1986) for isolation of mammalian centrosomes. Lysates were thawed on ice, and aliquots were removed and tested for aster
formation using hexylene glycol and polarized light microscopy as previously described. For each preparative gradient, 2-3 ml of crude lysate
(~9-18 × 106 centrosomes) was diluted with 0.60 vol of aster buffer (Aster Buffer: 20 mM sodium-Pipes, pH 7.2, 100 mM NaCl, 5.0 mM MgSO4), resuspended, and centrifuged (5,500 g/4°C/10 min) using a rotor (model JS
13.1; Beckman Instruments). Supernatants were collected, diluted to 50.2%
sucrose by addition of a stock solution of 70% (wt/wt) sucrose in PEM
(5 mM potassium-Pipes, pH 7.2, 1 mM EGTA, 1 mM MgSO4) and loaded
onto a two-step gradient consisting of 66% (3.0 ml) and 52.5% (5.0 ml) sucrose steps. Gradients were centrifuged at 70,000 g at 4°C for 90 min using
a rotor (model SW-28; Beckman Instruments). Fractions (1.0 ml) were
collected at 4°C by bottom puncture of centrifuge tubes, and sucrose density was determined with the use of a hand-held refractometer.
Fractions were tested for centrosome content based on their ability to
organize Mts in the form of radial astral arrays when reconstituted with tubulin media. Thus, 10 µl aliquots from each fraction were diluted into 40 µl
of tubulin media (0.5 mg/ml three-cycled sea urchin tubulin in RA buffer
containing 2 mM GTP) and incubated for 10 min at ambient temperature,
and asters were counted using a hemacytometer viewed with a polarized
light microscope. The fraction containing the highest density of centrosomes (centrosome fraction) was used directly, or snap frozen in liquid
nitrogen and stored at Immunofluorescence
Centrosome proteins were localized by immunofluorescence as previously
described (Mitchison and Kirschner, 1986 Electron Microscopy Tomography
Centrosome fractions were diluted into 2 vol of 3% glutaraldehyde in
PEM buffer and incubated on ice for 10 min. After fixation, centrosomes
were concentrated into a loose pellet by centrifugation at 70,000 g for 30 min at 4°C in a rotor (model TLA 100.3; Beckman Instruments). Pellets
were aspirated, postfixed with 1% OsO4, dehydrated, and embedded as
described previously for Spisula asters (Palazzo et al., 1988 Protein Analysis
To collect centrosomes, the centrosome fractions were diluted with equal
volume of PEM buffer followed by centrifugation at 128,000 g/4°C/20 min
using a rotor (model TLA 100.3; Beckman Instruments). For protein quantitation or SDS-PAGE analysis, centrosome pellets were first resuspended
in 25 mM Tris, pH 7.2, containing 0.5% SDS (TS). For two-dimensional
gel analysis, pellets were resuspended in urea sample buffer (O'Farrell,
1975 Denaturing Electrophoresis
Centrosome pellets were solubilized in 25-50 µl of TS. Equal volume of
2× SDS-sample buffer was added (Laemmli, 1970 Two-dimensional Electrophoresis
The Millipore (Bedford, MA) high-resolution electrophoresis system (Patton et al., 1990 Immunoblots
For immunoblot analysis, proteins resolved by SDS-PAGE were transferred to Immobilon-P membrane (Millipore Corp.) according to the
methods of Towbin et al. (1979) Quantitation of Centrosome Tubulin
To quantify tubulin in centrosome samples, Spisula glutamate-purified tubulin was used as a standard. The Spisula tubulin stock sample was determined to be 97% tubulin, with one contaminant representing <3% of the
protein. The stoichiometric ratio of Centrosome protein (5 µg) and 10 µl of each tubulin standard were
separated on 10% polyacrylamide gels and transferred to Immobilon-P
membrane (Millipore Corp.) as described in Towbin et al. (1979) Images of immunoblots were digitized using a BioImage analysis system (Milligen, Ann Arbor, MI) and analyzed using the one-dimensional module of the BioImage Visage software package. The integrated optical
density (IOD) of each standard was determined and plotted relative to the
tubulin concentration to determine the linear range. Tubulin standards
within the linear range were used for first-order regression analysis. The
IOD for the tubulin content of centrosome samples was obtained, and tubulin concentration was calculated using the equation x = b Isolation of Centrosomes
Centrosomes were isolated from Spisula oocyte lysates by a
discontinuous two-step sucrose gradient centrifugation system, consisting of 66 and 52.5% sucrose steps, similar to
that used for isolation of mammalian centrosomes (Mitchison and Kirschner, 1984
In these gradients, the highest density of centrosomes
was consistently found in a 59% sucrose fraction at the interface between the 52.5% step and the 66% cushion (centrosome fraction) (Fig. 1 A). Protein analysis revealed that
the centrosome fraction contained relatively little protein
compared to the rest of the gradient fractions collected
(Fig. 1 B). The number of centrosomes present in each fraction was determined by reconstituting gradient fractions with exogenous tubulin and counting the number of asters
that formed with a hemocytometer viewed by polarized
light microscopy (Fig. 2 A). Immunofluorescence analysis
of asters using antibodies that recognize
Using this isolation method, 4-10 × 106 centrosomes
could be obtained from a single 30-ml gradient, representing a yield of 2-3 × 106 centrosomes per ml of original lysate. When centrosome fractions from multiple gradients
were pooled and centrifuged, the final centrosome pellet
represented an ~3,000-fold purification from lysate. Results from a typical preparation are shown in Table 1. The
degree of purification was variable (~2,400-4,000 fold) depending on the protein concentration of the original lysates used, which ranged between ~75 mg/ml to ~150 mg/
ml. Based on the capacity of the SW28 rotor (six gradients), these preparations yielded ~300 µg of protein per
run, allowing the preparation and storage of more than
900 µg of centrosome protein per day.
Structural Analysis
IVEM analyses of serial semi-thick sections confirmed our
original finding (Palazzo et al., 1992
Protein Composition
Centrosome protein composition was analyzed using both
SDS-PAGE and two-dimensional gel electrophoresis. In
addition, immunoblot analysis was conducted to assay for
the presence of proteins known to be components of mitotic
centrosomes; the MPM-2 phosphoepitope proteins (Davis, et
al., 1983; Vandre et al., 1986 SDS-PAGE revealed that the centrosome fraction contained a number of proteins, including a prominent triplet
of proteins of ~20 kD (Fig. 4, lane 3). Comparison with
the lysate protein profiles revealed that these proteins
were highly enriched in the centrosome fraction (Fig. 4,
lanes 2 and 3). Thus, a number of specific proteins copurify with Spisula centrosomes. Immunoblot analysis revealed that relative to lysate, the centrosome fraction was
enriched in two MPM-2 phosphoepitope proteins of 230 and 115 kD (Fig. 4, lanes 5 and 6). Importantly, while
Quantitation of Centrosome Tubulins
Two-dimensional gel analysis of centrosome proteins visualized with silver stain revealed that the major form of tubulin to copurify with centrosomes was
Table II.
Quantitation of ; Kellogg et al., 1994
). The organization of these arrays is determined by Mt-organizing
centers (Pickett-Heaps, 1969
). In animal cells, the major
Mt-organizing center is the centrosome (Brinkley et al.,
1981
). Despite the critical role of the centrosome in many cellular processes, surprisingly little is known about the organelle's molecular composition or how Mt nucleation is
regulated biochemically. A major barrier has been the inability to isolate sufficient quantities of functional centrosomes for direct biochemical analysis (Brinkley, 1985
).
Recently, methods have been developed for the isolation of centrosomes from mammalian cell lines (Mitchison and
Kirschner, 1984
, 1986
; Bornens et al., 1987
), insects
(Moritz et al., 1995a), and the major Mt-organizing center
in fungal cells, the spindle pole body, from yeast (Rout
and Kilmartin, 1990
). These procedures provide an important first step for the characterization of proteins required
for centrosome function.
; Gould and Borisy, 1977
). The PCM is largely proteinaceous (Kuriyama, 1984
; Klotz et al., 1990
) and serves
as the site of origin for Mts (Gould and Borisy, 1977
; Soltys and Borisy, 1985
; Moritz et al., 1995a). At this time, relatively few PCM proteins have been identified (Sellitto et al.,
1992
; Kellogg et al., 1994
). An extensively characterized PCM protein required for Mt nucleation is
-tubulin, a
member of the tubulin superfamily first identified in Aspergillus nidulans (Oakley and Oakley, 1989
). A large body
of genetic and biochemical evidence indicates that
-tubulin plays a crucial role in Mt nucleation. First,
-tubulin is
localized to centrosomes and spindle pole bodies and is required for spindle assembly and progression through mitosis (Oakley and Oakley, 1989
; Oakley et al., 1990
; Stearns et al., 1991
; Zheng et al., 1991
). Second,
-tubulin antibodies inhibit centrosome-dependent Mt nucleation (Joshi et
al., 1992
). Third, recruitment of
-tubulin to sperm basal bodies is necessary for the assembly of an Mt nucleation-competent paternal centrosome (Felix et al., 1994
; Stearns and
Kirschner, 1994
). Fourth,
-tubulin binds tightly to the minus ends of Mts with a stoichiometry that suggests that one
-tubulin is bound per tubulin subunit exposed at the minus ends of Mts (Li and Joshi, 1995
). Finally,
-tubulin is a
component of nucleation-competent complexes, 25-nm-diam
"rings," recently isolated from Xenopus oocyte extracts
(Zheng et al., 1995
), and ring structures of similar diameter
have been identified as components of isolated Drosophila
centrosomes (Moritz et al., 1995b). Based on these studies,
it is clear that
-tubulin is a critical PCM component required
for centrosome-dependent Mt nucleation. However, the
role, if any, of conventional
- or
-tubulin in centrosomedependent Mt nucleation has yet to be investigated.
- and/or
-tubulin) with centrosome
components. Since
-tubulin was originally discovered as a
second-site suppressor of a
-tubulin mutation in Aspergillus nidulans (Oakley and Oakley, 1989
), it was proposed
that
-tubulin interacts with Mts via a physical interaction
with the
-tubulin subunit of the tubulin heterodimer (Mandelkow and Mandelkow, 1994
; Oakley, 1994
). In addition,
evidence gained from the binding of GTP-analogs covalently attached to fluorescent beads suggests that
-tubulin
is the terminal subunit at the plus end of the Mt; however,
it was proposed that the minus end may have an alternative structure, perhaps consisting of
:
heterodimer
(Mitchison, 1993
). Thus, it is important to investigate
whether conventional (
,
) tubulins, and in particular
-tubulin, are important for centrosome-dependent Mt
nucleation. In addition, numerous other questions remain
regarding the cell cycle-dependent regulation of centrosome assembly and duplication and the regulation of
Mt nucleation during meiosis and mitosis.
), they represent a pure synchronous culture of cells. Importantly, fertilization or parthenogenetic activation induces the synchronous assembly and maturation of functional centrosomes within minutes (Allen,
1953
; Rebhun, 1959
; Kuriyama, 1984
; Palazzo et al., 1992
),
and extracts prepared from activated oocytes assemble asters (Weisenburg and Rosenfeld, 1975; Palazzo et al., 1988
),
offering the possibility of a biochemical approach to understanding the regulation of centrosome assembly and
maturation. Previously, this system was used to study centriole assembly and maturation in vitro (Palazzo et al., 1992
).
Here we report methods for isolating homogeneous centrosomes from a specific time point in the meiotic cell cycle
for biochemical and structural analysis, and the discovery
that centrosomes contain an unexpected stoichiometric ratio of
/
tubulin.
Materials and Methods
). Aster formation in lysates was assessed with polarized light microscopy by adding 3% hexylene glycol to small aliquots and
warming to 24°C (Palazzo et al., 1988
). The remaining lysate was aliquoted, snap frozen, and stored at
80°C. Frozen lysates retain the ability to assemble asters after years of storage.
). Aliquots of ~2 mg/ml protein were stored in
100 mM potassium-Pipes, pH 6.9, 1 mM EGTA, 5 mM MgSO4, 2 mM
GTP (reassembly buffer) at
80°C. Three-cycled tubulin was diluted with
reassembly buffer to 0.7 mg/ml for all functional assays unless otherwise
stated.
. Lysates were thawed and diluted with 0.8 vol of dilution buffer (100 mM
potassium-Pipes, pH 7.2, 4 mM EGTA, 1 mM MgSO4, 1 mM DTT) containing 1 mM GTP, 10 mg/ml leupeptin, and 0.2 mM phenylmethylsulfonyl
fluoride. Diluted lysate was gently resuspended on ice and centrifuged
(39,000 g/2°C/30 min) using a rotor (model JA20; Beckman Instruments,
Fullerton, CA) to clarify. Supernatants were collected and three-cycled
tubulin prepared as previously described (Suprenant, 1989
).
), followed by successive cycles of polymerization/depolymerization. The final
five-cycled Mts were pelleted through a cushion of 30% glycerol/reassembly buffer. Pellets were aspirated dry, snap frozen in liquid nitrogen, and
stored at
80°C.
80°C for further use.
; Palazzo et al., 1992
) with the
following modifications: Aliquots of the centrosome fraction were reconstituted with tubulin media and incubated for 30 min at room temperature. Samples were fixed for 5 min at room temperature by adding 100 vol
of 1% glutaraldehyde (Ted Pella, Inc., Redding, CA) in PBS (10 mM
Na2HPO4, 1.8 mM KH2PO4, 136 mM NaCl, 2.6 mM KCl, pH 7.2). Fixed
samples were layered onto a 30% glycerol/PBS cushion, and centrosomes
were centrifuged onto polylysine-coated coverslips at 8,000 g using a rotor
(model JS13.1; Beckman Instruments) (Mitchison and Kirschner, 1984
).
The coverslips were incubated in ice-cold methanol for 5 min and then
washed three times with PBS to rehydrate. All subsequent steps used PBS
as buffer. Coverslips were blocked with 5% BSA for 30 min, followed by
application of the primary antibody. Tubulins were detected using the
following antibodies: DM1A (
-tubulin; Amersham Corp., Arlington
Heights, IL), TU27 (
-tubulin; a gift of Dr. A. Frankfurter, University of Virginia, Charlottesville, VA), or EAD24 (
-tubulin). Detection of
antigen/antibody complexes was accomplished using either anti-mouse FITC or anti-rabbit rhodamine conjugates (Calbiochem, La Jolla, CA).
Images were acquired with a laser scanning confocal microscope (model
MRC-100; BioRad Labs, Hercules, CA).
, 1992). Semithick (200-300 nm) serial sections were then cut from each preparation.
Each section was subsequently mounted in the center of a formvar-coated
slot grid on which 15-nm colloidal gold had been lightly deposited to facilitate subsequent micrograph alignment. Sections were next stained with
uranyl acetate and lead citrate. Selected regions within a section, containing the area to be reconstructed, were irradiated at 400 kV and normal beam intensity in an intermediate voltage electron microscope (IVEM)
(model JEM-4000 FX; JEOL U.S.A., Inc., Peabody, MA) until the initial
mass loss was completed (~10 min). They were then photographed on SO
163 film, using a tilt-rotation stage, around two orthogonal (x and y) axes
at increments of 2° over a ± 60° range. To limit specimen deformation
during sequential photography, the specimen was further irradiated only
during image recording. 122 tilt images were used for each reconstruction,
with 61 images around the x-axis and 61 images around the y-axis. Images
were then scanned so that each pixel was 2.6 nm and each image was 800 × 800 pixels. Three-dimensional tomographic reconstructions were then calculated by methods previously described in detail (Penczek et al., 1995
;
see also McEwen et al., 1993
). Images were displayed in negative form to
enhance the contrast.
). Protein content was determined with a bicinachromic acid protein
assay according to manufacturer's instruction (Pierce, Rockford, IL).
), samples were boiled,
and proteins were separated on either 10 or 4-20% gradient (BioRad
Labs) polyacrylamide gels (Laemmlli, 1970). Proteins were visualized by
staining with Coomassie G-250 (Neuhoff et al., 1988
).
) was used for two-dimensional gel electrophoresis. Centrosome pellets (containing 10-20 × 106 centrosomes) were solubilized in
50 µl of urea sample buffer (10 mM Tris, pH 8.8/0.06% SDS/0.1 M
-mercaptoethanol/100 mM DTT/9 M urea/4% NP-40/10 mM CHAPS). Approximately 5 µg of protein (~90,000 centrosomes) was loaded onto each
isoelectric focusing gel and proteins focused for 18,000 volt h using 4.1%
gels containing ampholytes with the pH ranges of 3-10, 5-7, and 4-8 (Millipore Corp.) at a ratio of 1:1:2, respectively. In the second dimension, proteins were separated on 10% SDS-PAGE gels (O'Farrell, 1975
), and
proteins were detected by either silver stain (Patton et al., 1990
) or by immunoblot. Apparent molecular weights and isoelectric points were determined using external standards (mol wt; BioRad Labs) (pI; Pharmacia
LKB Nuclear, Gaithersburg, MD).
. Membranes were blocked with 5% BSA
in TBS containing 0.2% Tween-20 (TBS/T) for 1 h, followed by incubation
with primary antibodies DM1A (
-tubulin; Amersham Corp.), DM1B
(
-tubulin; Amersham Corp.), C4 (actin; ICN Biomedicals, Costa Mesa,
CA), or EAD24 (
-tubulin) and HRP-IgG-conjugated secondary antibody (Promega Corp., Madison, WI). All antibodies were diluted in 0.5×
blocking buffer. Blots were washed between hybridizations with TBS/T.
Chemiluminescence (ECL; Amersham Corp.) was used to detect the antibody-antigen complexes according to product instructions.
/
tubulin was determined by densitometric measurement of Coomassie-stained tubulin separated in 7.5%
polyacrylamide gels and found to be 1:1 in these samples. Therefore, the
concentration of either
- or
-tubulin for each standard was considered
to be half of the protein concentration. Tubulin (2 mg/ml in reassembly
buffer containing 2 mM GTP) was diluted to 1 mg/ml on ice with ultrapure
H2O, followed by serial dilution in SDS-sample buffer to give standards
with the following concentrations: 0.1, 0.03, 0.01, 0.003, and 0.001 mg/ml.
. After
transfer, gels were stained as described in Neuhoff et al. (1988)
to confirm
that all the protein had been electroeluted to the membrane. Immunoblots were prepared and visualized by chemiluminescence as described
previously.
y/m, where
y is equal to the IOD value of the centrosome tubulin signal, b is equal to
the y-intercept (integrated optical density), and m is equal to the slope.
Since only half of the tubulin in each standard represented
- or
-tubulin
(e.g.,
-tubulin = a/a + b = 0.5), the tubulin concentration in centrosome
samples obtained by this regression analysis was adjusted by multiplying
the value by 0.5.
Results
). The centrosome content of gradient fractions was determined using a functional reconstitution assay based on the ability of centrosomes to organize
Mts into astral arrays (Mitchison and Kirschner, 1984
;
Palazzo et al., 1988
, 1992). Thus, aliquots of gradient fractions were diluted into tubulin-containing media, and aster
formation was visualized by polarized light microscopy (see Fig. 2 A).
Fig. 2.
In vitro aster formation using centrosome-containing
sucrose fractions. Aliquots of centrosome fractions were reconstituted with tubulin media to assemble asters. (A) Aster-forming
activity was quantified using polarized light microscopy. (B) Asters
were composed of microtubules (green) stained by -tubulin antibody TU27, which project from discrete central foci (red) stained
by
-tubulin antibody EAD24. (C) EM analysis revealed that
centrosome fractions contained bona fide centrosomes composed
of centrioles (arrows) surrounded by a mass of pericentriolar material ~2 µm in diameter. Bar, (A and B) 10 µm; (C) 2.0 µm.
[View Larger Version of this Image (49K GIF file)]
-tubulin (TU27)
or
-tubulin (EAD24) revealed that these reconstituted asters contain Mts emanating from a discrete
-tubulin staining center (Fig. 2 B). IVEM verified that centrosome fractions contained bona fide centrosomes (Fig. 2 C), which
were easily identified based on their centriole content
(Palazzo et al., 1992
). As expected, each centrosome contained a single centriole surrounded by PCM (Fig. 2 C).
Fig. 1.
Sedimentation of centrosomes in preparative (30 ml)
discontinuous sucrose gradients. Gradient fractions were analyzed for centrosome content using a functional reconstitution assay. Centrosomes were isolated from Spisula oocyte lysates as described in Materials and Methods. 5 µl of each fraction was
combined with 20 µl of tubulin media containing 0.5 mg/ml sea
urchin three-cycled tubulin and incubated at 22°C to allow aster
formation. (A) Concentration of asters (centrosomes) /ml was determined using a hemacytometer and polarized light microscope
and plotted against fraction density (% sucrose). The peak of aster forming activity, reflecting the number of centrosomes per
fraction, corresponded to the 59-60% fraction (59.7 ± 0.628, n = 5). (B) To determine the protein concentration relative to centrosome content, fractions were dialyzed against PEM buffer and
then against PEM/0.2% SDS for 2 h. Protein was precipitated
with 20% TCA on ice and pelleted in a microfuge. Pellets were
resuspended in 0.1 N NaOH and the protein concentration determined using a Coomassie assay. Aster-forming activity corresponded to a small peak of precipitable protein. The protein content of centrosome-containing fractions represented a small
fraction of the total protein recovered from the gradient.
[View Larger Version of this Image (14K GIF file)]
) that centrosomes isolated from Spisula oocytes shortly after activation contain
a single centriole embedded in the middle of an extensive
sphere of PCM (e.g., Fig. 3, A and G). When viewed by tomography, this PCM was seen to be studded throughout
its volume with numerous, conspicuous ring-shaped structures (Fig. 3, C-F, arrowheads). The depth (z-axis dimension) of each ring was less than its 25-28-nm diameter, and most were found to be completely contained within the
150-200-nm-thick (after irradiation) volume of each tomogram. These rings were similar, if not identical, to those
recently detected by IVEM tomography in the PCM of
isolated Drosophila centrosomes (Moritz et al., 1995a).
IVEM also revealed that the great majority of centrosomes isolated from Spisula oocytes shortly after activation contained an unusual vesicular structure at the base of
the centriole (Fig. 3, G and H). It is possible that this structure is involved in assembling the second centriole, which
we have previously shown to occur near this time point.
Fig. 3.
IVEM-tomography of centrosomes. (A) IVEM micrograph, (B) 2.6-nm-thick slice, and (C-F) rendered projections through
the volume of a tomographic reconstruction from a 0.25-µm section through a Spisula centrosome isolated from a 4-min extract. C and D
and E and F show highly magnified sections from this tomogram in untilted (C and E) and tilted (D and F) views. Numerous ringshaped structures (e.g., arrowheads), completely contained within the section, are found throughout the centrosome, including regions
near the centriole (C and D) and near its periphery (E and F). A 2.6-nm-thick slice from a second tomogram, obtained from a 0.25-µmthick section cut from one end of the centriole in an isolated centrosome, is pictured in G and at higher magnification in H. Note that a
conspicuous electron-opaque "vacuole" is associated with the centriolar end. Bars: (A, B, and G) 0.50 µm; (C-F, and H) 250 nm.
[View Larger Version of this Image (106K GIF file)]
; Ohta et al., 1993
),
-tubulin,
-tubulin (Bornens, 1987), and
-tubulin (Stearns et al.,
1991
; Zheng et al., 1991
).
-tubulin copurified with centrosomes as expected, it did not appear to be an MPM-2 protein (Fig. 4, lane 6). Finally, relatively little
- or
-tubulin was found in the centrosome
fraction (Fig. 4, lanes 9-12).
Fig. 4.
Protein composition of centrosomes. Spisula
oocyte lysate and isolated
centrosomes were compared. 5 µg of total protein
was loaded per lane. Proteins
were separated in 4-20% linear gradient minigels and
stained with colloidal Coomassie G-250. Molecular mass standards are shown in
lanes 1 and 4. A number of
proteins are enriched in centrosome fractions (lane 3)
over lysate (lane 2). These include a triplet of 20 kD, and
proteins of 25, 50, 65, 90, 115, 125, 210, 230, and 300 kD, respectively. Immunoblots were probed with antibodies
specific for the following:
MPM-2 phosphoepitope (lanes 5 and 6), -tubulin, (lanes 7 and 8),
-tubulin (lanes 9 and 10), and
-tubulin (lanes 11 and 12). Two
MPM-2 phosphoepitope proteins of 115 and 230 kD (lanes 5 and 6) and
-tubulin (lanes 7 and 8) are enriched in centrosome fractions
over lysate. Conventional tubulins (
- and
-tubulin) are not detected in centrosome fractions under these conditions (lanes 9-12). LYS,
lysate; CE, isolated centrosome fraction.
[View Larger Version of this Image (46K GIF file)]
-tubulin (Fig. 5 A).
The staining intensity of
-tubulin was higher than that observed for either
- or
-tubulin (Fig. 5 A). Densitometric
analysis of silver-stained gels revealed that centrosomes
contain ~10-fold more
-tubulin than
-tubulin, consistent
with the tubulin ratios reported for
-tubulin ring complexes isolated from Xenopus oocytes (Zheng et al., 1995
).
Surprisingly, the results of two-dimensional gel analysis suggested that centrosomes contain more
- than
-tubulin (Fig. 5 A).
Fig. 5.
Analysis of centrosome tubulins. Centrosome preparations were
analyzed by two-dimensional gel electrophoresis (A)
and quantitative immunoblot
(B-D). Three tubulin types
were found in centrosomes, ,
, and
(A).
-Tubulin
has a major isoform with a pI
of 5.52 and is the major form
of tubulin found in centrosome fractions. SDS-gel
analysis of glutamate-purified Spisula oocyte tubulin stained with Coomassie blue
(B, lane 1) provided an
- and
-tubulin standard (B, lane
2). Stoichiometrically equivalent amounts of
- and
-tubulin (B, lane 1) were probed simultaneously with antibodies specific to either
-tubulin
(DM1A) or
-tubulin (DM1B)
(B, lane 2). In these samples,
the
-tubulin-specific signal
is more intense than the
-tubulin-specific signal, indicating that DM1A is a more sensitive probe than
DM1B (B, lane 2). In contrast, probing centrosome fractions resulted in a higher
-tubulin-specific (DM1B) signal than the
-tubulin-specific (DM1A) signal
(B, lane 3). The tubulin content of centrosomes was determined using quantitative immunoblot analysis. (D) 5 µg of centrosome protein (lane 1) and series dilution of purified Spisula tubulin standards (lane 2, 1 mg/ml; lane 3, 0.3 mg/ml; lane 4, 0.1 mg/ml; lane 5, 0.03 mg/ ml; lane 6, 0.01 mg/ml) were separated in 10% polyacrylamide gels and processed for immunoblot analysis using DM1A and DM1B (see Materials and Methods for details). The IOD of tubulin stain for centrosome samples and each standard lane in D was determined by
densitometry and plotted (C). The IOD of only the top band of the two stained by DM1B in D, lane 1, was used in this analysis, the
lower band being an artifact in this particular blot. The linear range (C, solid lines) for DM1A (C, filled squares) and DM1B (C, open
circles) ranges from 0.01 to 0.1 mg. The IOD obtained for the tubulin signals in the centrosome sample for both DM1A and DM1B lay
within the linear range of detection (C, arrows). First order regression analysis was used to calculate the tubulin content of centrosomes
(results presented in text and Table II). The r2 value for the regression for DM1A was 0.962, and for DM1B was 0.893.
[View Larger Version of this Image (57K GIF file)]
- and
-Tubulin in Centrosome
Fractions
The intriguing possibility that the stoichiometric ratio of
/
tubulins was not 1:1 in centrosomes was tested further.
To conduct this analysis, the ratio of
/
tubulin present in
purified Spisula tubulin was first determined, and the staining intensity was assessed for
- (DM1A) and
- (DM1B)
tubulin-specific antibodies to be used as probes for quantitative immunoblot analyses. Comparison of the IOD values obtained from SDS-PAGE gels stained with Coomassie blue revealed that glutamate-purified Spisula tubulin
contained stoichiometrically equivalent amounts of
- and
-tubulin as expected (Fig. 5 B, lane 1). Furthermore,
Western blot analysis revealed that the relative staining intensity of DM1A and DM1B for equimolar protein was
similar (Fig. 5 B, lane 2), although DM1A was found to be
a slightly more sensitive probe than DM1B. However,
Western blot analysis of centrosome proteins resulted in
significantly higher staining intensity for DM1B than for
DM1A (Fig. 5 B, lane 3), confirming that centrosomes
contain more
-tubulin than
-tubulin.
To extend this analysis further, the stoichiometric ratio
of /
tubulin in centrosomes was determined by quantitative immunoblot analysis. Because of the differences in the
staining intensity observed for DM1A and DM1B when
challenged with equivalent protein (Fig. 5 B, lanes 1 and
2), identical immunoblots were prepared and probed with
each antibody separately. Gels were loaded with Spisula
glutamate-purified tubulin diluted in series (from 1.0 µg
to 0.01 µg/lane) to provide standards, and centrosome proteins were loaded onto adjacent lanes on the same gels.
Proteins were separated, transferred to immobilon membranes, probed with antibodies in parallel and blots visualized by chemiluminescence (Fig. 5 D), as described in
Materials and Methods. The signal for
- or
-tubulin produced by chemiluminescence was digitized, and the staining intensity (IOD) of tubulin in standards and centrosome samples was measured by quantitative densitometry
(Fig. 5 C). The IOD values obtained were plotted relative
to tubulin concentration, and the linear range of DM1A
and DM1B staining for tubulin was determined (Fig. 5 C).
The standard curves of both DM1A and DM1B staining
showed saturation at tubulin concentrations greater than 0.3 µg, but both standard curves were linear for tubulin
concentrations between 0.1 and 0.03 µg (Fig. 5 C). In all
cases, the IOD values obtained for centrosomal tubulin
bands stained by DM1A or DM1B lay within the linear region of these curves (Fig. 5 C; note arrows). The linear
range of each standard curve was used to derive a first order equation that was used to calculate the tubulin concentration in the centrosome protein sample as described in
Materials and Methods. The results, summarized in Table
II, show that
- and
-tubulin together represent 0.3% of
the total centrosome protein. Calculations indicate that
centrosomes contain 0.10 ± 0.021 pmol of
-tubulin (n = 5)
and 0.48 ± 0.099 pmol of
-tubulin (n = 5) per centrosome. Most importantly, the stoichiometric ratio of
/
in
centrosomes was not 1:1, but found to be an average of 1:4.6 in these experiments.
Despite its critical role in a variety of cellular processes
(Brinkley, 1985), the centrosome remains a relatively enigmatic organelle. A major barrier to understanding how it
functions has been the difficulty in obtaining highly purified and homogeneous preparations for biochemical analyses (Brinkley, 1985
). Recently, procedures have been developed for isolating centrosomes from mammalian cell lines
(Mitchison and Kirschner, 1984
, 1986
; Bornens et al., 1987
),
Drosophila embryos (Moritz et al., 1995a), and spindle
pole bodies from yeast (Rout and Kilmartin, 1990
). As described here, surf clam (Spisula solidissima) oocytes possess a number of characteristics that make them uniquely
suited as a model system for centrosome research. First,
Spisula is commercially fished, and substantial quantities
of pure oocytes (100 g) can be quickly and easily harvested. In addition, Spisula oocytes and embryos develop
synchronously (Allen, 1953
; Rebhun, 1959
; Palazzo et al.,
1988
), allowing for the acquisition of large numbers of
cells from specific time points in the meiotic and mitotic cell cycle. Importantly, Spisula oocytes are arrested naturally in prophase of meiosis I and enter M-phase within
minutes of oocyte activation (Allen, 1953
; Rebhun, 1959
).
Finally, parthenogenetic activation of oocytes induces centrosome maturation, which includes centriole duplication
(Palazzo et al., 1992
) and assembly of the meiosis I spindle
within 15 min (Allen, 1953
; Rebhun, 1959
).
Crude homogenates (Weisenberg and Rosenfeld, 1975)
and lysates (Palazzo et al., 1988
) prepared from parthenogenetically activated Spisula oocytes spontaneously assemble robust asters, which are easily discernible by conventional polarized light microscopy. Moreover, treating
lysates prepared from unactivated oocytes with high-speed
supernatants prepared from parthenogenetically activated oocytes induces centriole duplication and centrosome maturation within lysates (Palazzo et al., 1992
). The unique
properties of Spisula oocytes, together with these advancements, suggested that this system could be developed as a
powerful model for studying how centrosome function is
regulated. To this end, it becomes important to develop
methods for isolating centrosomes from various time points
in the cell cycle for analyses. Here we have described a
method for isolating centrosomes from one specific time
point, 4 min after parthenogenetic activation.
Asters are clearly present in Spisula oocytes 4 min after
activation (Allen, 1953; Rebhun, 1959
) and in lysates prepared from oocytes at this stage in development (Weisenberg and Rosenfeld, 1975
; Palazzo et al., 1992
). Past analyses
have shown that these asters contain bona fide centrosomes composed of a single centriole surrounded by PCM
(Weisenberg and Rosenfeld, 1975
; Palazzo et al., 1992
).
Spisula centrosomes were isolated from these lysates by
sucrose-density gradient centrifugation using a procedure
patterned after the methods of Mitchison and Kirschner
(1986). Centrosomes in sucrose fractions were detected by
simply diluting gradient fractions in tubulin-media under
conditions that support Mt polymerization and observing
aster formation by polarized light microscopy. Sucrose gradient fractions that contained aster-forming activity
were analyzed to determine if they contained bona fide
centrosomes by several methods. First, we assayed for centrosome-dependent Mt nucleation (aster formation) using
a functional reconstitution assay. Furthermore, double immunofluorescence revealed that fractions capable of inducing aster formation contained discrete structures that
stained for
-tubulin, a known component of centrosomes
(Stearns et al., 1991
; Zheng et al., 1991
). Finally, EM studies revealed that these fractions contained numerous centrioles surrounded by abundant PCM. Together, these observations indicate that we have successfully isolated
centrosomes from activated Spisula oocytes.
Because the Mt nucleation potential of isolated Spisula
centrosomes is so robust, centrosomes could be quantified at
every step of the purification process by simply counting
asters. Our isolation procedure yields an average of ~3.0 × 106 centrosomes/ml of lysate, with recovery as high as
70%. This recovery is comparable to that reported by Mitchison and Kirschner (1986) and Bornens et al. (1987), but
our system yields significantly more protein for analyses.
Indeed, as much as 53 µg of centrosome protein, representing a 2,500-4,000-fold purification from lysate, can be
collected from a single preparative gradient. Since six gradients can be run simultaneously, and three preparations
can be run in a 9-h period, milligram quantities of centrosomal protein can be obtained in a single day.
Our biochemical analyses of Spisula centrosomes were
facilitated by our ability to obtain relatively large quantities
of these organelles in a highly enriched state. We found
vandre that a minimum of thirty proteins ranging in molecular mass from 20-300 kD copurify with Spisula centrosomes,
including -tubulin and a prominent triplet of proteins of
~20 kD. Immunoblot analyses revealed that the 230- and
115-kD proteins appear to contain mitosis-specific, or in
this case meiosis-specific, phosphoepitopes (Vandre et al., 1986
). Also, the 230- and 115-kD proteins appear to contain
mitosis-specific, or in this case meiosis-specific, phosphoepitopes (Vandre et al., 1986
) since they cross-reacted
with the MPM-2 monoclonal antibody (Davis et al., 1983
).
Actin was also relatively abundant in our preparations,
suggesting that Spisula centrosomes contain one or more
actin-binding proteins. Finally, we found that
-tubulin was
the major tubulin component of Spisula centrosomes, which also contained more minor amounts of
- and
-tubulin.
-Tubulin (Oakley and Oakley, 1989
) is a highly conserved
component of centrosomes (Stearns et al., 1991
; Zheng et
al., 1991
; Sunkel et al., 1995
), and it is required for centrosomal Mt nucleation in diverse organisms. However,
the respective roles of the
- and
-tubulins in this nucleation process remain ambiguous. Based on extragenic suppression of a
-tubulin mutation by the mipA allele in Aspergillus, Oakley and Oakley (1989)
proposed that
- and
-tubulin interact physically to initiate Mt nucleation. Recently, an Mt nucleation-competent
-tubulin complex, also
containing
- and
-tubulins in stoichiometrically equivalent amounts, was identified in Xenopus cytoplasmic extracts (Zheng et al., 1995
). Other multiprotein complexes
containing
,
, and
-tubulins have also been reported for
other cell types (Marchesi and Ngo, 1993
; Melki et al., 1993
;
Raff et al., 1993
; Stearns and Kirschner, 1994
), but the stoichiometry of the various tubulins in all of these complexes remains to be determined. Regardless, given the recent discovery in Drosophila of
-tubulin-containing ring-structures, with the appropriate diameter of a 13-protofilament Mt (Moritz et al., 1995a,b), it is possible that
- and/or
-tubulin
are also components of
-tubulin-based Mt-nucleating sites.
We found that - and
-tubulins were minor components of Spisula centrosomes, representing <0.3% of the
total centrosome protein. This is dramatically lower than
the
and
tubulin content reported for mammalian centrosomes (3-6%; Mitchison and Kirschner, 1984
, 1986
;
Bornens et al., 1987
) and yeast spindle pole bodies (Rout and Kilmartin, 1990
). Two-dimensional gel analysis revealed that
-tubulin was at least 10-fold more abundant
in Spisula centrosomes than conventional tubulins. This
finding is similar to that reported for the
-tubulin-containing complexes isolated from Xenopus extracts (Zheng
et al., 1995
). However, we unexpectedly found that
-tubulin is significantly more abundant in Spisula centrosomes than
-tubulin. Since, to our knowledge, there are no known
posttranslational modifications of the epitope recognized
by the
-tubulin antibody (DM1A), it is unlikely that the
amount of
-tubulin in the centrosome is underreported
by our analysis. It is also unlikely that this disparity arises
from significant differences in the
/
tubulin ratios within
the centriole in each centrosome, since the
/
tubulin ratio in these organelles is expected to be 1:1 (Kochanski and Borisy, 1990
).
The results presented indicate that isolated Spisula centrosomes contain ring structures like those first reported
for Drosophila (Moritz et al., 1995b). Since the diameter of
these rings matches the diameter of microtubules and isolated ring complexes from Xenopus oocytes increase the
rate of microtubule polymerization (Zheng et al., 1995), it is
likely that these rings represent oligomeric protein complexes that serve as initiation sites for centrosome-dependent
microtubule assembly. The data presented here indicates
that while isolated Spisula centrosomes contain similar ring
structures, they also contain an unusual ratio of
/
tubulin that is not 1:1, but rather 1:4 or 1:5. This indicates that
in isolated centrosomes, at least some of the
-tubulin is not complexed in the form of conventional
/
heterodimers. This suggests the possibility that
-tubulin might
be a component of the oligomeric ring structure, possibly
associated with
-tubulin. Thus, the data presented is consistent with the hypothesis that
-tubulin plays an important role in centrosome structure and/or microtubule nucleation, a role that may be independent of
-tubulin.
Table I. Isolation of Centrosomes from Spisula Oocyte Lysates |
Received for publication 30 May 1996 and in revised form 16 January 1997.
Address all correspondence to Robert E. Palazzo, The Department of Physiology and Cell Biology, University of Kansas, Lawrence, KS 66045. Tel.: (913) 864-3872. Fax: (913) 864-5321.The authors would like to thank V. Mermall for comments, C. Lyddane and G. Osorio for assistance with centrosome preparation, EM, and confocal microscopy, and M. Marko for assistance with IVEM tomography. TU27 was the generous gift of A. Frankfurter. MPM-2 antibody was a gift of J. Kuang (University of Texas, M.D. Anderson Cancer Center).
Supported in part by the National Institutes of Health (NIH) (GM43264), the American Cancer Society (JFRA-314) and the Robert Day Allen Fellowship of the Marine Biological Laboratory (to R.E. Palazzo), the Ida H. Hyde Scholarship (to J.M. Vogel), NIH grant GM 40198 (to C.L. Reider), National Science Foundation grant BIR 9219043, and RR 01219 awarded by the Biomedical Research Technology Program, National Center for Research Resources (Department of Health and Human Resources/Public Health Service), to support the Wadsworth Center's Biological Microscopy and Image Reconstruction facility as a National Biotechnological Resource.
IVEM, intermediate voltage transmission electron microscopy; IOD, integrated optical density; MPM, M-phase- specific phosphoepitope; Mts, microtubules; PCM, pericentriolar material.