1 Department of Cell Biology, Kyoto University Faculty of Medicine,
Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan
2 Solution Oriented Research for Science and Technology, Japan Science and
Technology Corporation, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan
* Author for correspondence (e-mail: htsukita{at}mfour.med.kyoto-u.ac.jp)
Accepted 19 November 2002
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Summary |
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Key words: Centriolar satellites, PCM-1, Centrosome, Pericentrin, -Tubulin
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Introduction |
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The molecular components of centrosomes have been identified by several
distinct methods (Kimble and Kuriyama,
1992; Bornens and Moudjou,
1999
). As one of these methods, autoantibodies detected in
patients suffering from autoimmune diseases have often been utilized to
identify the centrosomal components
(Kimble and Kuriyama, 1992
;
Rattner et al., 1998
). A
high-titer serum from a patient with systemic sclerosis and Raynaud's
phenomenon was identified that contained autoantibodies that specifically
recognized centrosomes when mammalian cells were processed for
immunofluorescence microscopy (Osborn et
al., 1982
; Balczon and West,
1991
). This serum mainly detected three bands around 39, 185 and
230 kDa by immunoblotting (Balczon and
West, 1991
), and then a cDNA encoding the
230 kDa polypeptide
was isolated by screening a cDNA expression library. Immunofluorescence
microscopy with a polyclonal antibody specific for this polypeptide revealed
that this molecule was concentrated in the centrosomes, and then this molecule
was designated as pericentriolar material-1 (PCM-1)
(Balczon et al., 1994
).
However, the detailed localization of PCM-1 in centrosomes remained
unclarified.
In the previous study, we attempted to identify the centrosomal components
by raising monoclonal antibodies against a crude fraction of isolated
pericentriolar material from Xenopus egg extracts, and we obtained a
monoclonal antibody that specifically recognized Xenopus PCM-1
(XPCM-1) (Kubo et al., 1999).
Interestingly, immunoelectron microscopy revealed that PCM-1 was not
distributed diffusely at the pericentriolar region but localized exclusively
on centriolar satellites. Using a green fluorescent protein (GFP) fusion
protein with PCM-1, we found that PCM-1-positive centriolar satellites moved
along microtubules towards their minus ends (i.e. toward the centrosomes) in
live cells as well as in-vitro-reconstituted asters. These findings for the
first time defined centriolar satellites at the molecular level and explained
their pericentriolar localization. Furthermore, we found that anti-PCM-1
antibodies specifically labeled fibrous granules in ciliogenetic epithelial
cells. These findings suggested that centriolar satellites and fibrous
granules are identical novel non-membranous organelles containing PCM-1.
In this study, we further examined PCM-1 for a better understanding of the structure and functions of centriolar satellites/fibrous granules. We first found that PCM-1-positive granules were not necessarily concentrated around centrioles but were scattered throughout the cytoplasm in various types of cells. Therefore, in this study, we tentatively call them `PCM-1 granules'. Interestingly, a detailed examination in cultured cells revealed that PCM-1 granules disappeared during mitosis and re-appeared when the cells proceeded into the interphase. We found that PCM-1 granules were assembled and disassembled in a cell-cycle-dependent manner by regulating the activity of PCM-1 to self-aggregate. Furthermore, we found that PCM-1 granules were distinct from granules containing pericentrin, and that these two distinct types of granular structures frequently associated with each other within the cytoplasm. We believe that the present data provide an important clue to understand the structure and functions of the PCM-1 granule, a novel non-membranous granular organelle.
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Materials and Methods |
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Xenopus kidney epithelial cell line A6 was grown at 23°C without CO2 atmosphere in Leivobitz's L-15 medium (GIBCO BRL) supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U ml-1 penicillin and 0.2 mg ml-1 kanamycin). Mouse L fibroblasts, mouse Eph-4 epithelial cells and mouse CSG epithelial cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FCS.
Constructs and transfection
Full-length XPCM-1 (aa 1-2031) was fused with red-shifted GFP (rsGFP)
(pQBI25; Quantum Biotechnologies) at its C terminus (GFP-full) as described
previously (Kubo et al.,
1999). To cut off regions from the GFP-full expression vector to
construct a series of deletion mutants of XPCM-1, MluI sites or
KpnI sites were introduced into both ends of the regions by
site-directed mutagenesis. The cDNA between KpnI sites or
MluI sites was cut off by KpnI or MluI digestion
followed by self-ligation. All expression vectors were confirmed by
sequencing. A6 cells were transfected using LipofectAMINE plus reagent
(Invitrogen) and observed 2-3 days after transfection.
Immunofluorescence microscopy
Cells cultured on poly-L-lysine coated coverslips were fixed with methanol
for 5 minutes at -20°C, washed in PBS, incubated with 0.12% glycine/PBS
for 20 minutes and then processed for immunofluorescence microscopy as
described previously (Kubo et al.,
1999). In some experiments, A6 cells were incubated in L-15 medium
containing 0.4 µM nocodazole for 1 hour, washed twice with fresh medium,
incubated in fresh medium for 5 minutes and then fixed with methanol. After
washing with PBS, the cells were soaked in 20% FCS in DMEM for 30 minutes and
incubated with primary antibodies for 1 hour in a moist chamber. The cells
were then washed with PBS and incubated with fluorescently labeled secondary
antibodies for 1 hour. Rhodamine-conjugated donkey anti-rabbit IgG antibody
(Chemicon), Cy3-conjugated donkey anti-rat IgG antibody (Jackson Laboratory),
Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes) and
Cy5-conjugated donkey anti-mouse IgG antibody (Chemicon) were used as
secondary antibodies. Samples were then washed with PBS, rinsed in distilled
water, mounted in 40% w/w Mowiol (Calbiochem) and then observed using a
DeltaVision microscope (Applied Precision) equipped with an Olympus IX70
microscope (Olympus, Tokyo, Japan) and a cooled charge-coupled device (CCD)
system. Whole-cell images were obtained with 0.2 µm interval in z
section, deconvolved and integrated with DeltaVision software (Applied
Precision).
Mice were fixed by perfusing 3.7% formaldehyde/PBS from the heart. The
intestine, kidney, liver and brain were dissected and rinsed in 3.7%
formaldehyde/PBS for 30 minutes. Samples were mounted in Tissue-Tek and frozen
using liquid nitrogen. Frozen sections 12 µm thick were cut on a
cryostat, mounted on poly-L-lysine-coated glass coverslips, air-dried and
soaked in PBS containing 1% Triton X-100 for 10 minutes. They were then rinsed
in PBS and processed for immunofluorescence microscopy as described above.
Granule counting from deconvolved images
Images of whole cells were obtained with 0.2 µm interval in z
section and deconvolved with DeltaVision software (Applied Precision). To
evaluate the immunostaining of granules, a pixel more than three times
brighter than the average of the background fluorescent intensity was judged
as positive. Using two-dimensional polygon finder of DeltaVision software, we
regarded an aggregate consisting more than ten positive pixels (1 pixel = 44.7
nm in diameter) as a single PCM-1 or pericentrin granule, and with
three-dimensional (3D) volume builder of DeltaVision software, we
reconstituted the 3D images of granules to count the number of granules. In
some types of cells, PCM-1 granules were tightly accumulated around
centrosomes, so it was difficult to count the number of these granules
directly. In such a case, we first measured the average fluorescent intensity
of individual PCM-1 granules (FIg) in the cytoplasm, and then calculated the
number of PCM-1 granules around centrosomes through dividing the total
fluorescent intensity of PCM-1 around centrosomes by FIg.
Yeast two-hybrid
Protein interactions were examined using the MATCHMAKER LexA Two-Hybrid
System (Clontech). The cDNA encoding aa 1-484 or aa 745-1273 of XPCM-1 was
fused to the DNA-binding domain (DNA-BD) in the pLexA vector and the cDNA
encoding aa 1-711 or 745-1273 of XPCM-1 was fused to the activation domain
(AD) in the pB42AD vector. DNA-BD and AD constructs were transformed into
yeast strain EGY48 (p8op-lacZ). For the positive control, pLexA-53 and
pB42AD-T (murine p53 and SV40 large T-antigen were fused to the DNA-BD and AD,
respectively) provided by the supplier were transformed. For the negative
control, either pLexA-53 or pB42AD-T was transformed instead of XPCM-1
polypeptide fused to DNA-BD or AD, respectively. The activation of reporter
genes (lacZ and LEU2) was tested by replica plating the
transformants on SD plate (with galactose and raffinose to induce the
expression of AD fusion proteins, with X-gal to test for lacZ
expression, with or without leucine to test for LEU2 expression). SD
with glucose was used as a control to verify that the reporter activation was
correlated with the induction of AD fusion protein expression.
In vitro binding assay using fusion proteins
The cDNAs encoding aa 745-1271 and aa 1020-1271 of XPCM-1 were fused with
3'-untranslated region of XPCM-1 at their C termini and cloned into
baculovirus transfer vectors, pAcGHLT-B and -C (Pharmingen), respectively, to
generate GST fusion proteins. Recombinant baculoviruses were generated by
co-transfecting the transfer vector with BaculoGold DNA (Pharmingen) into Sf9
cells, and the resulting viruses were amplified by sequential infection into
Sf9 cells. The infected cells were harvested, lysed in IP lysis buffer (10 mM
Tris, 130 mM NaCl, 1% Triton X-100, 10 mM NaF, 10 mM sodium phosphate, 10 mM
sodium pyrophosphate, pH 7.5) on ice for 45 minutes and centrifuged at 45,000
g for 30 minutes. Pellets were denatured with 4 M urea for 20
minutes at 4°C. After urea was removed by dialysis, the samples were
centrifuged at 20,000 g for 30 minutes. The supernatant
containing the renatured recombinant proteins was applied onto glutathione
Sepharose 4B columns (Amersham Pharmacia), which were then washed with PBS
containing 400 mM NaCl. Xenopus interphase egg extracts, prepared as
described previously (Shiina et al.,
1992), were incubated with the beads for 3 hours at 4°C and
the beads were washed with PBS containing 200 mM NaCl and boiled in SDS-PAGE
sample buffer. SDS-PAGE and immunoblotting were performed as described
previously (Kubo et al.,
1999
).
Electron microscopy
For ultrathin section electron microscopy, A6 and Sf9 cells or pellets of
isolated aggregates were fixed with 2% glutaraldehyde in 0.1 M cacodylate
buffer for 2-3 hours at 4°C, washed with 0.1 M cacodylate buffer, followed
by incubation with 2% glutaraldehyde in 0.1 M cacodylate buffer containing
0.5% (0.1% for pellets) tannic acid for 1-2 days at 4°C. Samples were then
processed for ultrathin section electron microscopy as described previously
(Kubo et al., 1999).
For immunoelectron microscopy, A6 cells cultured on glass coverslips were
fixed with 0.125% glutaraldehyde in 80PEM buffer
(Kubo et al., 1999) containing
2% Triton X-100 for 10 minutes at room temperature, and processed for
immunoelectron microscopy as described previously
(Kubo et al., 1999
). Goat
anti-rabbit IgG coupled to 10 nm gold particles (Nycomed Amersham) was used as
a secondary antibody. Samples were examined with an electron microscope (JEM
1010; JEOL) at an accelerating voltage of 100 kV.
Observation of GFP fusion proteins in live cells
A6 cells were transiently transfected with various truncated forms of
XPCM-1, which were fused with GFP, and cultured for 2 days. These
transfectants were observed using a Delta Vision microscope (Applied
Precision). Each image was acquired with 1-second exposure of the CCD
camera.
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Results |
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We examined in detail the occurrence and distribution of PCM-1 granules in
various types of mouse organs. Their frozen sections were double stained with
anti-mPCM-1 pAb and anti--tubulin mAb. In all the types of cells we
examined, PCM-1 granules were detected, although their number per cell varied
significantly depending on the cell types. As previously reported
(Kubo et al., 1999
), in
ciliated epithelial cells in the oviduct and trachea, a large number of PCM-1
granules were accumulated around the basal bodies of cilia (data not shown).
Interestingly, even in intestinal and renal epithelial cells, which did not
bear cilia, PCM-1 granules were concentrated abundantly in the apical region
of the cytoplasm (Fig. 1c,d).
In hepatocytes, however, a fairly small number of PCM-1 granules were detected
and counter staining with anti-occludin mAb revealed that PCM-1 granules were
scattered around the bile canaliculi, where the centrosomes were located
(Fig. 1e). In contrast, in
nerve cells in the brain, PCM-1 granules were scattered over the cytoplasm
showing no significant concentration around the
-tubulin-positive
centrosomes (Fig. 1f).
These findings indicated that PCM-1 granules are novel non-membranous
organelles that occur ubiquitously in various types of cells. Furthermore, the
immunofluorescence images in Fig.
1a-f raised a simple question to what extent PCM-1 granules are
distributed around centrosomes. To answer this question, we quantitatively
evaluated the spatial relationship between PCM-1 granules and centrosomes
using four distinct types of cultured cells
(Fig. 1g). We counted the
number of PCM-1 granules inside and outside a 3 µm radius of the
-tubulin-positive centrioles as described in detail in Materials and
Methods. The total number of PCM-1 granules per cell did not appear to be
different significantly among cell types (
300-500 granules
cell-1) but, as expected from
Fig. 1a-f, their spatial
relationship with centrosomes varied significantly depending on cell types.
For example, in L fibroblasts and CSG epithelial cells, only 13±2%
(mean±s.e.m.; n=16) and 14±2% (n=18) of PCM-1
granules occurred around centrosomes, respectively, whereas, in A6 epithelial
cells, 64±5% (n=8) was found within a 3 µm radius of
centrioles. In this sense, it would be misleading, if PCM-1 granules are
called `centriolar satellites'.
Domains of PCM-1 responsible for its self-aggregation
Next, we examined how PCM-1 is involved in the formation of PCM-1 granules.
As previously reported, when the GFP fusion protein with full-length
Xenopus PCM-1 (XPCM-1-GFP) was exogenously expressed at a relatively
low level in Xenopus A6 cells, XPCM-1-GFP was recruited to PCM-1
granules containing endogenous XPCM-1 (data not shown). Then, we constructed
GFP fusion proteins with various deletion mutants of XPCM-1 and introduced
them into A6 cells. Interestingly, we found that some deletion mutants formed
large aggregates within the cytoplasm when overexpressed; one example of the
formation of such aggregates by a XPCM-1 deletion mutant (aa 745-1273) is
shown in Fig. 2A.
Immunostaining of these cells with anti-XPCM-1 pAb, which recognized
endogenous XPCM-1 but not the exogenous XPCM-1 mutant, revealed that not only
exogenous XPCM-1 mutant but also endogenous XPCM-1 were incorporated into
these aggregates. On ultrathin-section electron microscopy, these large
aggregates were observed as homogeneous electron-dense structures lacking
delineating membranes (Fig.
2B). These findings suggested that PCM-1 has an ability to
self-aggregate to form granular structures.
|
Fig. 2C summarizes the
ability of the formation of large aggregates for various deletion mutants of
XPCM-1. Two non-overlapping segments (aa 201-494 and aa 745-1128) formed large
aggregates independently when overexpressed, and recruited endogenous XPCM-1
to them. The Coils program (Lupas et al.,
1991) predicted multiple short coiled-coil regions within both
segments (data not shown). These findings suggested that XPCM-1 molecules
interact with each other through at least two independent regions.
Furthermore, considering that either full-length XPCM-1 or a truncated XPCM-1
carrying the C-terminal region (aa 487-2031) did not form large aggregates, it
was likely that the C-terminal region of XPCM-1 had the ability to suppress
the formation of large aggregates.
Then, we examined whether the intermolecular interaction of XPCM-1 is direct or indirect. First, we performed yeast two-hybrid analyses. The cDNA encoding aa 1-484 or aa 745-1273 of XPCM-1 was fused to DNA-BD in the pLexA vector and the cDNA encoding aa 1-711 or aa 745-1273 to AD in the pB42AD vector. DNA-BD and AD constructs were then transformed into yeast. As shown in Fig. 3A, aa 1-484 and aa 745-1273 bound directly to aa 1-711 and aa 745-1273, respectively. Although the possibility has not been theoretically excluded that some yeast protein functions as an intermediate partner in these binding, these findings strongly favored the notion that the intermolecular interaction of XPCM-1 is direct. Next, we overexpressed a GST fusion protein with XPCM-1 fragment (aa 745-1271) in insect Sf9 cells. Electron microscopy revealed that, in Sf9 cells, large aggregates were also formed in the cytoplasm, as observed in A6 cells (Fig. 3Ba). When these cells were lysed with a detergent followed by appropriate centrifugation, these aggregates were partially isolated as pellets (Fig. 3Bb). Interestingly, when these pellets were separated by SDS-PAGE, the GST-XPCM-1 fragment was detected as only one major component (Fig. 3C), indicating that these aggregates were formed through the direct intermolecular binding of XPCM-1 without an intermediate partner. Finally, we constructed a column consisting of the GST-XPCM-1 fragment (aa 745-1271 and aa 1020-1271) to which the whole lysate of Xenopus interphase egg extract was applied. The bound proteins were then eluted and immunoblotted with anti-XPCM-1 pAb. As shown in Fig. 3D, endogenous full-length XPCM-1 bound to aa 745-1271 but not to aa 1020-1271. This binding might explain how endogenous PCM-1 is recruited to the large aggregates induced by the overexpression of exogenous truncated PCM-1. These findings led us to speculate that the PCM-1 granules are formed by the self-aggregation of PCM-1.
|
Cell cycle-dependent assembly and disassembly of PCM-1 granules
PCM-1 was previously reported to concentrate at the centrosomes during
interphase but not during the mitotic phase, although PCM-1 granules have not
yet been identified at that time (Balczon
et al., 1994). Consistently, A6 cells at the interphase were
characterized by numerous PCM-1 granules gathering around the centrosomes,
whereas, during the mitotic phase, the PCM-1 granules became mostly
undetectable (Fig. 4A). This
finding suggested that, during mitosis, the self-aggregation of PCM-1 is
suppressed to disassemble PCM-1 granules. To evaluate this speculation, the
behavior of the large aggregates of GFP fusion proteins with truncated PCM-1
constructs was pursued during mitosis. One example of the aggregates of aa
745-1128 (Fig. 2C) is shown in
Fig. 4B. When A6 transfectants
proceeded into the mitotic phase with concomitant rounding-up, the aggregates
rapidly reduced in size. Furthermore, at the end of mitosis, the aggregates
grew again to their original size. These findings indicated that, during the
mitotic phase, there is some molecular mechanism that suppresses the
self-aggregation of PCM-1, i.e. it facilitates the disassembly of PCM-1
granules.
|
Relationship between PCM-1 granules and pericentrin granules
PCM-1 was reported to associate with pericentrin
(Li et al., 2001), a component
of pericentriolar material, which forms a large protein complex with the
-tubulin ring complex (Dictenberg et
al., 1998
). Therefore, we next examined the relationship between
PCM-1 granules and pericentrin/
-tubulin. When A6 transfectants bearing
large aggregates of a GFP fusion protein with a truncated XPCM-1 (aa 745-1128;
Fig. 2C) were double stained
with anti-pericentrin pAb and anti-
-tubulin mAb, both pericentrin and
-tubulin signals were clearly detected from the aggregates
(Fig. 5A), suggesting that, in
these cells, pericentrin and
-tubulin were sequestered from centrosomes
(i.e. the ability of the centrosomes to nucleate microtubules would be
suppressed). To evaluate this speculation, A6 cells with or without the large
aggregates of truncated XPCM-1 were incubated in a medium containing 0.4 µM
nocodazole for 1 hour to depolymerize microtubules, washed with fresh medium
twice, incubated in fresh medium for 5 minutes to repolymerize the
microtubules and then processed for immunofluorescence microscopy with
anti-
-tubulin mAb. As shown in Fig.
5B, as expected, a large number of microtubules elongated from the
centrosomes in A6 cells without large aggregates of truncated XPCM-1, whereas,
in A6 cells bearing large aggregates, only a small number of microtubules were
associated with the centrosomes. These findings confirmed the interaction
between PCM-1 and pericentrin/
-tubulin within the cells.
|
In addition to the pericentriolar localization, pericentrin was also
reported to form granular structures scattered in the cytoplasm moving along
microtubules (Young et al.,
2000) like PCM-1 granules, although these pericentrin granules
have not yet been examined in detail by electron microscopy. Therefore, we
compared the localization of endogenous PCM-1 with that of endogenous
pericentrin in the cytoplasm of CSG cells in detail using sectioning
microscopy. As shown in Fig.
5C, PCM-1 granules and pericentrin granules were distributed in
the cytoplasm as distinct structures, which were frequently associated with
each other in a granule-to-granule manner. Indeed, when we examined the
frequency for this association quantitatively in the non-centrosomal region of
the cytoplasm using sectioning microscopy, 17±4% (mean±s.e.m.;
54±6/380±45 granules cell-1; n=12) of PCM-1
granules and 48±2% (54±6/113±14 granules
cell-1; n=12) of pericentrin granules were associated with
each other. These findings suggested that PCM-1 granules and pericentrin
granules occur in the cytoplasm independently, and that they dynamically
interact with each other.
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Discussion |
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Yeast two-hybrid analyses revealed that PCM-1 molecules self-associate directly through their two distinct regions (aa 201-494 and aa 745-1128). Furthermore, when the PCM-1 fragments containing each or both of these regions was overexpressed, they formed large aggregates within the cytoplasm. These aggregates were similar to PCM-1 granules in their electron density at the electron microscopic level, and the isolated aggregates were mainly composed of PCM-1 with no major additional components. It remains unclear what molecules are contained in the in situ PCM-1 granules in addition to PCM-1 until they were isolated from cells to the homogeneity, but it is safe to say that PCM-1 constitutes the scaffolds of PCM-1 granules through its self-aggregation. Interestingly, detailed transfection experiments with various PCM-1 mutants suggested that the C-terminal region of PCM-1 molecules suppressed their self-aggregation. Thus, we were led to speculate that this C-terminal region is responsible for determining the size of individual PCM-1 granules.
The PCM-1 immunofluorescence signal was reported to disappear from the
centrosomes when the cells entered the mitotic phase, although the expression
level of PCM-1 appeared to be kept constant throughout the cell cycle
(Balczon et al., 1994).
Furthermore, previous electron microscopic observations demonstrated that
centriolar satellites were not detected around the centrioles in dividing
cells (Rattner, 1992
).
Therefore, taking our present observations together, we concluded that PCM-1
granules are disassembled at the mitotic phase. Interestingly, the large
aggregates induced by mutant PCM-1 were also decreased in size during the
mitotic phase. These findings suggested that, at the mitotic phase, the
self-aggregation (i.e. the intermolecular association) of PCM-1 was suppressed
within the cells to disassemble PCM-1 granules. The molecular mechanism of
this suppression should be interesting; for example, the possible involvement
of mitotic phase-specific phosphorylation of PCM-1 should be examined.
Furthermore, the physiological relevance of the disassembly of PCM-1 granules
should also be clarified. An interesting speculation is that at the mitotic
phase PCM-1 or other unidentified components of PCM-1 granules might be
released into the cytoplasm in a soluble form, which might play some important
role in mitotic-phase-specific events.
Some fractions of pericentrin were reported to exist in the cytoplasm as
granular structures, although most fractions were concentrated in the
pericentriolar region throughout cell cycle
(Dictenberg et al., 1998;
Young et al., 2000
). Close
inspection by immunofluorescence microscopy revealed that pericentrin granules
were distributed in the cytoplasm as distinct structures from PCM-1 granules,
and were frequently associated with PCM-1 granules in a granule-to-granule
manner. Consistent with this finding, PCM-1 was reported biochemically to bind
directly to pericentrin (Li et al.,
2001
). Pericentrin granules were reported to be associated with
dynein and
-tubulin, and to be transported along microtubules toward
their minus ends to supply pericentrin and
-tubulin to the centrosomes
(Young et al., 2000
;
Zimmerman and Doxsey, 2000
).
PCM-1 granules were also shown to be transported along microtubules in the
same direction (Kubo et al.,
1999
). Therefore, it is tempting to speculate that PCM-1 granules
might function as cargo carrying some centrosomal components other than
pericentrin/
-tubulin, and that these two distinct types of granules
interact mutually and dynamically on the way to the centrosomes. However, the
information on the components of PCM-1 granules is still fragmentary and it is
still premature to discuss further the physiological functions of PCM-1
granules.
As a continuation of our previous study, we have here further characterized PCM-1 granules (i.e. centriolar satellites/fibrous granules) in molecular terms. These data should be indispensable for future studies of the functions of PCM-1 granules, including the knockout of the PCM-1 gene.
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Acknowledgments |
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