Department of Anatomy and Cell Biology, The University of Calgary,
Calgary, Alberta, Canada, T2N 4N1
* Present address: Department of Biochemistry, Dartmouth Medical School,
Hanover, NH 03755, USA
Author for correspondence (e-mail:
rattner{at}ucalgary.ca
)
Accepted 16 February 2002
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Summary |
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Key words: CEP110, Ninein, CEP250, c-Nap1, Centrosome
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Introduction |
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The centrosome in mammalian cells has been thought to consist of an
amorphous spherical mass of centrosomal proteins known as the pericentriolar
material or PCM organized around a pair of centrioles or centriole duplex
(reviewed by Mack et al.,
2000). Recently, we have shown that a specific subset of PCM
proteins are not arranged in this manner but are rather organized in a highly
specific and reproducible configuration within the centrosome. In this report,
we will refer to this configuration as the `centrosome tube'. This
configuration appears as either an `O' or a `U' profile when the centrosome is
stained with specific antibodies and viewed by conventional indirect
immunofluorescence microscopy (IIF) (Ou
and Rattner, 2000
). The `O' and `U' profiles can actually be seen
in images present in many studies in the literature using a variety of
antibodies to stain the centrosomes [(e.g.
Young et al., 2000
)
Fig. 1A;
(Mogensen et al., 2000
) Figs
1,
2, ninein], although the
significance of the images was not apparent. Using digital confocal
microscopy, we found that these profiles reflect an underlying tubular protein
configuration that is closed at one end and open at the other
(Ou and Rattner, 2000
). The
centriole proteins CEP250/c-Nap1 and Nek2, which have been shown to associate
specifically with the proximal ends of both mother and daughter centrioles
(Fry et al., 1998
), have been
mapped to the closed end of the tube, suggesting that the centriole is
preferentially placed towards this end of the tube. Recently, using
immuno-electron microscopy, we have found that the parental centriole actually
occupies most of the tube lumen. Thus, the tube represents an arrangement of a
subset of PCM proteins surrounding the parental centriole (Y.Y.O. and J.B.R.,
unpublished).
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|
Centrosome duplication usually occurs at the open end of the centrosome
tube although occasionally it can also be seen at other sites along the side
of the mother centrosome tube. Since centrosome duplication occurs at the site
of the daughter centriole duplex, this variation in the duplication sites may
reflect the movement seen between parent and daughter centrioles that has been
detected in living cells (Piel et al.,
2000). Importantly, the tubular configuration of this subset of
proteins is duplicated in concert with centrosome duplication. Thus, the
ability to visualize the tubular configuration can be used as a light
microscope landmark from which individual centrosomal proteins can be mapped
both spatially and temporally during centrosome duplication and the cell
cycle. Such data may provide insights into the protein dynamics,
protein-protein interactions, and protein functions within the centrosome.
Furthermore, it may also allow the determination of the precise effects on
centrosome structure and duplication of experimental alterations in the
abundance of specific proteins. In this study, we carried out fine-mapping and
functional studies of two centrosomal proteins CEP110 and ninein.
CEP110 is a novel centrosomal protein that was identified in our laboratory
by screening a human cDNA expression library using a centrosome-reactive human
autoimmune serum (Guasch et al.,
2000). A portion of the gene encoding CEP110 was found to be fused
in-frame with a portion of the gene that encodes the protein kinase domain of
a receptor tyrosine kinase for the fibroblast growth factor (FGFR1) in a
myeloproliferative disorder associated with the chromosomal translocation
t(8;9)(p12:q33) (Guasch et al.,
2000
). The finding that the chimeric CEP110-FGFR1 protein has a
constitutive kinase activity led to the proposal that the malignancy that
arises from the CEP110-FGFR1 translocation may involve a combination of
constitutive activity of the FGFR1 kinase domain and the disruption of normal
centrosomal function due to the alteration of the CEP110 protein.
Conventional IIF using antibodies raised against CEP110 suggests that
CEP110 has a unique cell-cycle-dependent distribution when compared with most
other centrosomal proteins (Guasch et al.,
2000). CEP110 is detected in the G1 centrosome. However, following
centrosome duplication and separation, CEP110 is observed in association with
only one of the two centrosomes, presumably the mature one. This pattern
persists until the onset of prophase, at which time CEP110 is also detected at
the second centrosome. Whereas many centrosomal proteins such as pericentrin
and
-tubulin rapidly accumulate at the centrosome as the cell
approaches mitosis (Dictenberg et al.,
1998
; Khodjakov and Rieder,
1999
), CEP110 declines at this time and reappears during the
transition from telophase to G1. The distinctive distribution pattern of
CEP110 led us to speculate that this protein may have a function in early
centrosome organization and in centrosome maturation.
To obtain more information about the role of CEP110 within the centrosome,
we fine-mapped its location within the centrosome during the cell cycle. We
show that CEP110 was associated with both the open and the closed ends of the
tubular conformation of the mother centrosome at G1. Subsequently, CEP110 was
also present at the region where the onset of centrosomal duplication occurs.
In daughter centrosomes with a complete tubular configuration of proteins,
CEP110 was present only at the closed end. We compared the distribution of
CEP110 to that of other centrosomal proteins and found that ninein mapped to
regions similar to those of CEP110. Ninein is a coiled-coil protein of 220-240
kDa (Bouckson-Castaing et al.,
1996; Hong et al.,
2000
) that previously has been localized to the centrioles and
shown to function as a microtubule minus-end capping and anchoring protein
(Piel et al., 2000
;
Mogensen et al., 2000
). The
cDNA encoding the human homologue of ninein was initially identified in our
laboratory while screening a cDNA expression library with the autoimmune serum
M4491 (Mack et al., 1998
). The
present study indicates that both CEP110 and ninein appeared at the open end
of the daughter centrosome tube only after cell division, when the daughter
centrosome started to function as a mother centrosome. The change in
distribution of these two proteins following cell division suggests that
protein addition at the open end of the centrosome tube is a hallmark of
centrosome maturation. In addition, both proteins co-localized with the
centriolar protein CEP250/c-Nap1 (Mack et
al., 1998
; Fry et al.,
1998
; Ou and Rattner,
2000
) at the closed end of the centrosomal tube, which suggests
that both CEP110 and ninein are associated with the centrioles in both mother
and daughter centrosomes. Finally, microinjection of antibodies against CEP110
into HeLa cells at metaphase disrupted the reassembly of the tubular
configuration of proteins within the centrosome seen throughout interphase,
prevented the centrosomal localization of many centrosome proteins, and
interfered with the centrosome's ability to function as a MTOC. Centrosomal
architecture was not disrupted if antibodies were microinjected into cells
with fully formed centrosomes but the MTOC function of the centrosomes was
disrupted. Taken together, our findings suggest that protein addition at the
open end of the tubular configuration of the daughter centrosome is required
for the completion of centrosome maturation. Furthermore, both CEP110 and
ninein are required for the reformation of the tubular configuration of
proteins within the centrosome following cell division as well as being
required for the centrosome to function as a MTOC.
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Materials and Methods |
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Antibodies
The centrosome-reactive autoimmune serum M4491 was obtained from the serum
bank of the Advanced Diagnostic Laboratory at the University of Calgary.
Characterization of this serum has been reported previously
(Mack et al., 1998;
Ou and Rattner, 2000
).
Anti-CEP110 antibodies were generated by immunizing rabbits with the
recombinant protein as previously described
(Guasch et al., 2000
).
Anti-ninein and anti-CEP250/c-Nap1 antibodies have been described previously
(Ou and Rattner, 2000
). All
the antibodies were affinity-purified as described previously
(Guasch et al., 2000
) and
their specificities were confirmed by western blot analysis using whole cell
extracts of HeLa cells (Mack et al.,
1998
; Guasch et al.,
2000
; Ou and Rattner,
2000
). Anti-pericentrin antibodies were purchased from Babco
(Richmond, CA). Anti-
-tubulin antibodies were purchased from Accurate
Chemical & Scientific Corp. (Westbury, NY). Cy3-,
Cy5- and Alex488-labelled secondary antibodies were
purchased from Jackson ImmunoResearch (West Grove, PA), Sigma (St Louis, MO)
and Molecular Probes (Eugene, OR), respectively.
Microinjection protocol
Microinjections were performed under phase contrast using a Leitz
microinjector. All antibodies and normal control sera were diluted in sterile
Dulbecco's phosphate buffered saline (D-PBS). For control experiments,
nonspecific rabbit or mouse IgG (protein A sepharose-purified) were diluted to
1.5 µg/µl and the normal pre-immune rabbit or mouse sera were diluted at
1:5 in D-PBS. Antibodies specific to both CEP110 and ninein were diluted with
D-PBS and used at a final concentration of 1.5 µg/µl. In some
experiments, the injected cells were incubated with 0.1 µg/ml of Colcemid
(GibcoBRL) and then reversed from the Colcemid block by washing three times
and incubating with Colcemid-free medium until fixation.
Digital confocal microscopy and deconvolution
Digital confocal microscopy (immunofluorescence microscopy in conjunction
with digital optical sectioning) was performed as described previously
(Ou and Rattner, 2000).
Briefly, images were obtained by using the Leica PL APO 100x/1.40-0.7
oil objective lens and a 1.995x magnification tube attached to a CCD
camera. Deconvolved images were obtained using Vaytek (Fairfield, IA) Digital
Confocal Microscope 4.0 for DOS software. The deconvolved images were rescaled
to cover the entire 255-value gray range
(Hendzel et al., 1998
). The
images were further processed and aligned using Adobe PhotoShop 5.0. In some
cases, whole cell images were taken using DIC microscopy to illustrate the
position of the centrosome(s) within the cells.
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Results |
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To fine-map the distribution of CEP110 prior to the onset of centrosome
duplication, mitotic cells were collected by selective detachment, re-plated
onto coverslips and incubated for 4 hours prior to use. The G1 status of the
cells was confirmed by co-staining the cells with an antibody against CENP-F
as described previously (Ou and Rattner,
2000). In unduplicated mother centrosomes, CEP110 was observed at
two regions with respect to the centrosome tube, one at the closed end and the
other at the open end (Fig.
2a). Staining at the open end of the tube appeared as two foci in
optical sections passing longitudinally through the centrosome tube
(Fig. 2a) and as an `O' profile
in cross-sectional views of this region. These images indicate that, in
addition to the closed end, CEP110 is distributed within a narrow ring at the
open end of the tube (Fig.
2b).
In random cultures, centrosomes initiating duplication can be identified by
the appearance of a structure at the margin of the centrosome
(Ou and Rattner, 2000). When
cells displaying this stage of duplication were stained for CEP110, this
protein was found at the site of duplication
(Fig. 2c,d) as well as at the
open and closed ends of the mother centrosome tube as described above
(Fig. 2c,d). CEP110 was
localized to a single site within the daughter centrosome throughout the
centrosome duplication process. Thus, in late S phase cells that had two
separated centrosomes, the mother centrosome had two regions of CEP110
staining, whereas the daughter centrosome had only one site
(Fig. 2e). These observations
indicate that CEP110 is a component of the closed end of the centrosome tube
in both the mother and daughter centrosomes but it populates only the open end
of the mother centrosome tube.
A population of CEP110 co-localizes with the centriole component
within the centrosome
In our previous study, we demonstrated that the centriole components
CEP250/c-Nap1 and Nek2 are usually located at the closed end of the mother
centrosome tube and also at the closed end of the daughter centrosome tube
with a complete tubular configuration (Ou
and Rattner, 2000). Further, we showed that, following centriole
separation at G1-S, the daughter centriole moves along the mother centrosome
and comes to reside at the site of centrosome duplication
(Ou and Rattner, 2000
). Since
a subset of CEP110 reactivity was also found at these locations, we next
determined whether CEP110 co-localizes with the centrioles by triple-staining
cells with the autoimmune serum M4491, the antibody to CEP110 and an antibody
to CEP250/c-Nap1 [a protein confined to the centrioles
(Mack et al., 1998
;
Fry et al., 1998
;
Ou and Rattner, 2000
)].
Fig. 3 illustrates that CEP110
co-localized with CEP250/c-Nap1 at the site of the centriole component in the
mother centrosome (Fig. 3a,b,
arrowhead). This relationship is also seen at the site of the centrioles in
the forming daughter centrosome (Fig.
3a, arrow) and daughter centrosomes with a fully-formed daughter
centrosome tube (Fig. 3b,
arrow). These observations indicate that CEP110 is a centriole-associated
protein.
|
Ninein and CEP110 co-localize to similar centrosomal domains
The distribution of the centrosomal protein ninein during the cell cycle
(Piel et al., 2000;
Mogensen et al., 2000
), as
well as our preliminary mapping data using antibodies to centrosomal proteins
(Ou and Rattner, 2000
),
suggested that ninein might have a localization within the centrosome similar
to that of CEP110. Recently, ninein has been found, by immunofluorescence, to
associate primarily with the mother centriole and has been localized, by
immunoelectron microscopy, to both the appendages associated with the mother
centriole and to the minus ends of microtubules
(Piel at al., 2000
;
Mogensen at al., 2000
).
We examined the distribution of ninein within the tubular configuration of the centrosome during the cell cycle, and found that ninein and CEP110 localized to similar sites (Figs 4, 5). Thus, both CEP110 and ninein mapped to the site of CEP250/c-Nap1 in the centriole of mother, daughter and duplicating centrosomes, as well as being located within a ring at the open end of the tube in mother centrosomes. Interestingly, when the open end of the tube was visualized in cross-section, CEP110 was positioned closer to the central lumen of the centrosome, while ninein was more abundant at the periphery of the tube (Fig. 5). These observations indicate that, although CEP110 and ninein display similar staining patterns at the open end of the centrosome, these two proteins localize to distinct subdomains.
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Microinjection of anti-CEP110 or anti-ninein antibodies into
metaphase HeLa cells disrupts the reassembly of the centrosome
The tubular configuration of a subset of proteins within the centrosome is
reassembled at the end of mitosis, at which time CEP110 appears at the open
end of the daughter centrosome. If CEP110 is required during the post-mitotic
reassembly of the centrosome, we would expect to see abnormal organization of
proteins within the centrosome in cells where the CEP110 distribution is
perturbed. To test this hypothesis, we microinjected anti-CEP110 antibodies
into metaphase HeLa cells and examined the centrosome morphology in cells that
had been fixed at 6, 12 and 24 hours following microinjection, and stained
with the autoimmune serum M4491 as well as with Alexa488-labelled
anti-rabbit secondary antibody to visualize cells that were microinjected.
Control cells were injected with pre-immune serum or protein A-affinity
purified rabbit immunoglobins. At each time point, aberrant centrosome tube
morphology could be observed in cells that had been injected with the
anti-CEP110 antibodies (e.g. Fig.
6a-c). At 24 hours after microinjection, 17% (n=42) of
cells had multiple foci of centrosomal proteins scattered in the cytoplasm
(Fig. 6a), 26% had incompletely
formed or irregularly shaped centrosome tubes
(Fig. 6b) and the remainder,
57%, had a normal or nearly normal tubular conformation
(Fig. 6c). Interestingly, in a
majority of the injected cells, the staining intensity with M4491 appeared
weaker than that in sham-injected control cells (compare
Fig. 6c and d). In cells
microinjected with control antibodies (n=60), more than 95% of cells
had centrosomes with a normal tubular morphology (e.g.
Fig. 6d). Microinjection of
metaphase HeLa cells (n=28) with anti-ninein antibodies resulted in
defects in centrosome tube assembly that were comparable with those obtained
in the anti-CEP110 injected cells (data not shown).
|
We also injected anti-CEP110 antibodies into early interphase cells that were connected by an intercellular bridge but had a reformed nucleus. In this case, more than 95% (n=40) of the injected cells had centrosomes with a normal tubular morphology when examined at 6 and 12 hours after injection (e.g. Fig. 9e,f). These results were comparable with those obtained from sham-injected cells (data not shown). We conclude that both CEP110 and ninein are required for the reassembly of the centrosome following mitosis. Further, although antibody injection can disrupt centrosome tube reassembly following mitosis, it is ineffective in disrupting mature centrosomes or inhibiting centrosome duplication.
|
Disruption of centrosome organization following the injection of
anti-CEP110 antibodies affects the localization of a number of centrosomal
proteins
The finding that the tubular configuration within the centrosome is
disrupted following the injection of anti-CEP110 antibodies raises questions
as to the fate of specific centrosomal components under these conditions. To
address this question, HeLa cells injected with anti-CEP110 antibodies at
mitosis were fixed 24 hours post injection and triple-stained with the
autoimmune serum M4491, a secondary antibody (against anti-CEP110 antibody to
reveal the injected cells) and each of the following antibodies:
anti-CEP250/c-Nap1, anti-pericentrin, anti--tubulin and anti-ninein
antibodies. The results demonstrated that, in cells in which the assembly of
the tubular configuration of a subset of proteins within the centrosome was
disturbed, all these centrosomal components (CEP250/c-Nap1, pericentrin,
-tubulin and ninein) were dispersed in the cytosol (e.g.
Fig. 7).
|
Injection of anti-CEP110 or anti-ninein antibodies into PtK2 cells
affects the microtubule organizing ability of the centrosome
Our results indicated that the presence of both CEP110 and ninein are
required for the proper reassembly of the tubular configuration of the
centrosomes and consequently the localization of specific centrosomal proteins
to the G1 centrosome. To determine whether the absence of CEP110 and ninein
also affects the ability of the G1 centrosomes to function as a discrete MTOC,
we assayed for microtubules in PtK2 cells following antibody injection using
two protocols. In the first protocol, mitotic or post-mitotic cells were
injected with either anti-CEP110 or anti-ninein antibodies, fixed 6 hours
post-injection and stained with an anti-ß-tubulin antibody. In the second
protocol, the cells were injected in the same manner, but 0.1 µg/ml of
colcemid was added to the culture 6 hours after the injection. The cells were
incubated for 60 minutes at 37°C in the presence of the drug.
Subsequently, the drug was removed from the culture by washing with fresh
medium and incubating for an additional 20 minutes before fixation. The cells
were then stained with the anti-ß-tubulin antibody. The drug treatment
protocol was used to ensure that any microtubules detected upon fixation were
formed subsequent to antibody injection. The results were comparable using
both protocols and either anti-CEP110 or anti-ninein antibodies (Figs
8,
9). When mitotic cells were
used, microtubule formation at the centrosomes was observed in cells injected
with normal serum (Fig. 8a-b).
However, no localized microtubule formation was detected in cells injected
with anti-CEP110 antibodies (Fig.
8c-d). Similarly, post-mitotic cells injected with anti-ninein
antibodies also showed altered MTOC function
(Fig. 9). We conclude that the
presence of CEP110 and ninein are essential not only for the organization of
the daughter centrosomes following mitosis but also for the centrosome's
ability to function as a MTOC.
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Discussion |
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CEP110 and ninein distribution denotes centriole association and a
region associated with centrosome maturation
In this study, we have expanded our previous localization data by mapping
CEP110 in relation to the tubular configuration of a subset of proteins within
the centrosome. We demonstrated that CEP110 is localized to two regions
throughout the life of the mother centrosome, one at the position of the
centriole components CEP250/c-Nap1 and Nek2 and another at the open end of the
centrosomal tube. Since CEP250/c-Nap1 has been shown to localize to one end of
the centriole (Fry et al.,
1998), CEP110 must have a similar localization within the
centriole. Further, we showed that CEP110 appeared at only one site (the site
of CEP250/c-Nap1) within the daughter centrosome from the time of its initial
formation until prophase (Fig.
10). Our ability to refine and expand the protein localization
data obtained by conventional IIF using the techniques described in this study
highlights the usefulness of employing an architectural reference and confocal
microscopy to map centrosomal components.
We also investigated the position of ninein with respect to the centrosomal
tube. Ninein has previously been shown by immunofluorescence microscopy to be
associated primarily with the mother centriole during G1 and both centrioles
during S-G2 (Piel et al.,
2000). Further, it has been localized to the appendages
surrounding the mother centriole and also to the minus-ends of microtubules by
immunoelectron microscopy (Mogensen et
al., 2000
). Ninein, like CEP110, is found at the site of the
centriole component CEP250/c-Nap1 in both mother and daughter centrosomes and
at the open end of the mother centrosomal tube. Although the detection of
ninein in association with centrioles confirms previous findings
(Mogensen et al., 2000
), this
is the first report of ninein at a second site, the open end of the
centrosome. It should be noted that the distribution of ninein to two regions
of the centrosomes could be seen in previous localization studies as a
three-dot pattern (Mogensen et al.,
2000
), the significance of which was not obvious before our
experiments. As shown in our study, one dot maps to the region containing
CEP250/c-Nap1, while the other two dots map to the open end of the centrosomal
tube.
Since both CEP110 and ninein localize to the site of CEP250/c-Nap1 in mother and daughter centrosomes throughout the cell cycle, these two proteins are likely to be constitutive components of this centriole region. Importantly, the addition of these proteins to the open end of the daughter centrosome tube coincides with the transition of the daughter centrosome to a mature centrosome which is capable of duplication and acts as a microtubule organizing center. It is possible that the tubular structure of the daughter centrosome must be opened during mitosis so that CEP110 and ninein can be added and maturation of the centrosome completed (Fig. 10). Thus, our study, for the first time, identifies a specific region of the centrosome that is modified in its protein composition during the process of centrosome maturation, a process that is completed following cell division.
The identification and localization of two populations of both CEP110 and
ninein within the centrosome has some functional implications. First, the
function of CEP110 and ninein is, at least in part, centriole-based. Second,
previous studies have suggested that ninein plays a role in microtubule
anchoring at the centriole and acts as a cap for microtubule ends
(Mogensen et al., 2000). If
so, the presence of ninein at the open end of the centrosomal tube suggests
that microtubule anchoring or capping functions may occur within this region
as well, at least at some point during the cell cycle. The absence of ninein
from the daughter centrosomes would therefore limit their ability to anchor or
cap microtubules at the open end of the tube. Such a relationship may explain
in part why previous studies (e.g. Piel et
al., 2000
) failed to identify microtubules in association with
immature centrosomes. The observations described here raise the possibility
that ninein plays a larger, and perhaps more diverse, role within the
centrosome than previously thought, and that this role, in part, is a hallmark
of mature centrosomes.
Centrosome targeting signal and protein association at the
centrosome
Protein localization data, such as those presented here, raise important
questions about the mechanisms that target proteins to the centrosome and,
more specifically, the mechanisms that localize proteins to specific regions
within the centrosome. In the search for a centrosomal targeting sequence,
Gillingham and Munro identified a 90-residue PACT domain in the centrosomal
proteins pericentrin and AKAP450, as well as in two other proteins of unknown
function from Drosophila and fission yeast
(Gillingham and Munro, 2000).
This domain was capable of targeting a reporter protein, green fluorescent
protein, to the centrosome. Both pericentrin and AKAP450 are very large
proteins predicted to form a coiled-coil over most of their length
(Doxsey et al., 1994
;
Dictenberg et al., 1998
;
Witczak et al., 1999
), and the
PACT domain is outside of both their coiled-coil regions and their protein
kinase A binding site.
Examination of the protein sequences of CEP110 and ninein failed to reveal
the PACT domain. In the case of CEP110, we have previously shown that the
centrosome targeting signal is located within one of its coiled-coil regions.
This region spans 170 amino acids at the C-terminus and contains a leucine
zipper motif (Guasch et al.,
2000). Leucine zippers, initially identified in transcription
factors, have been shown to mediate homo- or heterodimerization of proteins
and to play a role in protein-DNA binding
(Landschulz et al., 1988
).
This motif has also been found to mediate protein-protein interactions in a
wide array of proteins and play pivotal roles in regulating the activities of
these proteins (e.g. Dubay et al.,
1992
; Vrana et al.,
1994
; Park and Seo,
1995
; Leung and Lassam,
1998
). However, our deletion experiments indicate that, in the
case of CEP110, the leucine zipper motif is not sufficient on its own to
support targeting of green fluorescent protein to the centrosome, which
indicates that flanking amino acids are also involved (Y.Y.O. and J.B.R.,
unpublished). It is likely that different centrosomal proteins contain
different centrosomal targeting motifs. This variation could, for example,
explain why some proteins are positioned throughout the entire centrosome
(e.g. pericentrin) whereas others are more domain-specific (e.g. CEP110).
The mapping data for CEP110 and ninein reported in this study raises the
possibility that they interact in vivo. We have been unable to detect such an
interaction using an in vitro TNT and immunoprecipitation assay. However, we
did find that both CEP110 and ninein bind to kendrin (Y.Y.O. and J.B.R.,
unpublished). Thus, these proteins can be added to a growing list of
pericentrin/kendrin-binding proteins, which include protein kinase A
(Diviani et al., 2000),
cytoplasmic dynein (Purohit et al.,
1999
), pcm-1 (Li et al.,
2001
), calmodulin (Flory et
al., 2000
) and
-tubulin
(Dictenberg et al., 1998
;
Young et al., 2000
).
Interestingly, we found that it is the centrosomal targeting region of CEP110
that interacts with kendrin, and this is the first motif identified to
interact directly with kendrin/pericentrin (Y.Y.O. and J.B.R.,
unpublished).
CEP110 and ninein are essential for centrosome assembly following
mitosis
Assembly of the tubular architecture within the centrosome occurs twice
during the cell cycle: once during centrosome duplication and once following
cell division. Our microinjection studies clearly indicate that both CEP110
and ninein are required for the reassembly of the tubular architecture of the
centrosome following cell division. The findings that CEP110 and ninein are
associated with the CEP250/c-Nap1 site within the centrioles when the tubular
configuration of the daughter centrosome is established and that neither
CEP110 nor ninein is present at the open end of the tubule at this time raise
the possibility that this centriole-associated population of CEP110 and ninein
may be especially critical for the assembly of the tubular conformation of
proteins. The simple interpretation of our microinjection results is that the
microinjected antibodies prevented the centriolar CEP110 and ninein from
participating in the normal formation of the tubular architecture of the
centrosome. This interpretation is consistent with the observations that the
centriole duplex functions to organize a centrosome in some cell types
(Bobinnec et al., 1998) (for
reviews, see Marshall, 1999
;
Karsenti, 1999
) and that
centrosome duplication is initiated at the site of the daughter centriole
(Ou and Rattner, 2000
).
Furthermore, by acting as a microtubule anchoring protein, the centriole-bound
CEP110 or ninein may act as a focus for the collection of centrosomal proteins
transiting along microtubules. This is supported by our results that show
that, in cells injected with an anti-CEP110 antibody, centrosomal material is
scattered throughout the cytosol and microtubule assembly at the centrosome is
disrupted. Thus, it appears that the assembly of the tubular conformation
within the centrosome, centrosome protein clustering, and microtubule
formation are interdependent.
Our inability to disrupt the centrosome structure by antibody microinjection at the G1 stage may indicate that both CEP110 and ninein are required for the assembly of the tubular conformation of proteins, but not for its stability once it has formed. Further, factors such as epitope accessibility, the abundance of existing protein, the rate of turnover of CEP110 and ninein in the centrosome, and the presence of a mature mother centrosome structure may contribute to our inability to disrupt daughter centrosome formation following antibody microinjection into interphase cells. However, it should be noted that our microinjection experiments indicate that both proteins are required for the G1 centrosome to function as a MTOC.
In summary, our study shows that proteins are arranged in a specific manner within the centrosome and this arrangement can be modified in both a spatial and temporal manner to accommodate centrosome function. Specifically, our data establishes that the open end of the centrosome tube is the site of protein addition associated with the final stages of centrosome maturation and CEP110 and ninein are two of the proteins added to that region.
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