1 Department of Biochemistry and Biophysics, University of California
San Francisco, 513 Parnassus Avenue, Box 0448, San Francisco, CA 94143,
USA
2 Howard Hughes Medical Institute, Department of Biochemistry and Biophysics,
University of California San Francisco, 513 Parnassus Avenue, Box
0448, San Francisco, CA 94143, USA
* Author for correspondence (e-mail: ofarrell{at}cgl.ucsf.edu)
Accepted 2 October 2002
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Summary |
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Key words: Centriole, Cdk1, Duplication, Nucleation, Cell cycle
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Introduction |
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A centriole is an open-ended cylinder composed of nine sets of
microtubules. The Drosophila melanogaster centriole is about 100 nm
in diameter and length (Vidwans et al.,
1999). During duplication, a daughter assembles perpendicular to
and off the side of the mother centriole producing an asymmetric arrangement
wherein the ends of the mother centriole are free while one end of the
daughter abuts the mother (Alberts et al.,
1994
). The daughter is initially shorter and lacks the distinct
microtubular appearance of a mature centriole and hence is referred to as
immature. It matures upon attainment of the microtubular arrangement and
length of the mother centriole. Finally, mother and daughter separate
(referred to as disengagement) and each can undergo another round of
duplication. These observed `rules' of daughter centriole assembly ensure that
the length of the centriole is maintained through generations of
duplication.
Centriolar structure and that of the mother-daughter pair raises issues
about the mode of duplication. Specifically, how is the structure of the
daughter centriole specified? Is centriole duplication replicative, like that
of DNA (i.e. does information present in the mother play an instructive role
in assembling the daughter?). Unlike the case of DNA, it is difficult to
imagine how information might be passed from a mother centriole to its
daughter because the orthogonal relationship of mother-daughter centriole
pairs would seem to preclude the juxtapositioning of substructures that might
allow templating. The process is more easily thought of as a nucleated
self-assembly in which the final structure of the centriole daughter is
dictated by interactions inherent to the assembly process. Even so, the
assembly of a centriole appears to depend on pre-existing centrioles
(Maniotis and Schliwa, 1991;
Sluder et al., 1989
) and
appears to be regulated in the context of the cell cycle
(Callaini et al., 1997
;
Vidwans et al., 1999
). These
regulatory inputs may impinge directly on the duplication process, for
example, by regulating activity of sites of assembly on a mother centriole or
limiting the time frame for growth of a daughter. At present, we have very
little experimental evidence to resolve these types of issues and define the
nature of the centriole duplication process.
We addressed some of these questions by manipulating a key cell cycle
regulator in the wing imaginal discs of Drosophila. The mitotic
kinase, Cdk1, is required for mitosis and for a checkpoint that normally
prevents another S phase prior to mitosis
(Hayashi, 1996;
Hayles et al., 1994
;
Knoblich et al., 1994
;
Weigmann et al., 1997
). Cdk1
inactivation in the normally diploid cells of wing discs prevents further
mitosis but allows multiple rounds of S phases
(Weigmann et al., 1997
).
Examination of centrioles by electron microscopy revealed centrioles that were
longer than normal, daughter centrioles that were longer than their mothers
and triplet centrioles. The presence of these centriole configurations
indicates that, at least under these circumstances: (1) more than one site on
a centriole can participate in daughter assembly; (2) separation of a
mother-daughter pair is not essential for new centriole synthesis; and (3)
unlike DNA duplication, templating by the mother does not specify the length
of the daughter. Because these outcomes followed inactivation of a cell cycle
regulator, Cdk1, rather than a disruption of centrosomal components, the
results suggest that, normally, regulatory controls, rather than structural
constraints dictate aspects of the stereotyped centriolar duplication
program.
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Materials and Methods |
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Preparation of wing imaginal discs for EM
Dissected larvae heads were fixed in 5% glutaraldehyde (diluted in 0.1 M
cacodylate buffer) (Vidwans et al.,
1999) for 1-2 hours at room temperature. They were then washed
overnight in cacodylate buffer at 4°C. This was followed by post-fixation
in 2% osmium tetroxide (diluted in 0.1 M cacodylate buffer) at room
temperature for 2 hours. The discs were then dehydrated by incubation for a
few minutes each in a series of ethanol washes increasing in strength (10%,
30%, 50% and 70%). We typically dissected away the wing imaginal discs from
the larvae heads in 70% ethanol. Then the imaginal discs were dehydrated and
treated as described for embryos (Vidwans
et al., 1999
).
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Results |
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It has been previously reported that inactivation of Cdk1 converts mitotic
wing disc cells into endoreplicating cells
(Weigmann et al., 1997).
Consistent with this, wing imaginal discs in which Cdk1 had been inactivated
(ts discs) were composed of fewer cells that were bigger and more brightly
stained with a DNA stain (presumably due to suppression of mitosis and
polyploidy, respectively) (Fig.
1). In contrast to naturally endoreplicating cells in which
centriole duplication is arrested and the centrioles retain normal morphology
(Mahowald et al., 1979
),
endoreplicating cells of ts discs exhibited a variety of centriole forms that
suggested continued and anomalous duplication cycles. Triplet forms provide
insights into the control of new daughter assembly (see below) while centriole
pairs with unusually long daughters provide insights into the processes that
terminate replicative growth of daughter centrioles.
|
Measurement of the mother centriole in control discs defines the average length of the mature centriole as 115 nm (Fig. 2A,B; Fig. 3). The daughter centriole of a pair is generally shorter than (70 nm on the average) or occasionally comparable in length to its mother (Fig. 3; Fig. 2A,B). This result is consistent with observations made in the embryos where daughter centrioles reach their mature length only upon entry into mitosis whereupon they soon separate from their mothers. In contrast, 15% of the centriole pairs from ts discs were longer than their mothers (Table 1). The average daughter centriole length was observed to be 114 nm a 63% increase over normal while mother centriole length averaged 134 nm a 17% increase (Figs 2, 3). These data suggest that the observed aberrant centriole pairs is attributable to excess growth of the daughter and not shrinkage of the mother.
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Mother centrioles with two daughters
About 12.5% (n=64) of centrioles in ts discs were triplets
containing three centrioles in one of two different configurations, neither of
which was observed in wild-type (n=31). In one configuration, two
daughter centrioles appeared to associate with a single mother. The
relationship between the centrioles in a triplet configuration was inferred
based on the asymmetric nature of centriole duplication (see Introduction). In
the examples shown in Fig. 4,
one centriole was identified as mother because neither of its ends abuts
another centriole. The other two centrioles extend perpendicular to this
mother centriole in the orientation typical of daughter centrioles. Based on
this observation, we conclude that, at least under these circumstances, a
daughter centriole can be assembled at more than one position on a mother
centriole.
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It is noteworthy that in each triplet, one of the daughters is full length (or excessively long) and exhibits the morphology of a mature centriole. The second daughter is short and has the indistinct morphology typical of an immature centriole.
Mother-daughter-granddaughter triplets
The second triplet configuration appeared to have a
mother-daughter-granddaughter relationship as illustrated in
Fig. 5 (inferred, again, based
on the asymmetric nature of centriole duplication). The mother centriole,
whose ends are free, has a single mature daughter arranged in the typical
position orthogonal to it. The third centriole is immature and is arranged
perpendicular to the daughter. Thus, in contrast to the triplets described
earlier, a new generation is nucleated off the side of a daughter within a
mother-daughter centriole pair. While in some of these triplets, the mother
and the daughter were not quite orthogonal
(Fig. 2B,C) in other triplets,
the mother and daughter were tightly paired and oriented
(Fig. 2D,E). The simplest
explanation for the precise relationship of this latter set of centriole pairs
is that, in this experimental setting, movement of the daughter centriole away
from the mother is not necessary to initiate assembly of a new
second-generation daughter.
|
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Discussion |
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Daughter centriole length is regulated
One of the observed aberrant centriole morphologies consisted of centriole
pairs in which the daughter centriole was longer than its mother. Our
observations differ from previous descriptions of centriole elongation in that
the extra growth appears to have occurred predominantly in the daughter
centrioles. In contrast, the dramatic (20x) elongation of centrioles in
Drosophila spermatogonial cells influences both mother and daughter
centrioles (A. D. Tates, Cytodifferentiation during spermatogenesis in
Drosophila melanogaster: an electron microscope study, PhD thesis,
Rijksuniversiteit, Leiden, 1971). Furthermore the production of a
pseudocillia, which occurs in several species, involves elongation of the
mother centriole. The growth of the mother centrioles by themselves or
concomitantly with daughter centrioles in these cases indicates that the
excess elongation is not connected with duplication.
While the inequity in length between mother and daughter centrioles upon inactivation of Cdk1 suggests that mis-specification of centriole length occurred during duplication, it should be noted that mother centrioles were also unusually long. We cannot exclude the possibility that extra centriole growth outside the context of duplication produced these longer mothers. However, measurements of centriole length showed that daughters are more dramatically affected than mothers. This is consistent with the possibility that the long mother centrioles are secondary and represent long daughters seen in a subsequent round of duplication.
If daughter centriole length were structurally constrained by the mother centriole, daughter length would be at most that of the mother. We emphasize the observation that daughter centrioles within pairs can exceed the length of their mothers. Consequently, we propose that maximum centriole length is not a direct function of the length of the parent centriole.
A centriole has multiple sites for daughter centriole assembly
A second type of aberrance we observed consisted of a mother centriole
sporting two daughters simultaneously. While it is possible that these triplet
centrioles are the result of reassociation of previously dissociated
centrioles, it appears implausible that any random mechanism would produce the
highly stereotyped and close association that is observed between the
centrioles of such triplets. Therefore, we suggest that these triplet
centrioles result from two rounds of centriole initiation. We emphasize that
this observation demonstrates that more than one site on a mother centriole
can be used for daughter centriole assembly in this experimental setting.
The angle between the long axes of the two daughter centrioles varied among the observed triplets. Although our data are not sufficient to define the number of possible angles between the two daughters, we note that this observation is compatible with a model in which there are nine potential sites (corresponding to the nine sets of microtubules that form the wall of the centriole) and that a site used for a round of initiation is selected randomly with respect to the site of centriole assembly in the previous round. Given that at least two sites on a mother centriole can be used for new centriole assembly, the normal limit of one daughter centriole per cycle is apparently not due to a structural asymmetry in the centriole that specifies a unique assembly site. Rather, our results suggest that regulatory interactions limit initiation of new daughter assembly to one per cycle.
The difference in the level of maturation of the two daughter centrioles suggests that they did not initiate, grow, and mature simultaneously, but instead represent successive generations of centriole assembly. Hence, even during defective duplication in the absence of Cdk1, it appears that there is still a temporally regulated cycle, and in an individual cycle, a single daughter centriole is initiated but this daughter fails to separate from its mother prior to initiation of a second cycle. In the second cycle, a second daughter assembles at a site distinct from the site of assembly of the first. Apparently, triplets arise because of a failure or defect in separation of mother and daughter centrioles, or a failure in coordination of a new round of centriole duplication with completion of the first. Note that even if transition to the second cycle of duplication is accompanied by a microscopically undetectable change in mother-daughter association, the precise structural association observed between the members of the triplet suggests that positions of the daughters mark their birth sites and hence demonstrate that the mother can use more than one site to initiate daughters.
Mother-daughter centriole pair separation is not essential for a new
round of assembly on the daughter centriole
What prevents a daughter centriole from inappropriately nucleating a
granddaughter? Prospective assembly sites on the daughter centriole are
unobstructed while in association with its mother. Triplets consisting of
mother-daughter-granddaughter centrioles indicate that, under our experimental
conditions, the transition from a daughter to a mother does not require
centriole pair separation. What conditions, if any, have to be met for a
daughter in a mother-daughter pair to nucleate its own daughter? Ordinarily,
the daughter centriole does not mature until entry into mitosis
(Callaini et al., 1997;
Vorobjev and Chentsov, 1982
),
hence the lack of maturity might limit a new generation of daughter centriole
assembly during G2. The daughters we observed that had nucleated
granddaughters were at least the length of a normal mother. We consequently
suggest that a daughter may have to attain mature length as a prerequisite for
unveiling sites for daughter assembly.
The impact of cell cycle regulators on centriole duplication
Drosophila centrioles at the developmental stages we studied are unique:
they are comprised of singlet microtubules and lack some of the sophisticated
appendages normally associated with more `mature' centrioles. It is possible
that `immature' Drosophila centrioles may be able to assemble non-canonical
replicative structures unlike `mature' centrioles that may be more constrained
(Garreau De Loubresse et al.,
2001). Thus these observations might be unique to `immature
centrioles'. However, cancerous cells are often associated with aberrant
centriole morphologies, which suggests that our observations might be
generalized to other systems and that such anomalous centriole duplication
might be one of the defects underlying the mitotic instability of cancer
cells. The possible involvement in genome destabilization emphasizes the
importance of understanding the basis of the regulation of daughter centriole
assembly.
The steps between inactivation of Cdk1 and the final anomalies are unknown and could involve a complex cascade and multiple defects. Nonetheless, our observations are consistent with alteration of the centriole cycle at a single step. As described above, the two daughters in a triplet configuration differ in maturity suggesting that they arise as a result of two duplication cycles without intervening disengagement of the products of the first cycle. Centriole pairs with excessively long daughters could also be due to continued (or renewed) replicative growth without disengagement. The simplest explanation for the observed anomalies is that coordination between disengagement and progression of duplication is lost because disengagement is defective or retarded. Alternatively, centriole maturation and duplication might lack normal constraints and occasionally get a step ahead of disengagement. In either case, it appears that disengagement is not totally blocked as centriole pairs continue to predominate.
Cell cycle regulators have previously been implicated in coordinating
centriole duplication with cell cycle progression. In S. cerevisiae,
mutation of Y19 of CDC28 (Cdk1) to E (cdc28-E19), a change that is presumed to
mimic inhibitory phosphorylation at this position, or overproduction of the
kinase phosphorylating this site blocked duplication of the centrosome analog,
the spindle pole body (SPB), at the stage of mother-daughter separation
(Lim et al., 1996).
Importantly, the block was specific in that other aspects of the cycle
continued. These results led the authors to conclude that Cdk1
dephosphorylation is required for SPB duplication. In Drosophila,
maturation of daughter centrioles requires Cdc25string, a
phosphatase that dephosphorylates Y-15 of Cdk1 (analogous to Y19 of Cdc28).
Dephosphorylation of Cdk1 might be an activating change or Cdk1-YPO4 might
impose a constraint on centriole duplication that is removed upon
dephosphorylation. Two types of observation favor the interpretation that
phosphorylated Cdk1 inhibits progress of the centriole cycle. First, in S.
cerevisiae the cdc28-E19 mutant has high kinase activity that is
sufficient to support cell cycle events other than SPB duplication, hence the
block to SPB duplication is not likely to be due to a lack of activity, but
could be due to the ability of Cdc28-E19 kinase to mimic a phosphorylated form
that can inhibit SPB duplication (Lim et
al., 1996
). Second, findings in S. cerevisiae and S.
pombe, as well as those reported here for Drosophila, suggest
that loss of function of Cdk1 releases constraints on centriole/SPB
duplication (Haase et al.,
2001
) (S. Uzawa and W. Z. Cande, personal communication), arguing
against a stimulatory role of cyclin/Cdk1. A plausible synthesis of these
results suggests that cyclin/Cdk1-YPO4 inhibits progress of the SPB/centriole
duplication cycle and its elimination leads to miscoordination of the
duplication program. However, because the results are based on genetic tests,
the connection between Cdk1 and the centriole cycle might be indirect and
complex, and diverse interactions might contribute to the results in the
different systems. More studies examining the role of the cyclin/Cdk1 complex
in regulating progress of the centriole duplication cycle are needed.
In summary, we provide evidence that regulatory rather than structural constraints limit the number of daughters assembled by a centriole, and argue that the `yardstick' that defines centriole length is independent of the mother centriole. Like the cell biologists of a century ago, we remain confounded by the duplication program of the centriole, but hope that identification of regulatory contributions to this duplication will shed light on the process.
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Acknowledgments |
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