Anomalous centriole configurations are detected in Drosophila wing disc cells upon Cdk1 inactivation

Smruti J. Vidwans1, Mei Lie Wong2 and Patrick H. O'Farrell1,*

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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 Materials and Methods
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The centriole, organizer of the centrosome, duplicates by assembling a unique daughter identical to itself in overall organization and length. The centriole is a cylindrical structure composed of nine sets of microtubules and is thus predicted to have nine-fold symmetry. During duplication, a daughter lacking discrete microtubular organization first appears off the wall of the mother centriole. It increases in length perpendicularly away from the mother and terminates growth when it matches the length of the mother. How a unique daughter of the correct length and overall organization is assembled is unknown. Here, we describe three types of unusual centriole configurations observed in wing imaginal discs of Drosophila following inactivation of Cdk1. First, we observed centriole triplets consisting of one mother and two daughters, which suggested that centrioles have more than one potential site for the assembly of daughters. Second, we observed centriole triplets comprising a grandmother, mother and daughter, which suggested that subsequent centriole duplication cycles do not require separation of mother and daughter centrioles. Finally, we observed centriole pairs in which the daughter is longer than its mother. These findings suggest that regulatory events rather than rigid structural constraints dictate features of the stereotyped duplication program of centrioles.

Key words: Centriole, Cdk1, Duplication, Nucleation, Cell cycle


    Introduction
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 Materials and Methods
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Successful duplication and segregation of cellular organelles, especially of those that exist in low copy number, is of paramount importance for cell duplication. The centrosome nucleates microtubules and organizes each of the two mitotic spindle poles. Centrosome number is dictated by duplication of its organizer, the centriole (Alberts et al., 1994Go). The centriole, and hence the centrosome, duplicates precisely once in each cell cycle, much like DNA. While the structure of DNA provided immediate insight into the mechanisms of its duplication, the physical processes underlying centriole duplication are unknown.

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., 1999Go). 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., 1994Go). 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, 1991Go; Sluder et al., 1989Go) and appears to be regulated in the context of the cell cycle (Callaini et al., 1997Go; Vidwans et al., 1999Go). 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, 1996Go; Hayles et al., 1994Go; Knoblich et al., 1994Go; Weigmann et al., 1997Go). Cdk1 inactivation in the normally diploid cells of wing discs prevents further mitosis but allows multiple rounds of S phases (Weigmann et al., 1997Go). 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.


    Materials and Methods
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 Materials and Methods
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Inactivation of Cdk1 in wing imaginal discs
Sevelen (wild-type) or Cdk1ts flies were maintained at 18°C. Embryos were collected and allowed to hatch and larvae allowed to develop at 18°C. Late second instar larvae were picked on a grape-juice plate and incubated at 30°C by transferring the grape juice plate to a humid 30°C incubator. Larvae were fixed 2 days after incubation at 30°C. Larval heads with imaginal discs attached to them were fixed in 7% formaldehyde (in PBS) for 20 minutes (for visualization under the light microscope) or as described below for EM.

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., 1999Go) 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., 1999Go).


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Inactivation of Cdk1 leads to aberrant centriole duplication in wing discs
We first examined centrioles from wild-type third instar wing discs (wt). EM analysis revealed that 20% of centrioles in interphase cells were singlets, whereas the rest were pairs with immature daughters (n=31, data not shown). Wing disc cells double in about 8 hours and spend approximately equal time in G1, S and G2 phases (Milan et al., 1996Go). It follows that about one-third of interphase cells from wt wing discs are in G1. In Drosophila embryos, G1 centrioles exist as singlets, daughter initiation accompanies S phase (Callaini et al., 1997Go) and maturation of daughter centrioles occurs in mitosis (Vidwans et al., 1999Go). If the timing of steps in the centriole cycle were conserved between Drosophila embryos and wing imaginal discs, then about two-thirds of interphase centriole pairs should contain immature daughters (corresponding to cells in S and G2) and the rest should be singlets. The observed preponderance of centriole pairs with immature daughters agrees with this expectation.

It has been previously reported that inactivation of Cdk1 converts mitotic wing disc cells into endoreplicating cells (Weigmann et al., 1997Go). 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., 1979Go), 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.



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Fig. 1. Wing discs from sevelen (wild-type) larvae or Cdk1ts larvae. Eggs were collected and aged to the late second instar larval stage at 18°C. Larvae were then shifted to 30°C for 2 days, after which they were fixed. Discs were dissected and stained for DNA with the dye, bis-benzidine (Hoechst). Wing discs in which Cdk1 has been inactivated are, typically, smaller in size compared with similarly treated sevelen discs and have fewer cells that are bigger than normal. Bar, 25 µm.

 

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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Fig. 2. Centriole length is misregulated upon inactivation of Cdk1. In all cases, the mother centriole is on the left and the daughter on the right. Furthermore, we have oriented images so that mothers in longitudinal section have their long axes running top-bottom and daughters in longitudinal section have their long axes facing left-right. Since the section is thinner than the centriole, a longitudinal section through a centriole appears as a pair of parallel lines (representing the walls of the centriole). A centriole in cross-section appears as a circle (Vidwans et al., 1999Go). The pairs of pictures in A, D and E are adjacent sections from a series. (A,B) Control centrioles from sevelen wing discs incubated at 30°C for 2 days. (C,D,E) Centrioles from Cdk1-inactivated wing disc cells. (A) Both the mother and the daughter in this centriole pair are in longitudinal section. Note that the daughter is shorter than its mother (compare the mother in the left section to daughter in the right). (B) Both the mother and the daughter centrioles are in longitudinal section, with the daughter shorter than its mother. (C) A longitudinal mother-daughter centriole pair with a long mother. Compare the length of this mother to those in A and B. (D) A mother in cross-section with a long daughter in longitudinal section. Compare the length of this mother to that in A and B. (E) A longitudinal centriole pair with long mother and daughter. The daughter is longer than its mother. Bar, 100 nm.

 


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Fig. 3. Distribution of lengths of mother and daughter centrioles from sevelen and Cdk1ts wing discs incubated at 30°C for 2 days. The x-axis is centriole length (measured in nm) and the y-axis is the number of centrioles at any given length. Note that the distribution of daughter centrioles from wild-type discs varies between 46 and 93 nm, while the corresponding distribution from ts discs varies between 46 and 162 nm. The wider distribution of daughter centriole lengths in ts discs is reflected in the higher average: 114 nm compared with 70 nm in wild-type. The distribution of mother centrioles from ts discs is also broader than wild-type (105 to 184 nm in ts discs compared with 93 to 139 nm in wild-type). The average mother centriole length in ts discs is 134 nm compared with 115 nm in wild-type.

 

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Table 1. Distribution of centrioles from Cdk1ts and sevelen wing discs incubated at 30°C for 2 days

 

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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Fig. 4. Mother centrioles with two daughters. Three examples of mother centrioles with two daughters. Each triplet is shown in two consecutive serial sections with a schematic to orient the reader. (A) A mother centriole (in the center) that is slightly angled with two longitudinal daughters, one to the left and the other to the right. (B) A mother in cross-section with two daughters, both in longitudinal section, one to the left and one (seen only in the section at the right) at the top. (C) A mother in cross-section with two longitudinal daughters, one to the right and the other to the left. Note that one of the daughters in each triplet is always longer than the other, suggesting that the two daughters were assembled at different times. Furthermore, note that the differences in the angle between the two daughter centrioles suggest that the initiation of a second daughter can occur at a variety of positions with respect to the first daughter. Bar, 100 nm.

 

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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Fig. 5. Examples of mother, daughter and granddaughter triplet centrioles. Each triplet is shown in two consecutive serial sections with a schematic on the right to orient the reader. All the centriole triplets are oriented with the mother on the left, the daughter to the right and the granddaughter to the top or bottom. To accomplish this orientation, adjustments were made in Adobe PhotoShop, some of which appear as discontinuities within the images (D,E). Note that mother and daughter in B are not quite orthogonal, while the arrangement in D and E appears to be orthogonal. Note that if mother daughter orientation is fixed, the granddaughter can emerge from different faces of the daughter, suggesting that the asymmetry is not programmed by the preceding generation of centriole. Bar, 100 nm.

 


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Centrioles are unique cytoskeletal organelles that duplicate precisely once in each cell cycle to generate daughters that are identical to themselves. Unlike the other replicative structure, DNA, the structure of the centriole does not provide insight into the mechanism of duplication. Rather, the structural relationships between mother and daughter centrioles highlight the mysterious features of centriole duplication. For example, while a centriole is purported to have ninefold symmetry along its long axis, it assembles only one daughter at a time. Additionally, the perpendicular arrangement of mother and daughter centrioles minimizes opportunities for structural templating; yet at the end of the duplication process the daughter is as long as its mother. And finally, a daughter centriole fails to initiate a new centriole until it has dissociated from its mother, even thought the associated mother does not occlude the future site of centriole initiation on the daughter. The absence of an obvious structural basis for these observed `rules' of centriole duplication suggests that these behaviors are guided by regulatory interactions rather than rigid structural constraints. If this were the case, one would expect that disruptions in the regulatory circuit might result in violations of the normal `rules' of centriole duplication. We gained insight into these issues when we observed aberrant centriole structures in Drosophila wing imaginal discs lacking Cdk1.

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., 1997Go; Vorobjev and Chentsov, 1982Go), 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., 2001Go). 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., 1996Go). 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., 1996Go). 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., 2001Go) (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.


    Acknowledgments
 
We thank T. Su, M. Winey, R. Feldman, A. Echard, E. Foley, P. DiGregorio, A. Shermoen, F. Banuett and D. Parry for comments on the manuscript. This work was supported by a Howard Hughes Medical Institute predoctoral fellowship to S.J.V. and NIH grant GM37193 to P.H.O'F.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K. and Watson, J. (1994). Molecular Biology of the Cell. New York: Garland Publishing.

Callaini, G., Whitfield, W. G. and Riparbelli, M. G. (1997). Centriole and centrosome dynamics during the embryonic cell cycles that follow the formation of the cellular blastoderm in Drosophila. Exp. Cell Res. 234,183 -190.[CrossRef][Medline]

Garreau de Loubresse, N., Ruiz, F., Beisson, J. and Klotz, C. (2001). Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies. BMC Cell Biol. 2, 4.[CrossRef][Medline]

Haase, S. B., Winey, M. and Reed, S. I. (2001). Multi-step control of spindle pole body duplication by cyclin-dependent kinase. Nat. Cell Biol. 3, 38-42.[CrossRef][Medline]

Hayashi, S. (1996). A Cdc2 dependent checkpoint maintains diploidy in Drosophila. Development 122,1051 -1058.[Abstract/Free Full Text]

Hayles, J., Fisher, D., Woollard, A. and Nurse, P. (1994). Temporal order of S phase and mitosis in fission yeast is determined by the state of the p34cdc2-mitotic B cyclin complex. Cell 78,813 -822.[Medline]

Knoblich, J. A., Sauer, K., Jones, L., Richardson, H., Saint, R. and Lehner, C. F. (1994). Cyclin E controls S phase progression and its down-regulation during Drosophila embryogenesis is required for the arrest of cell proliferation. Cell 77,107 -120.[Medline]

Lim, H. H., Goh, P. Y. and Surana, U. (1996). Spindle pole body separation in Saccharomyces cerevisiae requires dephosphorylation of the tyrosine 19 residue of Cdc28. Mol. Cell Biol. 16,6385 -6397.[Abstract]

Mahowald, A. P., Caulton, J. H., Edwards, M. K. and Floyd, A. D. (1979). Loss of centrioles and polyploidization in follicle cells of Drosophila melanogaster. Exp. Cell Res. 118,404 -410.[Medline]

Maniotis, A. and Schliwa, M. (1991). Microsurgical removal of centrosomes blocks cell reproduction and centriole generation in BSC-1 cells. Cell 67,495 -504.[Medline]

Milan, M., Campuzano, S. and Garcia-Bellido, A. (1996). Cell cycling and patterned cell proliferation in the wing primordium of Drosophila. Proc. Natl. Acad. Sci. USA 93,640 -645.[Abstract/Free Full Text]

Sluder, G., Miller, F. J. and Rieder, C. L. (1989). Reproductive capacity of sea urchin centrosomes without centrioles. Cell Motil. Cytoskeleton 13,264 -273.[Medline]

Vidwans, S. J., Wong, M. L. and O'Farrell, P. H. (1999). Mitotic regulators govern progress through steps in the centrosome duplication cycle. J. Cell Biol. 147,1371 -1378.[Abstract/Free Full Text]

Vorobjev, I. A. and Chentsov, Yu. S. (1982). Centrioles in the cell cycle. I. Epithelial cells. J. Cell Biol. 93,938 -949.[Abstract]

Weigmann, K., Cohen, S. M. and Lehner, C. F. (1997). Cell cycle progression, growth and patterning in imaginal discs despite inhibition of cell division after inactivation of Drosophila Cdc2 kinase. Development 124,3555 -3563.[Abstract/Free Full Text]


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