The conversion of centrioles to centrosomes: essential coupling of duplication with segregation

Centrioles are self-reproducing organelles that form the core structure of centrosomes or microtubule-organizing centers (MTOCs). However, whether duplication and MTOC organization reflect innate activities of centrioles or activities acquired conditionally is unclear. In this paper, we show that newly formed full-length centrioles had no inherent capacity to duplicate or to organize pericentriolar material (PCM) but acquired both after mitosis through a Plk1-dependent modification that occurred in early mitosis. Modified centrioles initiated PCM recruitment in G1 and segregated equally in mitosis through association with spindle poles. Conversely, unmodified centrioles segregated randomly unless passively tethered to modified centrioles. Strikingly, duplication occurred only in centrioles that were both modified and disengaged, whereas unmodified centrioles, engaged or not, were "infertile," indicating that engagement specifically blocks modified centrioles from reduplication. These two requirements, centriole modification and disengagement, fully exclude unlimited duplication in one cell cycle. We thus uncovered a Plk1-dependent mechanism whereby duplication and segregation are coupled to maintain centriole homeostasis.     

On the association of centrioles with the interphase nucleus.

Preparations of nuclei from rat liver and bovine spleen purified by centrifugation through dense sucrose solutions are shown to contain centrioles. These centrioles retain their in situ ultrastructure and are surrounded by a network of filaments adjacent to the nucleus and probably attached to it. The number of centrioles in isolated nuclei depends on the conditions of cell homogenization. Under certain conditions of homogenization, the fraction of purified nuclei contains almost all centrioles of the original tissue. The number of centrioles in isolated nuclei sharply decreases if the nuclei are rehomogenized under conditions that do not cause damage to nuclei. The number of nucleus-associated centrioles does not decrease after solubilization of nuclear membranes by Triton X-100. Nuclei retain the associated centrioles after treatmentwith RNase-free DNase I. It is concluded that in interphase the centrioles are associated with the nucleus and that this association which is probably mediated by filaments involves nuclear structures other than nuclear membranes or whole chromatin.     

Spatial relationships between centrioles and the centrosphere in monoasters induced by taxol inPhysarum polycephalum amoebae

Summary Monoasters induced by taxol in the amoebae of the MyxomycetePhysarum polycephalum show an unusual tridimensional location of the centrioles. Tridimensional reconstructions of individual monoasters with either two or four centrioles show that the position of centrioles is not random. The characteristics of these monoasters suggest that the centrioles are not linked to the mitotic center in the monoaster since mitotic centers completely devoid of centrioles in adjacent or central location are observed. However, the preferential centrifugal orientation of the centrioles in the centrosphere induced by taxol suggests that centrioles are initially located in the mitotic center in agreement with the attachment of the centrioles to the mitotic center during interphase.     

The de novo centriole assembly pathway in HeLa cells: cell cycle progression and centriole assembly/maturation

It has been reported that nontransformed mammalian cells become arrested during G1 in the absence of centrioles (Hinchcliffe, E., F. Miller, M. Cham, A. Khodjakov, and G. Sluder. 2001. Science. 291:1547-1550). Here, we show that removal of resident centrioles (by laser ablation or needle microsurgery) does not impede cell cycle progression in HeLa cells. HeLa cells born without centrosomes, later, assemble a variable number of centrioles de novo. Centriole assembly begins with the formation of small centrin aggregates that appear during the S phase. These, initially amorphous "precentrioles" become morphologically recognizable centrioles before mitosis. De novo-assembled centrioles mature (i.e., gain abilities to organize microtubules and replicate) in the next cell cycle. This maturation is not simply a time-dependent phenomenon, because de novo-formed centrioles do not mature if they are assembled in S phase-arrested cells. By selectively ablating only one centriole at a time, we find that the presence of a single centriole inhibits the assembly of additional centrioles, indicating that centrioles have an activity that suppresses the de novo pathway.     

Mode of centriole duplication and distribution

Centriole stability and distribution during the mammalian cell cycle was studied by microinjecting biotinylated tubulin into early G1 cells and analyzing the pattern of incorporation into centrioles. Cells were extracted and cold treated to depolymerize labile microtubules, allowing the fluorescent microscopic visualization of the stable centrioles. The ability to detect single centrioles was confirmed by use of correlative electron microscopy. Indirect hapten and immunofluorescent labeling of biotinylated and total tubulin permitted us to distinguish newly formed from preexisting centrioles. Daughter centrioles incorporated biotinylated tubulin, and at mitosis each cell received a centrosome containing one new and one old centriole. We conclude that in each cell cycle tubulin incorporation into centrioles is conservative, and centriole distribution is semiconservative.     

Kinetics and regulation of de novo centriole assembly. Implications for the mechanism of centriole duplication

BACKGROUND: Centriole duplication is a key step in the cell cycle whose mechanism is completely unknown. Why new centrioles always form next to preexisting ones is a fundamental question. The simplest model is that preexisting centrioles nucleate the assembly of new centrioles, and that although centrioles can in some cases form de novo without this nucleation, the de novo assembly mechanism should be too slow to compete with normal duplication in order to maintain fidelity of centriole duplication. RESULTS: We have measured the rate of de novo centriole assembly in vegetatively dividing cells that normally always contain centrioles. By using mutants of Chlamydomonas that are defective in centriole segregation, we obtained viable centrioleless cells that continue to divide, and find that within a single generation, 50% of these cells reacquire new centrioles by de novo assembly. This suggests that the rate of de novo assembly is approximately half the rate of templated duplication. A mutation in the VFL3 gene causes a complete loss of the templated assembly pathway without eliminating de novo assembly. A mutation in the centrin gene also reduced the rate of templated assembly. CONCLUSIONS: These results suggest that there are two pathways for centriole assembly, namely a templated pathway that requires preexisting centrioles to nucleate new centriole assembly, and a de novo assembly pathway that is normally turned off when centrioles are present.     

Nonequivalence of maternal centrosomes/centrioles in starfish oocytes: selective casting-off of reproductive centrioles into polar bodies

It is believed that in most animals only the paternal centrosome provides the division poles for mitosis in zygotes. This paternal inheritance of the centrosomes depends on the selective loss of the maternal centrosome. In order to understand the mechanism of centrosome inheritance, the behavior of all maternal centrosomes/centrioles was investigated throughout the meiotic and mitotic cycles by using starfish eggs that had polar body (PB) formation suppressed. In starfish oocytes, the centrioles do not duplicate during meiosis II. Hence, each centrosome of the meiosis II spindle has only one centriole, whereas in meiosis I, each has a pair of centrioles. When two pairs of meiosis I centrioles were retained in the cytoplasm of oocytes by complete suppression of PB extrusion, they separated into four single centrioles in meiosis II. However, after completion of the meiotic process, only two of the four single centrioles were found in addition to the pronucleus. When the two single centrioles of a meiosis II spindle were retained in the oocyte cytoplasm by suppressing the extrusion of the second PB, only one centriole was found with the pronucleus after the completion of the meiotic process. When these PB-suppressed eggs were artificially activated to drive the mitotic cycles, all the surviving single centrioles duplicated repeatedly to form pairs of centrioles, which could organize mitotic spindles. These results indicate that the maternal centrioles are not equivalent in their intrinsic stability and reproductive capacity. The centrosomes with the reproductive centrioles are selectively cast off into the PBs, resulting in the mature egg inheriting a nonreproductive centriole, which would degrade shortly after the completion of meiosis.     

GIANT CENTRIOLE FORMATION IN SCIARA

Although somatic tissues of Sciara contain 9-membered centrioles, germ line tissues develop giant centrioles with 60–90 singlet tubules disposed in an oval array. Some 9-membered centrioles still may be seen in second instar spermatogonia. Each of these centrioles is associated with a larger "daughter" or secondary centriole at right angles to it. Most centrioles of second instar spermatogonia consist of 20–50 singlet tubules arranged in an oval, sometimes associated with an even larger secondary centriole. The more recently formed centriole of a pair is distinguishable from its partner by a concentric band of electron-opaque material inside its tubules. If a pair of centrioles at right angles to each other is pictured as a "T" formed by two cylinders, the secondary centriole is always the stem of the T; the primary centriole is the top. The two centrioles are oriented at the pole of the mitotic spindle so that the tubules of the primary centriole are parallel to the spindle axis. Each daughter cell receives a pair of centrioles and, during interphase, each of these centrioles gives rise to a new daughter centriole. A Golgi area of characteristic morphology is found in association with centrioles shortly after two new ones have formed. We conclude that in Sciara a centriole may give rise to a daughter morphologically different from itself. Whether the daughter is a 9-membered or giant centriole depends on the tissue type and stage of development.     

The induction of de novo centrioles in sea urchin eggs: a possible common mechanism for centriolar activation among parthenogenetic procedures

Eggs from two species of sea urchins were subjected to a variety of novel two-step parthenogenetic activation procedures. These treatments readily resulted in the formation of cytasters and centrioles. Centrioles are restricted to the cytasters, as well as to the broad perinuclear zone, and it appears that all cytasters contain one or more centrioles. The mode of centriolar origin and maturation, as revealed by these new procedures, is identical to that of centrioles induced by other procedures. Both centriolar and cytastral development are retarded by the parthenogenetic treatments; also, mitosis is generally delayed. These results, along with those of others, reveal that parthenogenetic agents have opposing effects on eggs. The agents are stimulatory in that they activate latent centrioles and the cell cycle, in general. They are inhibitory in that they suppress a variety of physiochemical events. Of these events, only one appears to be common to all parthenogenetic agents: the inhibition of protein synthesis. Egg centrioles may be kept in a latent state by repressor proteins. If these proteins normally cycle rapidly, then an inhibition of their continuous synthesis will disrupt their normal replacement rate. As such, a delay in replacement will allow functioning repressor proteins, associated with latent centrioles, to become nonfunctional, which in turn allows latent centrioles to become derepressed. This deblocking reaction would then activate the appearance of de novo centrioles in eggs.     

Correlation between the number of centrioles and ploidy of mouse liver hepatocytes

Using the electron microscope it was shown that in interphase hepatocytes with ploidies equal to 2n, 2n.2, 4n, 4n.2 and 8n, the number of centrioles per cell exactly corresponded to the ploidy of the cell. Both in mononuclear and binuclear cells all the centrioles are accumulated in one complex in which each pair of centrioles forms a diplosome. In binuclear cells, the complex of diplosomes is situated at equal distances from each nucleus, thus making the cell centre. The involvement of the supernumerous centrioles in polyploid metaphase cells was detected for the regenerating liver of old mice. It was found that each mitotic pole had at least four centrioles. In the pole, a pair of centrioles forms diplosomes tightly connected to each other. It is suggested that the initially tetraploid cell might divide in this manner. In addition, a question is discussed on how the existence of centrioles can be associated with the mechanism of polyploidization.     

Destruction of maternal centrioles during fertilization of the brown alga, Scytosiphon lomentaria (Scytosiphonales, Phaeophyceae)

In brown algal fertilization, a pair of centrioles is derived from the male gamete, irrespective of the sexual reproduction pattern, i.e., isogamy, anisogamy, or oogamy. In this study, the manner in which the maternal centriole structure is destroyed in early zygotes of the isogamous brown alga Scytosiphon lomentaria was examined by electron microscopy. At fertilization, the zygote had two pairs of centrioles (flagellar basal bodies) derived from motile male and female gametes, and there was no morphological difference between the two pairs. The flagellar basal plate and the axonemal microtubules were still connected with the distal end of centrioles. Ultrastructural observations showed that the integrity of maternal-derived centrioles began to degenerate even in the 1-h-old zygote. At that time, the cylinder of triplet microtubules of the maternal centrioles became shorter from the distal end, and a section passing through the centrioles indicated that a part of the nine triplets of microtubules changed into doublet or singlet microtubules by degeneration of B and/or C tubules. In 2-h-old zygote, there was no trace of maternal centrioles ultrastructurally, and only the paternal centrioles remained. Further, reduction of centrin accompanying destruction of the maternal centrioles was examined in immunofluorescence microscopy. Centrin localized at the paternal and the maternal centrioles had the same fluorescence intensity in the early zygotes. At 4-6 h after fertilization, two spots indicating centrin localization showed different fluorescence intensity. Later, the weaker spot disappeared completely. These results showed that there is a difference in time between the destruction of the centriolar cylinders and the reduction of centrin molecules around them.     

Centrosome destined to decay in starfish oocytes

In contrast to the somatic cell cycle, duplication of the centrioles does not occur in the second meiotic cycle. Previous studies have revealed that in starfish each of the two centrosomes in fully-grown immature oocytes consists of two centrioles with different destinies: one survives and retains its reproductive capacity, and the other is lost after completion of meiosis. In this study, we investigated whether this heterogeneity of the meiotic centrioles is already determined before the re-initiation of meiosis. We prepared a small fragment of immature oocyte containing the four centrioles and fused it electrically with a mature egg in order to transfer two sets of the premeiotic centrioles into the mature cytoplasm. Two asters were present in this conjugate, and in each of them only a single centriole was detected by electron microscopy. In the first mitosis of the conjugate artificially activated without sperm, two division poles formed, each of which doubled in each subsequent round of mitosis. These results indicate that only two of the four premeiotic centrioles survived in the mature cytoplasm and that they retained their reproductive capacity, which suggests that the heterogeneity of the maternal centrioles is determined well before re-initiation of meiosis, and that some factor in the mature cytoplasm is responsible for suppressing the reproductive capacity of the centrioles destined to decay.     

Centriole modification in human aortic endothelial cells.

The structure of centrioles in endothelial cells of embryonic (22-24 weeks old) and definitive (2, 14-17, and 30-40 years) human aorta in situ and also in aortic endothelial cells dividing in organ and cell cultures (donor age 30-40 years) was studied. It was found that in the endothelial cells from definitive aorta the lengths of mother centrioles vary from 0.5 to 2 microns, whereas the length of daughter centrioles remains constant (0.4-0.5 microns). The distal part of the cylinder of long mother centrioles consists of microtubule doublets. In aorta of donors 30-40 years old in multinucleated cells and in one of 30 single-nucleated cells analyzed, C-shaped long centrioles were seen. These centrioles exhibit a doublet organization along all their length. Mitotic cells in organ and cell culture had a nonequal structure of spindle poles: at one pole, the long mother centriole was seen, while at the other a mother centriole of standard size was found. In such cells of organ culture long centrioles make contact with the remnant of primary cilia until the end of anaphase. In cell culture mitotic cells are also observed containing C-shaped centrioles. In these cells the number of mother centrioles is odd and their number is not equal to the number of daughter centrioles. The possible mechanism for transformation of endothelial centrioles and its role in the control of cell-cycle progression are discussed.     

The first spindle formation in brown algal zygotes

Regulation of the first spindle formation in brown algal zygotes was described. It is well known that there are three types of sexual reproduction in brown algae; isogamy, anisogamy and oogamy. Paternal inheritance of centrioles can be observed in all these cases, similar to animal fertilization. In isogamy and anisogamy, female centrioles (= flagellar basal bodies) selectively disappear and male centrioles remain after fertilization. In a typical oogamy (e.g. fucoid members), liberated egg does not have centrioles, and sperm centrioles are introduced in zygote. Participation of sperm centrioles to the spindle formation in zygotes was also described using Fucus distichus as a model system. Sperm centrioles function as a part of centrosome, namely microtubule organizing center, in zygote. Therefore, they have a crucial role in the spindle formation. Observations on the spindle formation in polygyny and karyogamy-blocked zygotes strongly suggest that egg nucleus can form a mitotic spindle by itself without centrosome, even though the resulting spindles are of abnormal shapes. %     

Rootletin interacts with C-Nap1 and may function as a physical linker between the pair of centrioles/basal bodies in cells

Rootletin, a major structural component of the ciliary rootlet, is located at the basal bodies and centrosomes in ciliated and nonciliated cells, respectively. Here we investigated its potential role in the linkage of basal bodies/centrioles and the mechanism involved in such linkages. We show that rootletin interacts with C-Nap1, a protein restricted at the ends of centrioles and functioning in centrosome cohesion in interphase cells. Their interaction in vivo is supported by their colocalization at the basal bodies/centrioles and coordinated association with the centrioles during the cell cycle. Ultrastructural examinations demonstrate that rootletin fibers connect the basal bodies in ciliated cells and are present both at the ends of and in between the pair of centrioles in nonciliated cells. The latter finding stands in contrast with C-Nap1, which is present only at the ends of the centrioles. Transient expression of C-Nap1 fragments dissociated rootletin fibers from the centrioles, resulting in centrosome separation in interphase. Overexpression of rootletin in cells caused multinucleation, micronucleation, and irregularity of nuclear shape and size, indicative of defects in chromosome separation. These data suggest that rootletin may function as a physical linker between the pair of basal bodies/centrioles by binding to C-Nap1.     

Centrioles: some self-assembly required

Centrioles play an important role in organizing microtubules and are precisely duplicated once per cell cycle. New (daughter) centrioles typically arise in association with existing (mother) centrioles (canonical assembly), suggesting that mother centrioles direct the formation of daughter centrioles. However, under certain circumstances, centrioles can also selfassemble free of an existing centriole (de novo assembly). Recent work indicates that the canonical and de novo pathways utilize a common mechanism and that a mother centriole spatially constrains the self-assembly process to occur within its immediate vicinity. Other recently identified mechanisms further regulate canonical assembly so that during each cell cycle, one and only one daughter centriole is assembled per mother centriole.     

Selective disappearance of maternal centrioles after fertilization in the anisogamous brown alga Cutleria cylindrica (Cutleriales, Phaeophyceae): Paternal inheritance of centrioles is universal in the brown algae

The behavior of centrioles in zygotes and female gametes developing parthenogenetically in the anisogamous brown alga Cutieria cyiindrica Okamura was studied using electron and immunofluorescence microscopy. Two pairs of centrioles, detected using anti-centrin antibody, were observed in the vicinity of the male and female nuclei, respectively, just after plasmogamy. The fluorescence intensity of one of the two centrin foci became weak 6 h after plasmogamy and finally disappeared. It was impossible to determine whether the male- or female-derived centrioles disappeared in zygotes, because there was nothing to detect morphological differences between the two centrioles. However, a prominent anti-centrin staining focus was located at the condensed male nucleus in zygotes in which karyogamy had not occurred yet. As a result, it was considered that the maternally inherited centrioles had selectively disappeared during development in C. cylindrica. The paternal inheritance of centrioles in zygotes was also confirmed by electron microscopy. Considering previous observations from oogamous and isogamous species of brown algae, we concluded that the paternal inheriance of centrioles could be universal in the brown algae.     

INTERCELLULAR MIGRATION OF CENTRIOLES IN THE GERMARIUM OF DROSOPHILA MELANOGASTER : An Electron Microscopic Study

A cluster of centrioles has been found in the early Drosophila oocyte. Since the oocyte is connected to 15 nurse cells by a system of intercellular bridges or ring canals, the possibility that the cluster of centrioles arose in the germarium from an intercellular migration of centrioles from the nurse cells to the oocyte was analyzed in serial sections for the electron microscope. Initially, all of the 16 cells of the future egg chambers possess centrioles, which are located in a juxtanuclear position. At the time the 16 cell cluster becomes arranged in a lens-shaped layer laterally across the germarium, the centrioles lose their juxtanuclear position and move towards the oocyte. By the time the 16 cell cluster of cells is surrounded by follicle cells (Stage 1), between 14 and 17 centrioles are found in the oocyte. Later, these centrioles become located between the oocyte nucleus and the follicle cell border and become aggregated into a cluster less than 1.5 µ in its largest dimension. The fate of these centrioles in the oocyte is not known. The fine structure of the germarium and the early oocyte is also described.     

Behavior of centrioles during meiosis in the male silkworm, Bombyx mori (Lepidoptera)

The behavior of centrioles during eupyrene and apyrene meiosis was examined in the silkworm, Bombyx mori , by transmission electron microscopy and indirect immunofluorescence for tubulin. In eupyrene spermatocytes the centrioles, accompanied by axonemes, attached temporarily to the nucleus at diplotene, then detached from the nucleus in diakinesis. After the separation, a beret-shaped structure consisting of a double membrane covered the proximal region of the pair of centrioles. The structure disappeared after breakdown of the nuclear membrane. The centriole, with the axoneme, reattached to the nucleus at telophase I. The process was repeated during meiosis II until the centrioles maintained their nuclear attachment in newly developed spermatids. In stark contrast to their eupyrene counterparts, apyrene spermatocytes were conspicuously devoid of any attachment of the centrioles to the nucleus. These eupyrene-specific and apyrene-specific relationships were consistently and repeatedly found between the nuclear membrane and centrioles, giving rise to suspicion that the behavioral phenomena may be related to differentiation of the dimorphic sperm types.     

De novo formation of centrosomes in vertebrate cells arrested during S phase

The centrosome usually replicates in a semiconservative fashion, i.e., new centrioles form in association with preexisting "maternal" centrioles. De novo formation of centrioles has been reported for a few highly specialized cell types but it has not been seen in vertebrate somatic cells. We find that when centrosomes are completely destroyed by laser microsurgery in CHO cells arrested in S phase by hydroxyurea, new centrosomes form by de novo assembly. Formation of new centrosomes occurs in two steps: approximately 5-8 h after ablation, clouds of pericentriolar material (PCM) containing gamma-tubulin and pericentrin appear in the cell. By 24 h, centrioles have formed inside of already well-developed PCM clouds. This de novo pathway leads to the formation of a random number of centrioles (2-14 per cell). Although clouds of PCM consistently form even when microtubules are completely disassembled by nocodazole, the centrioles are not assembled under these conditions.