Regulation of Tubulin Organization During Meiosis

Meiotic spindle formation in Spisula solidissima oocytes hasbeen studied in vivo and in vitro. Measurements were made ofpolymerized tubulin content during the first meiotic division.The amount of polymer was high prior to activation of the eggs,fell to a minimum of about 5 min after activation and at 15min (metaphase) returned to approximately its initial value.The polymerized tubulin in the unactivated eggs appears to beorganized into granular spheres about 10–20 microns indiameter attached to the egg cortex. This particle containsfew microtubules but is composed primarily of a granular matrixand fibrous material. The granular matrix may be a polymorphicaggregate of tubulin and could be a storage form of tubulinor an intermediate in microtubule assembly. The polymerization and organization of microtubules has beenstudied in vitro, using crude homogenates of Spisula oocytes.Microtubules can be formed in homogenates of both activatedand unactivated eggs; however, in homogenates of eggs in whichnuclear membrane breakdown has occurred, microtubules form arounda central phase dense particle resulting in a structure whichresembles a spindle aster. The central particle appears to bea microtubule organizing center (MTOC). The MTOC can be pelleledby centrifugation and will induce aster formation when remixedwith the supernatant. Aster formation can be obtained usingsupernatants prepared from either activated or unactivated eggs,while the pellet must be obtained from activated eggs. Thus,tubulin subunits appear to be capable of spindle formation atall stages, while MTOC formation or activation does not occuruntil about the time of nuclear membrane breakdown.     

CHANGES IN THE ORGANIZATION OF TUBULIN DURING MEIOSIS IN THE EGGS OF THE SURF CLAM, SPISULA SOLIDISSIMA

Polymerized tubulin can be stabilized in Kane's spindle isolation medium (HGL solution), isolated by differential centrifugation and then assayed by colchicine binding activity. In the eggs of the surf clam, Spisula solidissima, the level of particulate tubulin undergoes a series of specific changes during first meiotic division. In either unactivated ("interphase") eggs or metaphase eggs the amount of particulate tubulin was about 13% of the total at 23°C. The amount of particulate tubulin decreased shortly after activation, reaching a minimum value at about 5 min, the time of nuclear membrane breakdown. The particulate tubulin concentration then rose, reaching a maximum at metaphase, and then decreased again during anaphase, reaching a minimum at first polar body formation. In HGL homogenates of unactivated eggs a structure is present which has been shown to contain the interphase particulate tubulin (IPT). This structure consists essentially of a 10–20 µ granular sphere attached to a membranous material which is probably part of the egg cortex. These particles are absent at the time of nuclear membrane breakdown, when the level of particulate tubulin is minimal and when the first signs of spindle formation are visible. Electron microscopy of these particles by negative staining indicates that they are composed of microtubules associated with a granular matrix which may be a polymorphic aggregate of tubulin.     

Acetylated tubulin is found in all microtubule arrays of two species of pine

Summary The distribution of acetylated tubulin in microtubule arrays of conifer cells was investigated by immunofluorescence techniques with 6-11B-1, a monoclonal antibody specific for posttranslationally acetylated -tubulins. In methacrylate sections ofPinus radiata andPinus conforta root tip cells, acetylated tubulin was detected in mitotic spindles, phragmoplasts, and cortical microtubules. Furthermore, staining of isolated, intact cells ofP. radiata andP. contorta indicated that all microtubule structures, including preprophase bands, prophase, metaphase and anaphase spindles, and phragmoplasts, contained some acetylated tubulin, and that the intensity of staining with 6-11B-1 was variable. For example, preprophase bands were lightly labelled, kinetochore fibres of anaphase spindles and phragmoplasts were heavily stained, and metaphase spindles had a granular appearance suggesting discontinuous acetylation of their constituent microtubules. This first report of the presence of acetylated tubulin in conifer cells is in contrast to our results with two species of angiosperms where no acetylated tubulin was detected. The significance of this and the variability of the intensity of staining in conifer arrays is discussed in terms of microtubule dynamics.     

Interconversion of metaphase and interphase microtubule arrays, as studied by the injection of centrosomes and nuclei into Xenopus eggs

We have designed experiments that distinguish centrosomal , nuclear, and cytoplasmic contributions to the assembly of the mitotic spindle. Mammalian centrosomes acting as microtubule-organizing centers were assayed by injection into Xenopus eggs either in a metaphase or an interphase state. Injection of partially purified centrosomes into interphase eggs induced the formation of extensive asters. Although centrosomes injected into unactivated eggs (metaphase) did not form asters, inhibition of centrosomes is not irreversible in metaphase cytoplasm: subsequent activation caused aster formation. When cytoskeletons containing nuclei and centrosomes were injected into the metaphase cytoplasm, they produced spindle-like structures with clearly defined poles. Electron microscopy revealed centrioles with nucleated microtubules. However, injection of nuclei prepared from karyoplasts that were devoid of centrosomes produced anastral microtubule arrays around condensing chromatin. Co-injection of karyoplast nuclei with centrosomes reconstituted the formation of spindle-like structures with well-defined poles. We conclude from these experiments that in mitosis, the centrosome acts as a microtubule-organizing center only in the proximity of the nucleus or chromatin, whereas in interphase it functions independently. The general implications of these results for the interconversion of metaphase and interphase microtubule arrays in all cells are discussed.     

Immunoelectron microscopical detection of tubulins during spermiogenesis in phytophagous bugs (Hemiptera: Pentatomidae)

Summary

Ultrastructural and immunocytochemical studies were carried out in the tail region of spermatids and spermatozoa of the phytophagous bugs, Acrosternum aseadum and Euchistus heros. The axoneme presented a 9+9+2 microtubule pattern and bridges occurred between axonemal microtubules 1, 5, and mitochondrial derivatives. Two paracrystalline structures, embedded in an amorphous matrix, were observed in the mitochondrial derivatives. The axonemal microtubules contained alpha, acetylated and tyrosinated tubulin. Cytoplasmic microtubules contained alpha, beta and gamma tubulin. Moreover, the gamma tubulin was detected near the electron dense rod, an element associated with the centriole, suggesting that this structure may be a microtubule organizing center.     

Identification of a new protein localized at sites of cell-substrate adhesion

Tipulid spermatocytes form normally functioning bipolar spindles after one of the centrosomes is experimentally dislocated from the nucleus in late diakinesis (Dietz, R., 1959, Z. Naturforsch., 14b:749-752; Dietz, R., 1963, Zool. Anz. Suppl., 23:131-138; Dietz, R., 1966, Heredity, 19:161-166). The possibility that dissociated pericentriolar material (PCM) is nevertheless responsible for the formation of the spindle in these cells cannot be ruled out based on live observation. In studying serial sections of complete cells and of lysed cells, it was found that centrosome-free spindle poles in the crane fly show neither pericentriolar-like material nor aster microtubules, whereas the displaced centrosomes appear complete, i.e., consist of a centriole pair, aster microtubules, and PCM. Exposure to a lysis buffer containing tubulin resulted in an increase of centrosomal asters due to aster microtubule polymerization. Aster-free spindle poles did not show any reaction, also indicating the absence of PCM at these poles. The results favor the hypothesis of chromosome-induced spindle pole formation at the onset of prometaphase and the dispensability of PCM in Pales.     

Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin

Treatment of HeLa cells with Colcemid at concentrations of 0.06-0.10 mug/ml leads to irreversible arrest in mitosis. Colcemid-arrested cells contained few microtubules, and many kinetochores and centrioles were free of microtubule association. When these cells were exposed to microtubule reassembly buffer containing Triton X-100 and bovine brain tubulin at 37 degrees C, numerous microtubules were reassembled at all kinetochores of metaphase chromosomes and in association with centriole pairs. When bovine brain tubulin was eliminated from the reassembly system, microtubules failed to assemble at these sites. Similarly, when EGTA was eliminated from the reassembly system, microtubules failed to polymerize. These results are consistent with other investigations of in vitro microtubule assembly and indicate that HeLa chromosomes and centrioles can serve as nucleating sites for the assembly of microtubules from brain tubulin. Both chromosomes and centrioles became displaced from their C-metaphase configurations during tubulin reassembly, indicating that their movements were a direct result of microtubule formation. Although both kinetochore- and centriole- associated microtubules were assembled and movement occurred, we did not observe direct extension of microtubules from kinetochores to centrioles. This system should prove useful for experimental studies of spindle microtubule formation and chromosome movement in mammalian cells.     

Prophase microtubule arrays undergo flux-like behavior in mammalian cells

In higher eukaryotic cells, microtubules within metaphase and anaphase spindles undergo poleward flux, the slow, poleward movement of tubulin subunits through the spindle microtubule lattice. Although a number of studies have documented this phenomenon across a wide range of model systems, the possibility of poleward flux before nuclear envelope breakdown (NEB) has not been examined. Using a mammalian cell line expressing photoactivatable green fluorescent protein (GFP)-tubulin, we observe microtubule motion, both toward and away from centrosomes, at a wide range of rates (0.5–4.5 μm/min) in prophase cells. Rapid microtubule motion in both directions is dynein dependent. In contrast, slow microtubule motion, which occurs at rates consistent with metaphase flux, is insensitive to inhibition of dynein but sensitive to perturbation of Eg5 and Kif2a, two proteins with previously documented roles in flux. Our results demonstrate that microtubules in prophase cells are unexpectedly dynamic and that a subpopulation of these microtubules shows motion that is consistent with flux. We propose that the marked reduction in rate and directionality of microtubule motion from prophase to metaphase results from changes in microtubule organization during spindle formation.     

Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation

Centrioles organize microtubules in two ways: either microtubules elongate from the centriole cylinder itself, forming a flagellum or a cilium ("template elongation"), or pericentriolar material assembles and nucleates a microtubule aster ("astral nucleation"). During spermatogenesis in most species, a motile flagellum elongates from one of the sperm centrioles, whereas after fertilization a large aster of microtubules forms around the sperm centrioles in the egg cytoplasm. Using Xenopus egg extracts we have developed an in vitro system to study this change in microtubule-organizing activity. An aster of microtubules forms around the centrioles of permeabilized frog sperm in egg extracts, but not in pure tubulin. However, when the sperm heads are incubated in the egg extract in the presence of nocodazole, they are able to nucleate a microtubule aster after isolation and incubation with pure calf brain tubulin. This provides a two-step assay that distinguishes between centrosome assembly and subsequent microtubule nucleation. We have studied several centrosomal antigens during centrosome assembly. The CTR2611 antigen is present in the sperm head in the peri-centriolar region. gamma-tubulin and certain phosphorylated epitopes appear in the centrosome only after incubation in the egg extract. gamma-tubulin is recruited from the egg extract and associated with electron-dense patches dispersed in a wide area around the centrioles. Immunodepletion of gamma-tubulin and associated molecules from the egg extract before sperm head incubation prevents the change in microtubule-organizing activity of the sperm heads. This suggests that gamma-tubulin and/or associated molecules play a key role in centrosome formation and activity.     

In vitro reactivation of anaphase B in isolated spindles of the sea urchin egg

Spindles may be isolated from sea urchin eggs so that some mitotic processes can be reactivated in vitro. The isolation media allow spindles to remain stable for days. Transfer of the spindles to reactivation media results in loss of birefringence and breakdown of the matrix within which the microtubules function. If, however, tubulin and either guanosine triphosphate or adenosine triphosphate are present in these media so that tubulin can cycle, the spindles do not break down but grow in size and birefringence and show some of the movements of in vivo spindles. The most prominent is that of anaphase B if the mitotic apparatuses (MAs) have been isolated at a time when anaphase was initiated. When isolated during metaphase, MAs either do not show chromosome movement or, if they do, it is a random movement which causes redistribution of the chromosomes on the spindle surface. In either case, such metaphase spindles grow in size and birefringence. Thus under the proper conditions, cycling microtubules can interact with the spindle matrix to induce chromosome movements which resemble those seen in in vivo cells in the case of anaphase B and show some aspects of anaphase A in at least half the spindles isolated at metaphase, although such movements are not coordinated to show a true anaphase movement.     

Aster formation in eggs of Xenopus laevis. Induction by isolated basal bodies

We have assayed various materials for their ability to induce aster formation by microinjection into unfertilized eggs of Xenopus laevis. We have found that purified basal bodies from Chlamydomonas reinhardtii and Tetrahymena pyriformis induce the formation of asters and irregular cleavage furrows within 1 h after injection. Other microtubule structures such as flagella, flagellar axonemes, cilia, and brain microtubules are completely ineffective at inducing asters or cleavage furrows in unfertilized eggs. When known amounts of sonicated Tetrahymena and Chlamydomonas preparations are injected into unfertilized eggs, 50% of the injected eggs show a furrowing response at approximately 3 cell equvalents for Chlamydomonas and 0.1 cell equivalent for Tetrahymena. These results are close to those expected if basal bodies were the effective astral-inducing agent in these cells. Other materials effective at inducing asters in unfertilized eggs, such as crude brain nuclei, sperm, and a particulate fraction from brain known to induce parthenogenesis in eggs of Rana pipiens, probably contain centrioles as the effective agent. Our experiments provide the first functional assay to indicate that centrioles play an active role in aster initiation. None of the injected materials effective in unfertilized eggs produced any observable response in fully grown oocytes. Oocytes and eggs were found to have equal tubulin pools as judged by colchicine-binding activity. Therefore, the inability of oocytes to form asters cannot be due to a lack of an organizing center or to a lack of tubulin. Experiments in which D2O was found to stimulate aster-like fibrous areas in eggs but not oocytes suggest that the inability of oocytes to form asters may be due to an inability of tubulin in oocytes to assemble.     

Purification of meiotic spindles and cytoplasmic asters from mouse oocytes

The unfertilized mouse oocyte is arrested at second metaphase of meiosis with microtubules existing exclusively in the meiotic spindle. Multiple inactive cytoplasmic microtubule organizing centers (MTOCs) are also present. These MTOCs can be identified immunocytochemically with an autoimmune serum (No. 5051) directed against pericentriolar material (PCM) and also by their nucleating capacity in the presence of taxol which effectively lowers the critical concentration for tubulin polymerization. Taxol induces the formation of cytoplasmic microtubule asters around the PCM foci, a process which also occurs in untreated eggs after fertilization. The molecular characterization of these structures has not been undertaken previously, probably due to the very small amount of material available. We have developed a single-step purification procedure by which very clean preparations of meiotic spindles and cytoplasmic asters can be obtained, as judged by phase-contrast microscopy and transmission electron microscopy. The purified structures were shown to correspond to those observed in vivo: positive staining of the spindles was observed with anti-tubulin and anti-phosphoprotein (MPM2) antibodies, and positive staining of the MTOCs was observed with MPM2, No. 5051, and anti-calmodulin antibodies. As expected, tubulin was the major protein present in the preparations. Silver staining of SDS-PAGE also revealed the presence of a small number of other polypeptides (Mr of around 47, 35, and 25K). Amongst newly synthesized polypeptides associated with the preparation, two prominent high molecular weight proteins (greater than 200K) were enriched in addition to tubulin and polypeptides with Mr of around 52, 41, and 35K.     

Division polarity,development and configuration of microtubule arrays in Bryophyte meiosis

Summary Immunofluorescence and TEM studies of meiosis in two mosses (Bryophyta) provide evidence that the prophasic tetrahedral system of microtubules contributes directly to the metaphase I spindle. Intense staining of tubulin, conspicuously absent around the nuclear envelope, is first seen associated with plastids. By mid-prophase, microtubules radiate from the plastids to the nuclear envelope and become organized into six bands that interconnect the four plastids, forming a tetrahedral cytoskeleton surrounding the nucleus. During transition of prophase to metaphase, the four poles of the tetrahedral microtubule system converge in pairs toward opposite cleavage furrows. Opposite furrows occupy mutually perpendicular planes and the pair of microtubule focal points straddling one furrow lies at right angles to the pair straddling the opposite furrow. Additional microtubules terminate in numerous small clusters in the concave polar regions arching over the cleavage furrows. By early anaphase, the microtubule focal points lie very close to the division axis. We conclude that microtubules recruited from the prophasic quadripolar system are incorporated into the mature metaphase I spindle and the two principal focal points at each pole are those derived from poles of the prophasic quadripolar system.     

Evidence for microtubule subunit addition to the distal end of mitotic structures in vitro

HeLa cells blocked in metaphase with 0.04 micrograms/ml of the microtubule poison nocodazole were shown to contain large numbers of microtubules with typical mitotic organization but no cenriole. Lysis of nocodazole-poisoned cells in a microtubule reassembly buffer containing 0.5 M PIPES, 2.5% dimethyl sulfoxide, 1 mM EDTA, 1 mM MgCl2, 1 mM GTP, 1% Triton X-165, 0.5% sodium deoxycholate, 0.2% SDS, pH 6.9, preserved metaphase aster structures 5 micrograms in diameter surrounded only by a thin, fibrous cell remnant. Inclusion of 2 mg/ml porcine brain microtubule protein in the lysis buffer produced asters up to 20 micrometers in diameter with a birefringent retardation of 5-6 nm. In these large asters the central microtubules had normal morphology, but peripheral microtubules were clearly abnormal. Our interpretation is that in high PIPES lysis buffer, exogenous brain tubulin adds to the distal ends of preexisting aster microtubules to form abnormal microtubules. This observation supports the assumptions made by Borisy and by Summers and Kirschner in their interpretation of growth experiments to determine the microtubule polarity in mitotic structures.     

The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus

Early development in Xenopus is characterized by dramatic changes in the organization of the microtubule cytoskeleton. We have used whole-mount immunocytochemistry to follow the expression of the acetylated form of alpha-tubulin during early Xenopus development. In the egg and early embryo, the monoclonal anti-acetylated tubulin antibody 6-11B-1 stained meiotic and mitotic spindles, midbody microtubules, and what appears to be the central region of the sperm aster; the antibody did not stain the sperm aster itself or the cortical microtubule system associated with the rotation of the fertilized egg. Following gastrulation, acetylated tubulin disappeared from all but mitotic midbody microtubules. During the course of neurulation high levels of acetylated tubulin reappeared in the precursors of the ciliated epidermal cells (stage 15), transiently in neural folds (stage 16/17), in neuronal processes (stage 18/19), and in somas (stage 21). The changing pattern of anti-acetylated tubulin staining during Xenopus development raises intriguing questions as to the physiological significance of tubulin acetylation.     

Rotenone inhibition of spindle microtubule assembly in mammalian cells

Rotenone, a potent inhibitor of mitochondrial respiration is also an effective antimitotic agent. The addition of either rotenone or Colcemid to exponentially growing Chinese hamster ovary cells resulted in a dramatic increase in mitotic index after 90 min. When the cultures were washed free of the drugs, mitosis was completed and the cells progressed into G 1 at approximately the same rate. Further similarity of rotenone-arrested cells to Colcemid-induced mitotic inhibition was apparent at the ultrastructural level. Mitotic cells treated by either drug contained monopolar spindles with chromosomes grouped around centriole pairs near the cell center. Occasional microtubules were seen near the kinetochore and centrioles. These observations, along with the fact that rotenone inhibited the binding of ³H-colchicine to isolated bovine brain tubulin, suggested that rotenone inhibited mitosis by binding directly to tubulin to prevent microtubule assembly.     

Cortical differentiation of the animal pole during maturation division in fertilized eggs of Tubifex (Annelida,Oligochaeta): I. Meiotic apparatus formation

The fine structure of the animal pole cortex is examined in the fertilized Tubifex egg undergoing the formation of the second meiotic apparatus (MA). The fully formed MA which orients its axis at right angles to the surface is found at the animal pole about 40 min after formation of the first polar body. It is composed of a spindle and asters at its poles; a centriole is found in the inner aster, but not in the peripheral aster adjacent to the surface. During the formation of the MA, the animal pole surface is lined with a 0.15-μm-thick, electron-dense cortical layer, which is rich in microfilaments. The arrangement of the filaments in the layer changes from a parallel array to a meshwork with progressive formation of the MA. Microtubules of the peripheral aster terminate in the cortical layer. When a jet stream of glycerol/dimethyl sulfoxide solution is applied to an egg fragment glued on a polylysine-coated coverslip, an egg cortex-MA complex is isolated on the coverslip; the MA appears to be tethered to the egg surface by the structural connection between the filamentous cortical layer and microtubules of the peripheral aster. Cytochalasin B (50 μg/ml), when administrated at early phase of the MA formation, does not show any effect on the structure of the cortical layer and the MA; however, if eggs shortly before the termination of the first polar body formation are immersed in the same test solution, the cortical layer of the animal pole becomes thinner, and the filamentous material is not observed in it. Furthermore, in these eggs, the peripheral aster and the spindle are not structurally discernible because of the suppression of microtubule assembly, whereas microtubules on kinetochores and in the inner aster are normally developed. These results are discussed in relation to the role of the animal pole cortex in fixing of the MA to the egg surface and in forming of the MA.     

Taxol inhibits the nuclear movements during fertilization and induces asters in unfertilized sea urchin eggs

Taxol blocks the migrations of the sperm and egg nuclei in fertilized eggs and induces asters in unfertilized eggs of the sea urchins Lytechinus variegatus and Arbacia punctulata. Video recordings of eggs inseminated in 10 microM taxol demonstrate that sperm incorporation and sperm tail motility are unaffected, that the sperm aster formed is unusually pronounced, and that the migration of the egg nucleus and pronuclear centration are inhibited. The huge monopolar aster persists for at least 6 h; cleavage attempts and nuclear cycles are observed. Colcemid (10 microM) disassembles both the large taxol-stabilized sperm aster in fertilized eggs and the numerous asters induced in unfertilized eggs. Antitubulin immunofluorescence microscopy demonstrates that in fertilized eggs all microtubules are within the prominent sperm aster. Within 15 min of treatment with 10 microM taxol, unfertilized eggs develop numerous (greater than 25) asters de novo. Transmission electron microscopy of unfertilized eggs reveals the presence of microtubule bundles that do not emanate from centrioles but rather from osmiophilic foci or, at times, the nuclear envelope. Taxol-treated eggs are not activated as judged by the lack of DNA synthesis, nuclear or chromosome cycles, and the cortical reaction. These results indicate that: (a) taxol prevents the normal cycles of microtubule assembly and disassembly observed during development; (b) microtubule disassembly is required for the nuclear movements during fertilization; (c) taxol induces microtubules in unfertilized eggs; and (d) nucleation centers other than centrioles and kinetochores exist within unfertilized eggs; these presumptive microtubule organizing centers appear idle in the presence of the sperm centrioles.     

Protoplast isolation,development, and regeneration in different strains ofPilayella littoralis (L.) Kjellm. (Phaeophyceae)

Summary Using an antibody against tyrosinated tubulin and epifluorescence microscopy, mitosis was studied in three different microvessel endothelial cell types recently isolated from bovine corpus luteum. Dividing cells were flat and at certain stages individual microtubules could be followed for considerable lengths. The structure of the spindle apparatus and the course of mitosis were conventional. Microtubule asters were small from prophase until metaphase in all three cell types. However, whereas in two cell types telophase asters remained inconspicuous, prominent asters, of mostly straight microtubules, formed in telophase cells of a third cell type. Thus, aster size is heterogeneous between different endothelial cell types. Large microtubule asters are not regularly found in dividing cultured mammalian cells. The microendothelial cell types present themselves as appropriate systems for spindle research and especially for the study of aster microtubule dynamics and function.     

Structural and functional effects of hydrostatic pressure on centrosomes from vertebrate cells

In an attempt to better understand the role of centrioles in vertebrate centrosomes, hydrostatic pressure was applied to isolated centrosomes as a means to disassemble centriole microtubules. Treatments of the centrosomes were monitored by analyzing their protein composition, ultrastructure, their ability to nucleate microtubules from pure tubulin, and their capability to induce parthenogenetic development of Xenopus eggs. Moderate hydrostatic pressure (95 MPa) already affected the organization of centriole microtubules in isolated centrosomes, and also impaired microtubule nucleation. At higher pressure, the protein composition of the peri-centriolar matrix (PCM) was also altered and the capacity to nucleate microtubules severely impaired. Incubation of the treated centrosomes in Xenopus egg extract could restore their capacity to nucleate microtubules after treatment at 95 MPa, but not after higher pressure treatment. However, the centriole structure was in no case restored. It is noteworthy that centrosomes treated with mild pressure did not allow parthenogenetic development after injection into Xenopus eggs, even if they had recovered their capacity to nucleate microtubules. This suggested that, in agreement with previous results, centrosomes in which centriole architecture is impaired, could not direct the biogenesis of new centrioles in Xenopus eggs. Centriole structure could also be affected by applying mild hydrostatic pressure directly to living cells. Comparison of the effect of hydrostatic pressure on cells at the G1/S border or on the corresponding cytoplasts suggests that pro-centrioles are very sensitive to pressure. However, cells can regrow a centriole after pressure-induced disassembly. In that case, centrosomes eventually recover an apparently normal duplication cycle although with some delay.