Key Words: centrosome, centrioles, GFP, centrin, motility
We have generated several stable cell lines expressing GFP-labeled centrin. This fusion protein becomes concentrated in the lumen of both centrioles, making them clearly visible in the living cell. Time-lapse fluorescence microscopy reveals that the centriole pair inherited after mitosis splits during or just after telophase. At this time the mother centriole remains near the cell center while the daughter migrates extensively throughout the cytoplasm. This differential behavior is not related to the presence of a nucleus because it is also observed in enucleated cells. The characteristic motions of the daughter centriole persist in the absence of microtubules (Mts), or actin, but are arrested when both Mts and actin filaments are disrupted. As the centrioles replicate at the G1/S transition the movements exhibited by the original daughter become progressively attenuated, and by the onset of mitosis its behavior is indistinguishable from that of the mother centriole. While both centrioles possess associated -tubulin, and nucleate similar number of Mts in Mt repolymerization experiments, during G1 and S only the mother centriole is located at the focus of the Mt array. A model, based on differences in Mt anchoring and release by the mother and daughter centrioles, is proposed to explain these results.
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Introduction |
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During mitosis, animal cells inherit a single centrosome that contains a pair of centrioles, each of which is associated with a cloud of pericentriolar material (reviewed in
Centrioles replicate during S phase concurrent with DNA replication. During this time a small "procentriole" bud forms adjacent to the proximal wall of each parenting centriole, which then gradually elongates (
The function(s) of the centrioles within the centrosome remain unclear, although recent results demonstrate that they are required for organizing the centrosomal components into a single stable structure (
The centrosomal components involved in microtubule (Mt)1 nucleation (e.g., -tubulin, HsSpc98p) are localized within the pericentriolar material (PCM) associated with each centriole (
Centrin is a small (20 kD) protein that concentrates within the centriole distal lumen (
In this study, we used time-lapse fluorescence microscopy and serial section EM to define, for the first time, the in vivo behavior of centrioles during the vertebrate somatic cell cycle. The data reveal that the mother and daughter centrioles differ in their behavior and in their respective contributions to forming the interphase Mt array.
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Materials and Methods |
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Cell Culture, Cloning, and Synchronization
L929, NIH 3T3, and HeLa-B cells were grown in DME medium (GIBCO) supplemented with 10% fetal calf serum.
Centrin cDNA was sub-cloned in the pEGFP-N1 vector (Clontech) and cells were transformed by electroporation. Stable clones expressing the centrin/GFP fusion protein were then isolated from each of the three parental cell lines, by using the limited dilution method in the presence of 500 µg/ml G418. Multiple independent clones, expressing ~20 times the endogenous level of centrin, were kept for each of the three parental cell lines. The centrioles in all of the clones exhibited a similar behavior to that described here.
Synchronization in early G1 was accomplished by a single thymidine block of 20 h (5 mM thymidine for L929 and 2 mM for HeLa). Mitotic cells were then collected by shakeoff 8 or 20 h after releasing the block (for L929 and HeLa, respectively). Cells were then replated on coverslips and used 2 h later. Synchronization at the G1/S border was accomplished using the double-thymidine block technique. To determine the duration of S and G2 we incubated cells at various times after thymidine washout with 30 µM BrdU for 15 min and then analyzed them by FACS®. The maximum content of S cells (by BrdU incorporation) was reached 14 h after release. The maximum number of G2 cells (double DNA content and no BrdU incorporation) was observed ~5 h after release in L929 cells and 910 h in HeLa cells.
Cell Enucleation
Enucleation was performed as described by
Drug Treatments
We used a combination of ND (5 µM) and cold (40 min on ice) to depolymerize Mts. This treatment depolymerizes even the most stable Mts in L929 cells, and these do not reassemble when the cells are subsequently incubated in warm media containing 5 µM ND. To disrupt actin filaments cells were treated with 3 µg/ml of CD for 30 min. Latrunculin A (Molecular Probes) was used at 1 µM and added just before initiating observations. Butanedione Monoxime (BDM; Sigma) was used at 20 µM and cells were observed 30 min after treatment.
Microinjection of Rhodamine-Tubulin and Incorporation of the Shiga Toxin B Fragment
Rhodamine-tubulin (catalog number T331M; TEBU, Inc.) was microinjected using an automatic microinjector (Eppendorf). The B fragment of Shiga toxin was incorporated into cells using the method described by
Microscopy and Data Processing
For time-lapse imaging cells were plated on #1 1/2 coverslips (L929 cells were plated on coverslips coated with collagen and fibronectin to induce cell flattening). For brief (<1 h) experiments cells were maintained at 37°C in sealed chambers containing complete phenol red-free culture medium supplemented with 20 mM Hepes. Open chambers equilibrated in 5% CO2 and maintained at 37°C were used for longer experiments. Rhodamine-labeled cells were mounted in hermetically sealed chambers containing Oxyrase and lactic acid (
Time-lapse Z-sequences were collected on a Leica DMIRBE microscope controlled by Metamorph software (Universal Imaging). This microscope was equipped with a piezoelectric device for rapid and reproducible focal changes, a 100x 1.4 NA Plan Apo lens, and a cooled CCD camera (MicroMax 5 MHz; Roper Scientific). The final magnification on the camera chip was 84 nm/pixel. Using a DG4 illumination device (DeMey, J., and J.B. Sibarita, manuscript in preparation) we could collect a Z-sequence through an entire cell, of two different wavelengths, in under 2 s.
As a rule 610 sequential Z-axis images were collected in 0.5-µm steps every 230 s. However, as the cell rounded during late G2 and mitosis it was often necessary to collect as many as 30 Z-axis images. Centriole tracking was performed automatically by Metamorph in maximal-intensity projections computed from the original three-dimensional data sets.
Same Cell Correlative Video, Immunofluorescence, and/or Electron Microscopy
To identify cells followed in vivo thought subsequent preparative procedures we cultured them on Cellocate coverslips (Eppendorf). For indirect immunofluorescence studies they were then rapidly extracted with 0.2% NP-40 in BRB80 (80 mM KPIPES, pH 6.8, 1 mM MgCl2; 1 mM EGTA) for 30 s, followed by fixation in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde in PBS for 3 min. After reducing free aldehydes with 0.1% NaBH4 in PBS, the coverslips were incubated in primary antibodies followed by the appropriate secondary antibody coupled to either cyanine 3 (red channel; Jackson ImmunoResearch) or AMKA (blue channel; Jackson ImmunoResearch). The green channel was used to record the GFP signal which was preserved by our fixation protocol.
After immunostaining, cells that had been followed in vivo were relocated and imaged on a Leica DMRXA microscope. Image stacks (200-nm steps) were recorded using a piezoelectric objective positioning device and a MicroMAx CCD camera (Princeton Instruments). With a 100x 1.4 NA objective the final magnification on the chip was 67 nm/pixel. All centrin, ninein and -tubulin images shown in this paper are maximal intensity projections, while Mts are presented as self-luminous reconstructions.
Serial section EM of cells previously followed in vivo was performed as detailed by
Supplemental Material
Video supplements for Fig 2, Fig 4, Fig 5, and Fig 9 are at http://www.jcb.org/cgi/content/full/149/2/317/DC1. To ensure a good resolution of the movies, please check that the monitor of your computer is set on millions of colors or true colors (32 bits). All of the videos correspond to cells or cytoplasts displayed on figures or from which data were extracted, except for Fig 3 for which a movie showing a G2 cytoplast is added. Refer to the respective figure legend for further explanation.
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Video 1 corresponds to the cell shown on Fig 2 B.
Videos 2 and 3 correspond to the S and G2 cells shown on Fig 3 and video 4 is a G2 cytoplast shown for comparison: the behavior of the diplosomes in G2 is the same in cells and in cytoplasts.
Video 5 corresponds to the G1 cytoplast shown on Fig 4 A, rotated 90°. Videos 6 and 7 correspond to the G1 cytoplasts as shown in Fig 4 injected with rhodamine-tubulin or having incorporated Shiga toxin B fragment coupled with rhodamine, respectively.
Videos 810 correspond to the G1 cytoplasts treated with nocodazole and cold, cytochalasin D, or both, respectively, and whose centrioles trajectories are shown on Fig 5 B. Video 11 shows videos 810 one after the other in the same file, thus making easier the comparison between different treatments.
Videos 12 and 13 correspond to the fields containing three G1 cytoplasts shown respectively on the left and on the right in the left panel of Fig 9. Video 14 corresponds to the G1 cytoplast containing four centrioles shown on the right panel of Fig 9.
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Results |
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Distribution of GFP-Centrin
We established several stable (>50 passages) cell lines that express centrin as a NH2-terminal fusion with GFP. Multiple clones that exhibit growth rates similar to the parental cell lines were isolated from HeLa, NIH-3T3, and L929 cells. In this report, we illustrate our finding using data obtained from L929 clones because the mother and daughter centrioles in these cells are sufficiently separated during G1 so that their individual behavior can be easily observed. In G1 HeLa cells, centrioles remain relatively close to each other while in NIH 3T3 they are usually separated by distances greater than those seen in L929 cells. Despite these differences, the centrioles behave the same in all three cell lines. Thus, the phenomena described here are exhibited by a number of different vertebrate somatic cell lines.
In all clones the distribution of GFP-centrin was very similar to that previously described by indirect immunofluorescence (-tubulin, not shown).
In an asynchronous population the number of centrin-GFP dots varied from cell to cell. Most cells contained two individual dots, positioned at variable distances from one another, but some contained two pairs of dots. In the latter cells the two dots comprising each pair often differed in their intensity. Correlative LM/EM studies confirmed that individual dots seen at the LM level were single centrioles, while the paired dots corresponded to orthogonally oriented mother/daughter centriole pairs (i.e., a diplosome; not shown).
To determine how centrin/GFP labeling changes with respect to the centrosome cycle we investigated the centrin/GFP distribution in synchronized cell populations. When cells were synchronized by mitotic shakeoff and replated for 2 h, 94% of cells (n >200) contained two individual dots. By contrast when cells were synchronized by a double-thymidine block and then allowed to progress into S-phase, 87% cells contained two pairs of dots with one member of each pair significantly brighter than the other. Finally, when cells were released from a double-thymidine block and allowed to progress into G2, 69% of cells contained two pairs of dots each of which was approximately equal in intensity, 25% still exhibiting the S-phase pattern. It is noteworthy that the distance separating the two dots comprising each diplosome increased as the cells progressed through S-G2, likely reflecting the elongation of the pro-centriole. From these results we conclude that centrin/GFP can be used as a live cell marker for the formation and maturation of individual centrioles, and is thus a very good marker of cell cycle progression. The typical distribution of centrin/GFP during the cell cycle is summarized in Fig 1 (top row).
To investigate the behavior of the two centrioles within the centrosome, we used both whole cells and enucleated cells (cytoplasts). Cytoplasts were used to determine if the behavior of centrioles is influenced by the presence of nucleus. Additionally, cytoplasts are very flat and immobile, facilitating observation and analysis of centriole movements. In our hands, cytoplasts were viable for >50 h. The typical distribution of centrin/GFP in cytoplasts during different periods of cell cycle is summarized in Fig 1 (bottom row).
Centriole Behavior during the Cell Cycle
During mitosis each daughter cell inherits a single diplosome consisting of a closely associated mother and daughter centriole pair. We found that the mother and daughter centrioles comprising the diplosome separate into individual units during or just after telophase, well before the completion of cytokinesis (Fig 2). Most of the time, that separation correlates with cell spreading. Once separated the mutual distance between the two centrioles fluctuates, but the average value increases steadily up to a few microns. From that moment on and up to the completion of cytokinesis (i.e., breaking of the midbody), the two centrioles exhibit dramatically different behaviors. One remains almost stationary and near the geometrical center of the forming daughter cell while the other is wandering around and eventually migrates around the nucleus and towards the forming midbody (Fig 2 C). Although the movement of one centriole toward the midbody sometimes occurred only in one of the daughter cells, it was usually observed in both, just before the completion of cytokinesis.
We then asked if this differential behavior persists throughout interphase. We found that during G1 one centriole remained relatively stationary while the other wandered throughout the cytoplasm (Fig 3). This exaggerated motion of one centriole was greatly attenuated during S and G2 when the centrioles were in the process of replicating (Fig 3). However, the two forming diplosomes still exhibited different behaviors during S and G2; one remained stationary while the other exhibited rocking motions even when translational movements were suppressed in G2 (Fig 3S and G2). This movement ceased during late G2/prophase before nuclear envelope breakdown.
Since the centrosome has been shown to be tightly associated with the nucleus in some cells (
We next asked how Mts were distributed around the motile and nonmotile centrioles. To answer this question we followed living cytoplasts after having either microinjected them with rhodamine-labeled tubulin or incubated them with a living marker for the Golgi apparatus (the internalized B fragment of the Shiga toxin whom internalization leads, at the steady-state to an accumulation in the Golgi;
Next we conducted a serial section EM analysis of cells previously followed in vivo to determine which of the centrioles in our cells was the mother (Fig 4 C). In all seven cytoplasts containing one motile and one stationary centriole, the stationary centriole was always found to be the mother based on the presence of sub-distal appendages. By contrast, the centrioles in adjacent cytoplasts, in which all GFP-centrin dots remained relatively stationary, were always found to be replicating (n = 5).
Effects of Nocodazole and Cytochalasin D on Centriole Behavior during G1
To determine if the differential behavior of mother and daughter centrioles during G1 depends on the presence of Mts and/or actin filaments we analyzed centriole movements in G1 cytoplasts treated with either ND (5 µM), CD (3 µg/ml), or both. For these analyses we recorded time-lapse sequences at high temporal resolution (1 frame every 2 s). Even at this high framing rate we could follow centriole movement for ~10 min before the GFP signal photobleached significantly. For these studies we chose to analyze two parameters of centriole behavior: the number of fast movement periods per unit of time, and the area covered by centriole motions (i.e., the root mean square of the excursion: [< (X - < X >)2 > . < (Y - < Y >)2 >]1/2). The latter parameter is independent of the framing rate and allows one to determine if the centriole exhibited persistent motion.
The results of these studies are summarized in Fig 5. In most untreated cytoplasts the daughter centriole exhibited brief periods of rapid movement (numbers in Fig 5 A) interrupted by longer periods of slower motion. The typical trajectory of the two centrioles is illustrated in Fig 5 A. When compared with untreated cytoplasts, the trajectories of both centrioles became much smoother when Mts were completely disassembled by ND (Fig 5 B). Under these conditions the centrioles no longer exhibited sudden direction changes, but the daughter centriole still remained more motile than the mother centriole. Even after a 4-h incubation in ND most mother centrioles remained positioned near the geometrical center of the cell. Remarkably, the mother and the daughter centrioles had very correlated movements (Fig 5 C).
The extensive motion of daughter centrioles persisted in CD-treated cytoplasts, but the movements were more abrupt compared with untreated cytoplasts (Fig 5 B). The same effect was observed when cytoplasts were treated with Latrunculin A (actin inhibitor) or BDM (a myosin inhibitor). In BDM-treated cytoplasts this effect was even more prominent, suggesting that the dependency of centriole movement on microfilaments is mediated by myosin (data not shown).
When Mts and microfilaments were both disrupted by the combined action of ND and CD all centrioles ceased moving and exhibited only brief tumbling motions (Fig 5 B).
The Respective Contribution of the Mother and Daughter Centrioles in Forming the Interphase Mt Array
As noted above, we found that the nonmotile mother centriole was associated with a typical radial array of Mts whereas the motile daughter centriole had either no obvious direct interactions with the Mt array, or was associated with only a few Mts (Fig 4 B). The different density of Mts associated with the mother and daughter centrioles in our cells could be due to differences in their nucleating potential and/or their ability to anchor Mts. In an attempt to differentiate between these possibilities, we determined the relative content between the mother and daughter centrioles of the Mt-nucleating protein -tubulin and the Mt-anchoring protein ninein (
-tubulin associated with each centriole (or diplosome during S-G2) was very similar. However, ninein was associated primarily if not exclusively with the mother centriole during G1, but with both diplosomes during S-G2 (Fig 6).
As shown in Fig 7 A, only the ninein-containing centriole maintained an aster of Mts after treatment with low doses of ND. In agreement with -tubulin immunolocalization, both centrioles re-nucleated a comparable number of Mts after complete disassembly (Fig 7 B). Similar numbers of short Mts were found to be arranged radially around both centrioles 2 min after washing ND-treated cytoplasts. By contrast, Mt arrays were associated only with the ninein-containing mother centrioles 15 min after ND washout. After 15 min regrowth, a common and conspicuous radial array of Mts was observed if the two centrioles were close to each other (Fig 7 B, top), but some "free" cortical Mts were also observed when the two centrioles were split apart (Fig 7 B, bottom). Moreover, many free short Mts were observed at 5 min regrowth when centrioles were split.
Mts Organization and Centriole Behavior in Cytoplasts Containing One or More than Two Centrioles
To further investigate the specific contribution of the daughter and the mother centrioles to the formation of the interphase Mt array, we enucleated synchronized G1 cells in the presence of both ND and CD which produced many cytoplasts that contained only one centriole (either mother or daughter) or no centrioles at all (
This experiment also demonstrates that the mother centriole does not simply out-compete the daughter for those components (e.g., ninein) required for anchoring Mts. Rather, the younger daughter is not yet competent to recruit these components from the cytoplasmic pool and cannot mimic the mother in her absence.
In cytoplasts containing a single centriole, the centriole either remained relatively stationary or wandered throughout the cell (Fig 9). An IMF analysis of these cells revealed that the stationary centriole was always associated with ninein and a Mt array, whereas the motile centriole lacked both (data not shown). Nonmotile centrioles were always positioned near the geometric center of cytoplasts containing only one centriole, whereas motile ones were often located near the periphery. These latter centrioles exhibited motions roughly parallel to the cell edge, seldom directed towards the cell center. This behavior differs from that found in cytoplasts containing a full complement of centrioles (i.e., one mother and one daughter) in which the daughter centriole often exhibits motions towards and away from the centrally located mother. Finally, in cytoplasts containing two mother and two daughter centrioles, two remained relatively motionless while two were highly motile (Fig 9, right panel). As in our previous studies, the only centrioles associated with radial arrays of Mts were the immotile (mother) centrioles (not shown).
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Discussion |
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Many observations of the centrosome in the past, either in situ or after isolation, have suggested a great complexity of this organelle. But most often obtained on fixed cells, structural features have been difficult to integrate into a coherent and reliable model of the centrosome organization. Here, by observing the centrosome in living cells, and in spite of a much lower resolution than that of EM, one could get at a more integrated view of the centrosome. Our work reveals novel features of the centrosome dynamics. First, splitting of centrioles in each postmitotic centrosome, which corresponds to the moment when orthogonal orientation is lost, occurs soon after anaphase, long before cytokinesis is completed. Second, the two centrioles of postmitotic cells demonstrate differential movements, one maintaining a central and stable location, whereas the other has a wide and eccentric trajectory. Third, the motile centriole progressively slows down from the onset of centriole duplication at the G1/S border, up to late G2; although maintaining a stable location within the cell once duplicated (in G2), the former motile centriole and its associated pro-centriole conserve more independence with respect to the surrounding cytoplasm until the onset of mitosis where it stops rocking completely. We also demonstrate a specific contribution of each centriole to the activity of the centrosome: both centrioles nucleate Mts but only the mother centriole anchors them. A general conclusion consistent with all the observations reported in this work is that the behavior of individual centrioles is maturation-dependent, correlated with the generation process of these organelles. In other words, one centriole cannot replace the other one in a centrosome.
The Two Centrioles-Centrosome: A Constitutive Generational Asymmetry of the Centriole Pair Necessary for Centrosome Function
Centriole Movements and the Interaction of the Centrosome with the Surrounding Cytoplasm.
The analysis of the centrosome dynamics in vivo suggests the existence of an intercentriole link. Even when Mts were totally depolymerized, there was still a strong correlation between the smooth movements of the two centrioles, even when they were microns apart (see Fig 5 C). This is in agreement with the observation that, once isolated from cultured cells, centrosomes are always composed of the two centrioles associated with a complex filament network which seems to link them at the proximal ends (
Wide excursions of the daughter centriole are mainly due to acto-myosin activity (see quantification in Fig 5 B). This suggests that the motile centriole could be driven either by global cytoplasmic actin-dependent movements, or by a direct interaction with the acto-myosin system. Recent reports favor the last interpretation, as an accumulation of the myosin V isoform at the centrosome has been demonstrated (
A maturation-dependent anchorage of the centrosome within the cytoplasm, distinct from the Mt-dependent centering of the mother centriole, might involve the centrosomal matrix: when Mts were depolymerized in S or G2, the daughter centriole did not recover the motility observed in G1 (not shown). Interactions of the centrosomal matrix with other cytoskeletal components such as intermediate filaments or membranes will deserve further characterization. One may recall here that cells possessing a primary cilium provide an example of how the mother centriole could be anchored independently of the Mts. The distal end of the mother centriole interacts directly with the plasma membrane through its distal appendages. These structures which are observed in centrosomes isolated from cells which never grow a primary cilium (
The centering of the mother centriole depends, however, on its Mt-anchoring activity. When Mts were completely depolymerized over several hours, the immotile centriole could be eventually found away from the cytoplast center. The centering capacity of an aster of Mts nucleated either from a bead bearing Mt seeds, or from isolated centrosomes, has been demonstrated in vitro in an artificial cell (
The characteristic ninein staining of the mother centriole (see Fig 6) might correspond to sub-distal appendages of the mother centriole (see
The Release and Capture Model and the Regulation of Release Versus Capture by Centriole Splitting.
All the data presented in this work, particularly the strikingly different Mt arrays observed in cytoplasts containing either the mother or the daughter centriole (see Fig 8), can be accounted for by the working model depicted in Fig 10. It proposes that Mts are nucleated near centrioles (Fig 7 B), then released and transported either to the ninein-containing complexes associated with the mother centriole or to other anchoring sites, mainly near or at the plasma membrane. Released Mts would be transported by dynein motors for example, as it was shown that perturbing dynactin activity greatly disturbed the organization of the Mt array. It has also been proposed that the dynein/dynactin complex could have a role in anchoring the Mts at the centrosome (
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The release and capture model, first proposed by
Calcium-dependent regulation of the centrosome matrix (
From the extensive data on fixed cells from various cell lines in the literature, one can observe that there is often a correlation between the extent of centriole splitting and the amount of Mts involved in the aster. All cell lines in which most of the Mts are participating in the aster, such as PtK1, CV1, COS, have two centrioles near one another at the center of the aster. Another way to control Mt organization in differentiated cells could be to redistribute anchoring proteins from the centrosome to other sites (
Perspectives
The implication of the centrosome in cell motility is controversial as it has been shown that pieces of different kinds of cells can polarize and migrate (
Another situation in which the specific activity of each centriole might be required is cytokinesis during which a peculiar behavior of the centrosome organelle has been observed (
In conclusion, our work reveals an unexpected complexity in the behavior of the centrosome. The analysis of the centriole movements and of the effect of cytoskeletal drugs has implications for the integration of the centrosome organelle within the cell. A specific role for each centriole in the Mt organizing activity of the centrosome is also demonstrated, which strongly suggests that centriole splitting could be a way to control the cellular array of Mts. This could have important implications for understanding how the centrosome activity and the plasma membrane activity could be coupled with each other during cell locomotion or cell division.
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Footnotes |
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The online version of this article contains supplemental material.
Dr. Michel Bornens, Institut Curie, Section Recherche, UMR 144 du CNRS, 26, rue d'ULM, 75248 Paris Cedex 05, France. Tel.: 01 42 34 64 20. Fax: 01 42 34 64 21. E-mail: mbornens{at}curie.fr
1 Abbreviations used in this paper: CD, cytochalasin D; EM, electron microscopy; GFP, green fluorescent protein; LM, light microscopy; Mts, microtubules; ND, nocodazole; PCM, pericentriolar material.
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Acknowledgements |
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We thank Jan de Mey and Jean-Baptiste Sibarita for the setting of the video recording device which was very helpful; Olivier Cardoso and Virginie Emselem for helpful discussions on the analysis of the centriole movements; Andrew Matus for well-advised commentaries on this work; Yann Abraham for the GFP constructs and Claude Celati for her help in the cloning of stable cell lines; Bruno Goud and Ludger Johannes for the Shiga toxin B fragment; and Anne-Marie Tassin, Guy Keryer, Bruno Goud, Andrew Matus, and Don Cleveland for critical reading of the manuscript.
This work is supported by Centre National de la Recherche Scientifique, Institut Curie, Direction des Recherches, Etudes et Techniques and Association pour la Recherche sur le Cancer. It was also supported, in part, by the National Institute of Health grants GMS R01 59363 to A. Khodjakov and GMS R01 40198 to C.L. Rieder.
Submitted: 21 December 1999
Revised: 28 February 2000
Accepted: 2 March 2000
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References |
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