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

La Terra S, English CN, Hergert P, McEwen BF, Sluder G, Khodjakov A - J. Cell Biol. (2005)

Bottom Line: Here, we show that removal of resident centrioles (by laser ablation or needle microsurgery) does not impede cell cycle progression in HeLa cells.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.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.

ABSTRACT
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.

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Centriole de novo formation occurs in HeLa cells arrested in S but not in G1. (A) Resident centrosome was ablated (arrows, compare 00:00 and 00:01) in a cell pretreated with 5 μM lovastatin for ∼15 h. Time lapse recording of this cell revealed no formation of centrin/GFP aggregates for 46 h. (B) Similar procedure to A was followed, except this cell was pretreated with 2 mM hydroxyurea. Time lapse recording revealed that ∼5 h after ablation of the resident centrioles centrin/GFP, aggregates formed in the cytoplasm (arrows, 05:30). These aggregates moved continuously in the cytoplasm while their intensity gradually increased (05:30–50:00). Time is shown in hours:minutes. Bar, 5 μm.
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fig6: Centriole de novo formation occurs in HeLa cells arrested in S but not in G1. (A) Resident centrosome was ablated (arrows, compare 00:00 and 00:01) in a cell pretreated with 5 μM lovastatin for ∼15 h. Time lapse recording of this cell revealed no formation of centrin/GFP aggregates for 46 h. (B) Similar procedure to A was followed, except this cell was pretreated with 2 mM hydroxyurea. Time lapse recording revealed that ∼5 h after ablation of the resident centrioles centrin/GFP, aggregates formed in the cytoplasm (arrows, 05:30). These aggregates moved continuously in the cytoplasm while their intensity gradually increased (05:30–50:00). Time is shown in hours:minutes. Bar, 5 μm.

Mentions: We found that formation of centrin aggregates did not occur after ablating resident centrioles in cells arrested during G1 (Fig. 6 A; n = 5). In contrast, cells arrested in S (Fig. 6 B; n = 5) consistently formed numerous centrin aggregates after the resident centrosome was laser ablated. These dots gradually increased in intensity until they were indistinguishable from normal centrioles. The kinetics of this intensity increase was similar to those observed in the cycling cells during the first cell cycle. EM analysis revealed that centrin aggregates developed into morphologically recognizable centrioles in S-arrested cells (n = 2). In one cell, we found that some centrin aggregates corresponded to structures that appeared to be intermediate stages of centriole formation. EM tomography reconstructions of three of the centrin aggregates in this cell revealed that one of the aggregates corresponded to an electron dense amorphous cloud, with just two microtubule blades present within the cloud. The other two centrin dots corresponded to more completed, although still abnormal, centrioles. These structures contained four microtubule blades in one case and six to seven in the other; however, the triplet blades were not properly organized into closed cylinders (Fig. 7 and Video 4, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1).


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

La Terra S, English CN, Hergert P, McEwen BF, Sluder G, Khodjakov A - J. Cell Biol. (2005)

Centriole de novo formation occurs in HeLa cells arrested in S but not in G1. (A) Resident centrosome was ablated (arrows, compare 00:00 and 00:01) in a cell pretreated with 5 μM lovastatin for ∼15 h. Time lapse recording of this cell revealed no formation of centrin/GFP aggregates for 46 h. (B) Similar procedure to A was followed, except this cell was pretreated with 2 mM hydroxyurea. Time lapse recording revealed that ∼5 h after ablation of the resident centrioles centrin/GFP, aggregates formed in the cytoplasm (arrows, 05:30). These aggregates moved continuously in the cytoplasm while their intensity gradually increased (05:30–50:00). Time is shown in hours:minutes. Bar, 5 μm.
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Related In: Results  -  Collection

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fig6: Centriole de novo formation occurs in HeLa cells arrested in S but not in G1. (A) Resident centrosome was ablated (arrows, compare 00:00 and 00:01) in a cell pretreated with 5 μM lovastatin for ∼15 h. Time lapse recording of this cell revealed no formation of centrin/GFP aggregates for 46 h. (B) Similar procedure to A was followed, except this cell was pretreated with 2 mM hydroxyurea. Time lapse recording revealed that ∼5 h after ablation of the resident centrioles centrin/GFP, aggregates formed in the cytoplasm (arrows, 05:30). These aggregates moved continuously in the cytoplasm while their intensity gradually increased (05:30–50:00). Time is shown in hours:minutes. Bar, 5 μm.
Mentions: We found that formation of centrin aggregates did not occur after ablating resident centrioles in cells arrested during G1 (Fig. 6 A; n = 5). In contrast, cells arrested in S (Fig. 6 B; n = 5) consistently formed numerous centrin aggregates after the resident centrosome was laser ablated. These dots gradually increased in intensity until they were indistinguishable from normal centrioles. The kinetics of this intensity increase was similar to those observed in the cycling cells during the first cell cycle. EM analysis revealed that centrin aggregates developed into morphologically recognizable centrioles in S-arrested cells (n = 2). In one cell, we found that some centrin aggregates corresponded to structures that appeared to be intermediate stages of centriole formation. EM tomography reconstructions of three of the centrin aggregates in this cell revealed that one of the aggregates corresponded to an electron dense amorphous cloud, with just two microtubule blades present within the cloud. The other two centrin dots corresponded to more completed, although still abnormal, centrioles. These structures contained four microtubule blades in one case and six to seven in the other; however, the triplet blades were not properly organized into closed cylinders (Fig. 7 and Video 4, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1).

Bottom Line: Here, we show that removal of resident centrioles (by laser ablation or needle microsurgery) does not impede cell cycle progression in HeLa cells.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.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus