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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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Related in: MedlinePlus

Centrin aggregates are not associated with microtubules during the first cell cycle, but become positioned inside of microtubule foci after they coalesce into a common structure in the second cell cycle. (A) GFP/centrin, γ-tubulin, and α-tubulin distribution in a cell fixed during first cell cycle (∼49 h after centrosome ablation and 24 h after formation of detectable centrin aggregates). Although some of the centrin/GFP aggregates also contain γ-tubulin, none of them is associated with microtubule foci (arrows). (B and C) Two progeny of a cell born without a centrosome that were fixed in the second cell cycle, after the coalescence of the de novo–formed centrioles (∼48 h after centrosome ablation; 20 h after formation of centrin aggregates; and 15 h after second mitosis). In contrast to the centrin aggregates during the first cell cycle (A), de novo–formed centrioles after mitosis reside inside of microtubule foci and are associated with large amount of γ-tubulin (arrows). Bar, 10 μm. Maximal-intensity projections.
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fig4: Centrin aggregates are not associated with microtubules during the first cell cycle, but become positioned inside of microtubule foci after they coalesce into a common structure in the second cell cycle. (A) GFP/centrin, γ-tubulin, and α-tubulin distribution in a cell fixed during first cell cycle (∼49 h after centrosome ablation and 24 h after formation of detectable centrin aggregates). Although some of the centrin/GFP aggregates also contain γ-tubulin, none of them is associated with microtubule foci (arrows). (B and C) Two progeny of a cell born without a centrosome that were fixed in the second cell cycle, after the coalescence of the de novo–formed centrioles (∼48 h after centrosome ablation; 20 h after formation of centrin aggregates; and 15 h after second mitosis). In contrast to the centrin aggregates during the first cell cycle (A), de novo–formed centrioles after mitosis reside inside of microtubule foci and are associated with large amount of γ-tubulin (arrows). Bar, 10 μm. Maximal-intensity projections.

Mentions: It has been previously shown that the motilities of the daughter and mother centrioles during G1 correlate with their ability to organize microtubule networks. Although both daughter and mother centrioles are capable of nucleating similar numbers of microtubules, only the mother can organize microtubules into a typical radial array (Piel et al., 2000). As a result, mother centrioles always reside inside of microtubule foci, whereas the daughters, at least in some cell types (e.g., HeLa and L-929), are not associated with microtubule asters (Piel et al., 2000). We investigated at which point the de novo–formed centrioles become associated with microtubule foci. Immunofluorescence analysis revealed that, not surprisingly, the moving centrin aggregates/centrioles of the first interphase were not associated with microtubules, even though some of them were associated with bona fide PCM components, such as γ-tubulin (Fig. 4 A). During the first cell cycle, the interphase microtubule array in cells born without centrioles did not converge on common focal points and were instead randomized, showing only loose concentration at the perinuclear region. Importantly, this difference in microtubule organization was observed even in those cells where the duration of first cell cycle was for some reason prolonged, something that occurs with equal frequency in cells born with and without centrosomes.


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)

Centrin aggregates are not associated with microtubules during the first cell cycle, but become positioned inside of microtubule foci after they coalesce into a common structure in the second cell cycle. (A) GFP/centrin, γ-tubulin, and α-tubulin distribution in a cell fixed during first cell cycle (∼49 h after centrosome ablation and 24 h after formation of detectable centrin aggregates). Although some of the centrin/GFP aggregates also contain γ-tubulin, none of them is associated with microtubule foci (arrows). (B and C) Two progeny of a cell born without a centrosome that were fixed in the second cell cycle, after the coalescence of the de novo–formed centrioles (∼48 h after centrosome ablation; 20 h after formation of centrin aggregates; and 15 h after second mitosis). In contrast to the centrin aggregates during the first cell cycle (A), de novo–formed centrioles after mitosis reside inside of microtubule foci and are associated with large amount of γ-tubulin (arrows). Bar, 10 μm. Maximal-intensity projections.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171814&req=5

fig4: Centrin aggregates are not associated with microtubules during the first cell cycle, but become positioned inside of microtubule foci after they coalesce into a common structure in the second cell cycle. (A) GFP/centrin, γ-tubulin, and α-tubulin distribution in a cell fixed during first cell cycle (∼49 h after centrosome ablation and 24 h after formation of detectable centrin aggregates). Although some of the centrin/GFP aggregates also contain γ-tubulin, none of them is associated with microtubule foci (arrows). (B and C) Two progeny of a cell born without a centrosome that were fixed in the second cell cycle, after the coalescence of the de novo–formed centrioles (∼48 h after centrosome ablation; 20 h after formation of centrin aggregates; and 15 h after second mitosis). In contrast to the centrin aggregates during the first cell cycle (A), de novo–formed centrioles after mitosis reside inside of microtubule foci and are associated with large amount of γ-tubulin (arrows). Bar, 10 μm. Maximal-intensity projections.
Mentions: It has been previously shown that the motilities of the daughter and mother centrioles during G1 correlate with their ability to organize microtubule networks. Although both daughter and mother centrioles are capable of nucleating similar numbers of microtubules, only the mother can organize microtubules into a typical radial array (Piel et al., 2000). As a result, mother centrioles always reside inside of microtubule foci, whereas the daughters, at least in some cell types (e.g., HeLa and L-929), are not associated with microtubule asters (Piel et al., 2000). We investigated at which point the de novo–formed centrioles become associated with microtubule foci. Immunofluorescence analysis revealed that, not surprisingly, the moving centrin aggregates/centrioles of the first interphase were not associated with microtubules, even though some of them were associated with bona fide PCM components, such as γ-tubulin (Fig. 4 A). During the first cell cycle, the interphase microtubule array in cells born without centrioles did not converge on common focal points and were instead randomized, showing only loose concentration at the perinuclear region. Importantly, this difference in microtubule organization was observed even in those cells where the duration of first cell cycle was for some reason prolonged, something that occurs with equal frequency in cells born with and without centrosomes.

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