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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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Reformation of centrin/GFP aggregates and their behavior in HeLa cells born without a centrosome. Selected GFP fluorescence frames (maximal-intensity projections) from a multimode time lapse recording (same recording as in Fig. 1). The cell born without centrosome exhibits only diffuse cytoplasmic centrin/GFP localization for ∼24 h (A and B, top). Then, small centrin/GFP aggregates appear in the cytoplasm (C). These aggregates move continuously in the cytoplasm (see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1). After mitosis, which occurs at 32 h (Fig. 1 E) one of the progeny inherits seven of these aggregates, whereas the other inherits just one aggregate (D). The seven aggregates continue to move in the cytoplasm for ∼7–8 h (E), and then they coalesce into a common structure that remains relatively stationary in the middle of the cell (F and G, and Fig. 1I). The centrioles in the sister cell that inherited a normal centrosome (bottom) exhibit expected behavior, because they replicate (D) and then are properly distributed between the daughter cells after mitosis (E). Times are shown in hours:minutes. Bar, 5 μm.
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fig2: Reformation of centrin/GFP aggregates and their behavior in HeLa cells born without a centrosome. Selected GFP fluorescence frames (maximal-intensity projections) from a multimode time lapse recording (same recording as in Fig. 1). The cell born without centrosome exhibits only diffuse cytoplasmic centrin/GFP localization for ∼24 h (A and B, top). Then, small centrin/GFP aggregates appear in the cytoplasm (C). These aggregates move continuously in the cytoplasm (see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1). After mitosis, which occurs at 32 h (Fig. 1 E) one of the progeny inherits seven of these aggregates, whereas the other inherits just one aggregate (D). The seven aggregates continue to move in the cytoplasm for ∼7–8 h (E), and then they coalesce into a common structure that remains relatively stationary in the middle of the cell (F and G, and Fig. 1I). The centrioles in the sister cell that inherited a normal centrosome (bottom) exhibit expected behavior, because they replicate (D) and then are properly distributed between the daughter cells after mitosis (E). Times are shown in hours:minutes. Bar, 5 μm.

Mentions: Approximately 20–25 h into the first cell cycle, a number of minuscule aggregates of centrin/GFP appeared in the cytoplasm of cells that were born without centrioles. Initially, these aggregates were barely recognizable against the diffuse centrin/GFP background fluorescence (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1). Their intensity gradually increased until they reached the levels typical for normal centrioles in this cell line (Fig. 2 and Videos 1 and 2). The increase in intensity was usually completed just before or during first mitosis (30–35 h after the cell's birth). The number of aggregates was variable (from 2 to >10 per cell); however, once the initial aggregates became detectable, their number in an individual cell did not increase over time. This indicated that the formation of the aggregates in each cell occurred within a relatively short period of time instead of gradually accumulating as the cell progressed through the cell cycle.


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)

Reformation of centrin/GFP aggregates and their behavior in HeLa cells born without a centrosome. Selected GFP fluorescence frames (maximal-intensity projections) from a multimode time lapse recording (same recording as in Fig. 1). The cell born without centrosome exhibits only diffuse cytoplasmic centrin/GFP localization for ∼24 h (A and B, top). Then, small centrin/GFP aggregates appear in the cytoplasm (C). These aggregates move continuously in the cytoplasm (see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1). After mitosis, which occurs at 32 h (Fig. 1 E) one of the progeny inherits seven of these aggregates, whereas the other inherits just one aggregate (D). The seven aggregates continue to move in the cytoplasm for ∼7–8 h (E), and then they coalesce into a common structure that remains relatively stationary in the middle of the cell (F and G, and Fig. 1I). The centrioles in the sister cell that inherited a normal centrosome (bottom) exhibit expected behavior, because they replicate (D) and then are properly distributed between the daughter cells after mitosis (E). Times are shown in hours:minutes. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Reformation of centrin/GFP aggregates and their behavior in HeLa cells born without a centrosome. Selected GFP fluorescence frames (maximal-intensity projections) from a multimode time lapse recording (same recording as in Fig. 1). The cell born without centrosome exhibits only diffuse cytoplasmic centrin/GFP localization for ∼24 h (A and B, top). Then, small centrin/GFP aggregates appear in the cytoplasm (C). These aggregates move continuously in the cytoplasm (see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1). After mitosis, which occurs at 32 h (Fig. 1 E) one of the progeny inherits seven of these aggregates, whereas the other inherits just one aggregate (D). The seven aggregates continue to move in the cytoplasm for ∼7–8 h (E), and then they coalesce into a common structure that remains relatively stationary in the middle of the cell (F and G, and Fig. 1I). The centrioles in the sister cell that inherited a normal centrosome (bottom) exhibit expected behavior, because they replicate (D) and then are properly distributed between the daughter cells after mitosis (E). Times are shown in hours:minutes. Bar, 5 μm.
Mentions: Approximately 20–25 h into the first cell cycle, a number of minuscule aggregates of centrin/GFP appeared in the cytoplasm of cells that were born without centrioles. Initially, these aggregates were barely recognizable against the diffuse centrin/GFP background fluorescence (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1). Their intensity gradually increased until they reached the levels typical for normal centrioles in this cell line (Fig. 2 and Videos 1 and 2). The increase in intensity was usually completed just before or during first mitosis (30–35 h after the cell's birth). The number of aggregates was variable (from 2 to >10 per cell); however, once the initial aggregates became detectable, their number in an individual cell did not increase over time. This indicated that the formation of the aggregates in each cell occurred within a relatively short period of time instead of gradually accumulating as the cell progressed through the cell cycle.

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