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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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Centrin aggregates formed in cells born without centrosomes become morphologically recognizable as centrioles when the cell enters mitosis. (A and B) Fluorescence images of a metaphase cell before (A) and after (B) laser ablation of one of the two centrosomes (A and B, compare arrowheads). Insets are shown at a higher magnification. (C–E) The cell born without a centrosome (C and D, arrowheads) forms prominent centrin aggregates, enters mitosis, and forms a multipolar mitotic spindle. The left image in each panel shows phase-contrast microscopy; the right images show centrin/GFP fluorescence. (F) Serial section EM analysis of this cell reveals that each of the centrin aggregates corresponds to a single centriole, surrounded by a small amount of pericentriolar material (1–5; selected sections from a full series of 100-nm sections). Time is shown in hours:minutes. Bars: (A–E) 5 μm; (F) 500 nm.
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fig3: Centrin aggregates formed in cells born without centrosomes become morphologically recognizable as centrioles when the cell enters mitosis. (A and B) Fluorescence images of a metaphase cell before (A) and after (B) laser ablation of one of the two centrosomes (A and B, compare arrowheads). Insets are shown at a higher magnification. (C–E) The cell born without a centrosome (C and D, arrowheads) forms prominent centrin aggregates, enters mitosis, and forms a multipolar mitotic spindle. The left image in each panel shows phase-contrast microscopy; the right images show centrin/GFP fluorescence. (F) Serial section EM analysis of this cell reveals that each of the centrin aggregates corresponds to a single centriole, surrounded by a small amount of pericentriolar material (1–5; selected sections from a full series of 100-nm sections). Time is shown in hours:minutes. Bars: (A–E) 5 μm; (F) 500 nm.

Mentions: Correlative GFP fluorescence light microscopy/serial-section EM analyses revealed that these aggregates are amorphous at the EM level for ∼5–10 h after they became recognizable by light microscopy (n = 2). Nevertheless, by the time the cells reached first mitosis or the second cell cycle centrin aggregates corresponded to morphologically complete centrioles (Fig. 3) in all cells investigated (n = 3). Limited sample size did not allow us to identify intermediate stages of the transition from the amorphous centrin aggregates to complete centrioles in cycling cells. During first mitosis, centrioles did not pair to form diplosomes, but rather they organized spindle poles as individual centrioles (Fig. 3), surrounded by minimal amount of PCM.


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 formed in cells born without centrosomes become morphologically recognizable as centrioles when the cell enters mitosis. (A and B) Fluorescence images of a metaphase cell before (A) and after (B) laser ablation of one of the two centrosomes (A and B, compare arrowheads). Insets are shown at a higher magnification. (C–E) The cell born without a centrosome (C and D, arrowheads) forms prominent centrin aggregates, enters mitosis, and forms a multipolar mitotic spindle. The left image in each panel shows phase-contrast microscopy; the right images show centrin/GFP fluorescence. (F) Serial section EM analysis of this cell reveals that each of the centrin aggregates corresponds to a single centriole, surrounded by a small amount of pericentriolar material (1–5; selected sections from a full series of 100-nm sections). Time is shown in hours:minutes. Bars: (A–E) 5 μm; (F) 500 nm.
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Related In: Results  -  Collection

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fig3: Centrin aggregates formed in cells born without centrosomes become morphologically recognizable as centrioles when the cell enters mitosis. (A and B) Fluorescence images of a metaphase cell before (A) and after (B) laser ablation of one of the two centrosomes (A and B, compare arrowheads). Insets are shown at a higher magnification. (C–E) The cell born without a centrosome (C and D, arrowheads) forms prominent centrin aggregates, enters mitosis, and forms a multipolar mitotic spindle. The left image in each panel shows phase-contrast microscopy; the right images show centrin/GFP fluorescence. (F) Serial section EM analysis of this cell reveals that each of the centrin aggregates corresponds to a single centriole, surrounded by a small amount of pericentriolar material (1–5; selected sections from a full series of 100-nm sections). Time is shown in hours:minutes. Bars: (A–E) 5 μm; (F) 500 nm.
Mentions: Correlative GFP fluorescence light microscopy/serial-section EM analyses revealed that these aggregates are amorphous at the EM level for ∼5–10 h after they became recognizable by light microscopy (n = 2). Nevertheless, by the time the cells reached first mitosis or the second cell cycle centrin aggregates corresponded to morphologically complete centrioles (Fig. 3) in all cells investigated (n = 3). Limited sample size did not allow us to identify intermediate stages of the transition from the amorphous centrin aggregates to complete centrioles in cycling cells. During first mitosis, centrioles did not pair to form diplosomes, but rather they organized spindle poles as individual centrioles (Fig. 3), surrounded by minimal amount of PCM.

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