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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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HeLa cells progress through the cell cycle and form centrioles de novo after removal of the resident centrosome by needle microsurgery. A large piece of cytoplasm containing both centrioles (i.e., cytoplast; A–C, arrow) was separated from the rest of the cell by a microneedle (see Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1). The nucleus-containing karyoplast (A–F, arrowheads) entered mitosis ∼16 h after the operation (D). The division resulted in the formation of two daughter cells (E) that were followed for an additional 9 h, and then transferred onto a higher magnification microscope. Multimode phase-contrast (G) and 3-D fluorescence (H) imaging revealed that one of the daughter cells contained 2 and the other one 4 centrioles. Insets in H present centrioles at a higher magnification. Bar, 10 μm.
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fig9: HeLa cells progress through the cell cycle and form centrioles de novo after removal of the resident centrosome by needle microsurgery. A large piece of cytoplasm containing both centrioles (i.e., cytoplast; A–C, arrow) was separated from the rest of the cell by a microneedle (see Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1). The nucleus-containing karyoplast (A–F, arrowheads) entered mitosis ∼16 h after the operation (D). The division resulted in the formation of two daughter cells (E) that were followed for an additional 9 h, and then transferred onto a higher magnification microscope. Multimode phase-contrast (G) and 3-D fluorescence (H) imaging revealed that one of the daughter cells contained 2 and the other one 4 centrioles. Insets in H present centrioles at a higher magnification. Bar, 10 μm.

Mentions: We used the GFP centrin signal to identify cells with two centrioles (G1) that were located away from the nucleus. Such cells were then cut with a glass needle so that a piece of cytoplasm containing the centrosome was separated from the rest of the cell (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1; also see Hinchcliffe et al., 2001). We then followed each karyoplast by phase-contrast video microscopy for 22–72 h; at the end of the video records, we collected three-dimensional (3-D) fluorescence images. Nine karyoplasts were followed through the first division, and seven were fixed 9–12 h after its completion. The duration of mitosis was ∼2 h (range 1–6), which is in good agreement with the duration of the first mitosis in cells after laser ablation of centrosomes. Most karyoplasts formed more than one furrow during cytokinesis, which once again was reminiscent of the cytokinesis pattern in cells after centrosome laser ablation, and implied that the mitotic spindle in karyoplasts was multipolar. However, extra furrows regressed and all nine karyoplasts ultimately divided just two daughter cells. All daughter karyoplasts contained a variable number (1–5 per cell) of bright centrin/GFP aggregates indistinguishable from those observed after laser ablations (Fig. 9). Importantly, the number of centrin foci was different between daughter karyoplasts, indicating that the distribution of these structures during mitosis was random, as observed for centrioles formed de novo after laser ablation.


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)

HeLa cells progress through the cell cycle and form centrioles de novo after removal of the resident centrosome by needle microsurgery. A large piece of cytoplasm containing both centrioles (i.e., cytoplast; A–C, arrow) was separated from the rest of the cell by a microneedle (see Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1). The nucleus-containing karyoplast (A–F, arrowheads) entered mitosis ∼16 h after the operation (D). The division resulted in the formation of two daughter cells (E) that were followed for an additional 9 h, and then transferred onto a higher magnification microscope. Multimode phase-contrast (G) and 3-D fluorescence (H) imaging revealed that one of the daughter cells contained 2 and the other one 4 centrioles. Insets in H present centrioles at a higher magnification. Bar, 10 μm.
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Related In: Results  -  Collection

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fig9: HeLa cells progress through the cell cycle and form centrioles de novo after removal of the resident centrosome by needle microsurgery. A large piece of cytoplasm containing both centrioles (i.e., cytoplast; A–C, arrow) was separated from the rest of the cell by a microneedle (see Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1). The nucleus-containing karyoplast (A–F, arrowheads) entered mitosis ∼16 h after the operation (D). The division resulted in the formation of two daughter cells (E) that were followed for an additional 9 h, and then transferred onto a higher magnification microscope. Multimode phase-contrast (G) and 3-D fluorescence (H) imaging revealed that one of the daughter cells contained 2 and the other one 4 centrioles. Insets in H present centrioles at a higher magnification. Bar, 10 μm.
Mentions: We used the GFP centrin signal to identify cells with two centrioles (G1) that were located away from the nucleus. Such cells were then cut with a glass needle so that a piece of cytoplasm containing the centrosome was separated from the rest of the cell (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1; also see Hinchcliffe et al., 2001). We then followed each karyoplast by phase-contrast video microscopy for 22–72 h; at the end of the video records, we collected three-dimensional (3-D) fluorescence images. Nine karyoplasts were followed through the first division, and seven were fixed 9–12 h after its completion. The duration of mitosis was ∼2 h (range 1–6), which is in good agreement with the duration of the first mitosis in cells after laser ablation of centrosomes. Most karyoplasts formed more than one furrow during cytokinesis, which once again was reminiscent of the cytokinesis pattern in cells after centrosome laser ablation, and implied that the mitotic spindle in karyoplasts was multipolar. However, extra furrows regressed and all nine karyoplasts ultimately divided just two daughter cells. All daughter karyoplasts contained a variable number (1–5 per cell) of bright centrin/GFP aggregates indistinguishable from those observed after laser ablations (Fig. 9). Importantly, the number of centrin foci was different between daughter karyoplasts, indicating that the distribution of these structures during mitosis was random, as observed for centrioles formed de novo after laser ablation.

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