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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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Intermediate stages of centriole formation in S-arrested cells. Similar procedure to Fig. 6 B was performed, except this cell was fixed 24 h after ablating the resident centrosome. (A) GFP fluorescence revealed that several prominent centrin/GFP aggregates had formed in the cell. (B–D) Individual 3.2-nm-thick slices from the tomogram of a 100-nm-thick section revealed that some of these aggregates correspond to centriole-like structures that appear to be at different stages of assembly. (E) Tracing of microtubules (blue) centriolar blades and electron-opaque material from the tomogram. Note that while there are multiple microtubules, they do not converge on the forming centrioles. Bar, 500 nm.
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fig7: Intermediate stages of centriole formation in S-arrested cells. Similar procedure to Fig. 6 B was performed, except this cell was fixed 24 h after ablating the resident centrosome. (A) GFP fluorescence revealed that several prominent centrin/GFP aggregates had formed in the cell. (B–D) Individual 3.2-nm-thick slices from the tomogram of a 100-nm-thick section revealed that some of these aggregates correspond to centriole-like structures that appear to be at different stages of assembly. (E) Tracing of microtubules (blue) centriolar blades and electron-opaque material from the tomogram. Note that while there are multiple microtubules, they do not converge on the forming centrioles. Bar, 500 nm.

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)

Intermediate stages of centriole formation in S-arrested cells. Similar procedure to Fig. 6 B was performed, except this cell was fixed 24 h after ablating the resident centrosome. (A) GFP fluorescence revealed that several prominent centrin/GFP aggregates had formed in the cell. (B–D) Individual 3.2-nm-thick slices from the tomogram of a 100-nm-thick section revealed that some of these aggregates correspond to centriole-like structures that appear to be at different stages of assembly. (E) Tracing of microtubules (blue) centriolar blades and electron-opaque material from the tomogram. Note that while there are multiple microtubules, they do not converge on the forming centrioles. Bar, 500 nm.
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Related In: Results  -  Collection

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fig7: Intermediate stages of centriole formation in S-arrested cells. Similar procedure to Fig. 6 B was performed, except this cell was fixed 24 h after ablating the resident centrosome. (A) GFP fluorescence revealed that several prominent centrin/GFP aggregates had formed in the cell. (B–D) Individual 3.2-nm-thick slices from the tomogram of a 100-nm-thick section revealed that some of these aggregates correspond to centriole-like structures that appear to be at different stages of assembly. (E) Tracing of microtubules (blue) centriolar blades and electron-opaque material from the tomogram. Note that while there are multiple microtubules, they do not converge on the forming centrioles. Bar, 500 nm.
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