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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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Pedigree of a cell born with only one (daughter) centriole. In this cell, just one (mother) centriole within a diplosome was ablated (white arrows; insets present centrioles at a higher magnification). As a result, after the end of mitosis, one daughter cell was born with normal centrosome, but the other one inherited only one (daughter) centriole. This centriole replicated once during the first cell cycle, and the resulting diplosome was distributed to one of the progeny during second mitosis. As a result, one of the progeny now inherited a normal centrosome (one daughter and one mother centriole), whereas the other one was born without a centrosome. This cell eventually formed five centrioles de novo. Progeny of the control cell (sister of the one born without centriole) exhibited expected centriole replication pattern.
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fig8: Pedigree of a cell born with only one (daughter) centriole. In this cell, just one (mother) centriole within a diplosome was ablated (white arrows; insets present centrioles at a higher magnification). As a result, after the end of mitosis, one daughter cell was born with normal centrosome, but the other one inherited only one (daughter) centriole. This centriole replicated once during the first cell cycle, and the resulting diplosome was distributed to one of the progeny during second mitosis. As a result, one of the progeny now inherited a normal centrosome (one daughter and one mother centriole), whereas the other one was born without a centrosome. This cell eventually formed five centrioles de novo. Progeny of the control cell (sister of the one born without centriole) exhibited expected centriole replication pattern.

Mentions: Our observations indicated that de novo assembly of centrioles in HeLa cells occurs whenever resident centrioles disappear from the cell. The means in which the centrioles vanish from the cell do not appear to be important, for we observed de novo centriole assembly after ablation of resident centrioles as well as in cells that lost their centrioles via misdistribution during mitosis (Fig. 5). The fact that de novo centriole assembly pathway activates whenever resident centrioles are missing implies that cells posses a mechanism that somehow senses the presence of centrioles. In this respect, it is important to determine whether this mechanism monitors the presence of a mature (mother) centriole or whether it is satisfied by any centriole present in the cell. To address this issue, we laser ablated just one mother centriole within the diplosome at a spindle pole during mitosis. As mother centrioles contain greater amounts of centrin than the daughters, the relative intensity of the GFP signal allowed us to distinguish the mother centriole from the daughter in live cells (Fig. 8; Piel et al., 2000, 2001). As a result of such an operation, one daughter cell is born with just one immature centriole. We reasoned that, if inhibition of the de novo pathway requires the presence of the mother centriole, cells born with one immature centriole would exhibit the de novo assembly of multiple centrioles.


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)

Pedigree of a cell born with only one (daughter) centriole. In this cell, just one (mother) centriole within a diplosome was ablated (white arrows; insets present centrioles at a higher magnification). As a result, after the end of mitosis, one daughter cell was born with normal centrosome, but the other one inherited only one (daughter) centriole. This centriole replicated once during the first cell cycle, and the resulting diplosome was distributed to one of the progeny during second mitosis. As a result, one of the progeny now inherited a normal centrosome (one daughter and one mother centriole), whereas the other one was born without a centrosome. This cell eventually formed five centrioles de novo. Progeny of the control cell (sister of the one born without centriole) exhibited expected centriole replication pattern.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171814&req=5

fig8: Pedigree of a cell born with only one (daughter) centriole. In this cell, just one (mother) centriole within a diplosome was ablated (white arrows; insets present centrioles at a higher magnification). As a result, after the end of mitosis, one daughter cell was born with normal centrosome, but the other one inherited only one (daughter) centriole. This centriole replicated once during the first cell cycle, and the resulting diplosome was distributed to one of the progeny during second mitosis. As a result, one of the progeny now inherited a normal centrosome (one daughter and one mother centriole), whereas the other one was born without a centrosome. This cell eventually formed five centrioles de novo. Progeny of the control cell (sister of the one born without centriole) exhibited expected centriole replication pattern.
Mentions: Our observations indicated that de novo assembly of centrioles in HeLa cells occurs whenever resident centrioles disappear from the cell. The means in which the centrioles vanish from the cell do not appear to be important, for we observed de novo centriole assembly after ablation of resident centrioles as well as in cells that lost their centrioles via misdistribution during mitosis (Fig. 5). The fact that de novo centriole assembly pathway activates whenever resident centrioles are missing implies that cells posses a mechanism that somehow senses the presence of centrioles. In this respect, it is important to determine whether this mechanism monitors the presence of a mature (mother) centriole or whether it is satisfied by any centriole present in the cell. To address this issue, we laser ablated just one mother centriole within the diplosome at a spindle pole during mitosis. As mother centrioles contain greater amounts of centrin than the daughters, the relative intensity of the GFP signal allowed us to distinguish the mother centriole from the daughter in live cells (Fig. 8; Piel et al., 2000, 2001). As a result of such an operation, one daughter cell is born with just one immature centriole. We reasoned that, if inhibition of the de novo pathway requires the presence of the mother centriole, cells born with one immature centriole would exhibit the de novo assembly of multiple centrioles.

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