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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 born without centrosomes continue to progress through the cell cycle. (A and B) DIC (top) and fluorescence images (bottom) of a metaphase cell before (A) and after (B) laser ablation of one of the two centrosomes (A and B, compare arrowheads). Insets show centrioles at a higher magnification. (C–H) Selected phase-contrast frames from the multimode time lapse video recording of this cell. Arrowheads mark the cell born without centrosome, and arrows point at its sister that inherited the normal centrosome. Both cells undergo normal postmitotic flattening (C) and are morphologically similar to each other and nonirradiated cells (D). The cell born without a centrosome undergoes mitosis 32 h after the operation (E), and its sister follows just 4 h later (F). (I) GFP fluorescence (maximal-intensity projection) reveals that 48 h after the operation, one progeny of the cell born without a centrosome contains one centrin/GFP aggregate, whereas its sister contains seven centrin aggregates. Each of the two progeny of the sister cell that inherited a normal centrosome contains two centrin/GFP aggregate (centrioles). Insets show centrin aggregates at a higher magnification. Time is shown in hours:minutes. Bar, 5 μm.
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fig1: HeLa cells born without centrosomes continue to progress through the cell cycle. (A and B) DIC (top) and fluorescence images (bottom) of a metaphase cell before (A) and after (B) laser ablation of one of the two centrosomes (A and B, compare arrowheads). Insets show centrioles at a higher magnification. (C–H) Selected phase-contrast frames from the multimode time lapse video recording of this cell. Arrowheads mark the cell born without centrosome, and arrows point at its sister that inherited the normal centrosome. Both cells undergo normal postmitotic flattening (C) and are morphologically similar to each other and nonirradiated cells (D). The cell born without a centrosome undergoes mitosis 32 h after the operation (E), and its sister follows just 4 h later (F). (I) GFP fluorescence (maximal-intensity projection) reveals that 48 h after the operation, one progeny of the cell born without a centrosome contains one centrin/GFP aggregate, whereas its sister contains seven centrin aggregates. Each of the two progeny of the sister cell that inherited a normal centrosome contains two centrin/GFP aggregate (centrioles). Insets show centrin aggregates at a higher magnification. Time is shown in hours:minutes. Bar, 5 μm.

Mentions: We found that complete ablation of one of the two centrosomes in HeLa cells at metaphase or early anaphase did not affect cytokinesis, reconstitution of nuclei, and postmitotic flattening (Fig. 1). Time lapse microscopy revealed that the centrosomal and acentrosomal sisters are morphologically indistinguishable from each other and exhibit similar behavior, which is consistent with our previous observations in CV-1 and PtK1 cells (Khodjakov et al., 2000; Khodjakov and Rieder, 2001). However, in sharp contrast with nontransformed cells (e.g., CV-1, BSC-1, or PtK1) that arrest during G1 in the absence of centrosomes (Hinchcliffe et al., 2001; Khodjakov and Rieder, 2001), HeLa cells born without centrosomes progressed through the cell cycle with normal timing. These cells entered the next mitosis at 31.9 ± 7.1 h (n = 16) versus 30.6 ± 7.3 h (n = 14) for their centrosomal sisters. To standardize our descriptions of the progression through several consecutive cell cycles for these cells, we will hereafter refer to the completion of the mitosis during which the centrioles were ablated as the “birth” of the cell. After birth, the cells undergo a “first” cell cycle that culminates in the “first mitosis”, and then the “second cell cycle” and “second mitosis,” etc.


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 born without centrosomes continue to progress through the cell cycle. (A and B) DIC (top) and fluorescence images (bottom) of a metaphase cell before (A) and after (B) laser ablation of one of the two centrosomes (A and B, compare arrowheads). Insets show centrioles at a higher magnification. (C–H) Selected phase-contrast frames from the multimode time lapse video recording of this cell. Arrowheads mark the cell born without centrosome, and arrows point at its sister that inherited the normal centrosome. Both cells undergo normal postmitotic flattening (C) and are morphologically similar to each other and nonirradiated cells (D). The cell born without a centrosome undergoes mitosis 32 h after the operation (E), and its sister follows just 4 h later (F). (I) GFP fluorescence (maximal-intensity projection) reveals that 48 h after the operation, one progeny of the cell born without a centrosome contains one centrin/GFP aggregate, whereas its sister contains seven centrin aggregates. Each of the two progeny of the sister cell that inherited a normal centrosome contains two centrin/GFP aggregate (centrioles). Insets show centrin aggregates at a higher magnification. Time is shown in hours:minutes. Bar, 5 μm.
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Related In: Results  -  Collection

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fig1: HeLa cells born without centrosomes continue to progress through the cell cycle. (A and B) DIC (top) and fluorescence images (bottom) of a metaphase cell before (A) and after (B) laser ablation of one of the two centrosomes (A and B, compare arrowheads). Insets show centrioles at a higher magnification. (C–H) Selected phase-contrast frames from the multimode time lapse video recording of this cell. Arrowheads mark the cell born without centrosome, and arrows point at its sister that inherited the normal centrosome. Both cells undergo normal postmitotic flattening (C) and are morphologically similar to each other and nonirradiated cells (D). The cell born without a centrosome undergoes mitosis 32 h after the operation (E), and its sister follows just 4 h later (F). (I) GFP fluorescence (maximal-intensity projection) reveals that 48 h after the operation, one progeny of the cell born without a centrosome contains one centrin/GFP aggregate, whereas its sister contains seven centrin aggregates. Each of the two progeny of the sister cell that inherited a normal centrosome contains two centrin/GFP aggregate (centrioles). Insets show centrin aggregates at a higher magnification. Time is shown in hours:minutes. Bar, 5 μm.
Mentions: We found that complete ablation of one of the two centrosomes in HeLa cells at metaphase or early anaphase did not affect cytokinesis, reconstitution of nuclei, and postmitotic flattening (Fig. 1). Time lapse microscopy revealed that the centrosomal and acentrosomal sisters are morphologically indistinguishable from each other and exhibit similar behavior, which is consistent with our previous observations in CV-1 and PtK1 cells (Khodjakov et al., 2000; Khodjakov and Rieder, 2001). However, in sharp contrast with nontransformed cells (e.g., CV-1, BSC-1, or PtK1) that arrest during G1 in the absence of centrosomes (Hinchcliffe et al., 2001; Khodjakov and Rieder, 2001), HeLa cells born without centrosomes progressed through the cell cycle with normal timing. These cells entered the next mitosis at 31.9 ± 7.1 h (n = 16) versus 30.6 ± 7.3 h (n = 14) for their centrosomal sisters. To standardize our descriptions of the progression through several consecutive cell cycles for these cells, we will hereafter refer to the completion of the mitosis during which the centrioles were ablated as the “birth” of the cell. After birth, the cells undergo a “first” cell cycle that culminates in the “first mitosis”, and then the “second cell cycle” and “second mitosis,” etc.

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