The de novo centriole assembly pathway in HeLa cells: cell cycle progression and centriole assembly/maturation

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.

Figure 1.

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.

Figure 2.

Reformation of centrin/GFP aggregates and their behavior in HeLa cells born without a centrosome. Selected GFP fluorescence frames (maximal-intensity projections) from a multimode time lapse recording (same recording as in Fig. 1). The cell born without centrosome exhibits only diffuse cytoplasmic centrin/GFP localization for ∼24 h (A and B, top). Then, small centrin/GFP aggregates appear in the cytoplasm (C). These aggregates move continuously in the cytoplasm (see Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1). After mitosis, which occurs at 32 h (Fig. 1 E) one of the progeny inherits seven of these aggregates, whereas the other inherits just one aggregate (D). The seven aggregates continue to move in the cytoplasm for ∼7–8 h (E), and then they coalesce into a common structure that remains relatively stationary in the middle of the cell (F and G, and Fig. 1I). The centrioles in the sister cell that inherited a normal centrosome (bottom) exhibit expected behavior, because they replicate (D) and then are properly distributed between the daughter cells after mitosis (E). Times are shown in hours:minutes. Bar, 5 μm.

Figure 3.

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.

Figure 4.

Centrin aggregates are not associated with microtubules during the first cell cycle, but become positioned inside of microtubule foci after they coalesce into a common structure in the second cell cycle. (A) GFP/centrin, γ-tubulin, and α-tubulin distribution in a cell fixed during first cell cycle (∼49 h after centrosome ablation and 24 h after formation of detectable centrin aggregates). Although some of the centrin/GFP aggregates also contain γ-tubulin, none of them is associated with microtubule foci (arrows). (B and C) Two progeny of a cell born without a centrosome that were fixed in the second cell cycle, after the coalescence of the de novo–formed centrioles (∼48 h after centrosome ablation; 20 h after formation of centrin aggregates; and 15 h after second mitosis). In contrast to the centrin aggregates during the first cell cycle (A), de novo–formed centrioles after mitosis reside inside of microtubule foci and are associated with large amount of γ-tubulin (arrows). Bar, 10 μm. Maximal-intensity projections.

Figure 5.

Pedigree of a cell born without centrosome. In this particular experiment, the acentrosomal cell formed two centrioles during the first cell cycle (compare 22:00 with 29:00). When this cell underwent mitosis, both de novo–formed centrioles were distributed to one of the two progeny (a), leaving the other one acentrosomal (b). This cell activated the de novo pathway, which now resulted in the formation of eight new centrioles (b, compare 46:00 with 53:45). During third mitosis these 8 centrioles were equally distributed between the two progeny (ba and bb). Cell a, which inherited two de novo–formed centrioles as the result of the second mitosis, replicated these centrioles during the second cell cycle so that both progeny of this cell (aa and ab) inherited two centrioles (one mother and one daughter). Control cell (sister of the cell born without a centrosome) and its progeny exhibited the expected orderly replication of centrioles in both first and second cell cycles. Time is shown in hours:minutes. Bar, 5 μm.

Figure 6.

Centriole de novo formation occurs in HeLa cells arrested in S but not in G1. (A) Resident centrosome was ablated (arrows, compare 00:00 and 00:01) in a cell pretreated with 5 μM lovastatin for ∼15 h. Time lapse recording of this cell revealed no formation of centrin/GFP aggregates for 46 h. (B) Similar procedure to A was followed, except this cell was pretreated with 2 mM hydroxyurea. Time lapse recording revealed that ∼5 h after ablation of the resident centrioles centrin/GFP, aggregates formed in the cytoplasm (arrows, 05:30). These aggregates moved continuously in the cytoplasm while their intensity gradually increased (05:30–50:00). Time is shown in hours:minutes. Bar, 5 μm.

Figure 7.

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.

Figure 8.

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.

Figure 9.

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.

Figure 10.

Timeline of centrosome reformation in HeLa cells. When a cell is born without centrioles, it continues to progress through the cell cycle with normal kinetics. When cells enter S phase, multiple aggregates of centrin (precentrioles, small green dots) form. These precentrioles transform into morphologically complete centrioles (green open circles) by the time the cell enters its first mitosis. However, de novo–formed centrioles do not mature centrosomes until the ensuing G₁ in the second cell cycle. As cell enters the second cell cycle S phase, de novo–formed centrioles replicate and normal centriolar cycles resume.