HeLa cells born without centrosomes progress through the cell cycle and form centrioles de novo
All experiments were conducted in HeLa cells stably transfected with human centrin-1/GFP. This protein accumulates inside the centriole lumen from the earliest stages of centriole formation, which makes it a reliable marker for individual centrioles (Piel et al., 2000
). Using the GFP signal as a target, we laser ablated both centrioles (diplosome) associated with one of the two spindle poles during mitosis. At the completion of mitosis, this produces two genetically identical daughter cells: one with and one without a centrosome. Comparative analysis of the two sisters allowed us to differentiate between the effects specific to the centrosome ablation versus potential nonspecific effects of laser irradiation and long-term fluorescence imaging. It is important to emphasize that the laser beam destroys all structures/proteins within an ~0.4–0.4–0.6-μm (x-y-z) volume. When we aimed the laser at a centriole, the beam destroyed both the centriole and the PCM associated with the targeted centriole. Thus, ablation of both centrioles results in the structural and functional destruction of the entire centrosome (Khodjakov et al., 1997
; for review see Khodjakov and Rieder, 2004
). However, it is possible to ablate just a single centriole (along with its associated PCM) within a diplosome so that the other centriole (and its PCM) remain intact (see Materials and methods).
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 (). 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.
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). (more ...)
Approximately 20–25 h into the first cell cycle, a number of minuscule aggregates of centrin/GFP appeared in the cytoplasm of cells that were born without centrioles. Initially, these aggregates were barely recognizable against the diffuse centrin/GFP background fluorescence (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1
). Their intensity gradually increased until they reached the levels typical for normal centrioles in this cell line ( and Videos 1 and 2). The increase in intensity was usually completed just before or during first mitosis (30–35 h after the cell's birth). The number of aggregates was variable (from 2 to >10 per cell); however, once the initial aggregates became detectable, their number in an individual cell did not increase over time. This indicated that the formation of the aggregates in each cell occurred within a relatively short period of time instead of gradually accumulating as the cell progressed through the cell cycle.
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 ). The cell born without (more ...)
Correlative GFP fluorescence light microscopy/serial-section EM analyses revealed that these aggregates are amorphous at the EM level for ~5–10 h after they became recognizable by light microscopy (n = 2). Nevertheless, by the time the cells reached first mitosis or the second cell cycle centrin aggregates corresponded to morphologically complete centrioles () in all cells investigated (n = 3). Limited sample size did not allow us to identify intermediate stages of the transition from the amorphous centrin aggregates to complete centrioles in cycling cells. During first mitosis, centrioles did not pair to form diplosomes, but rather they organized spindle poles as individual centrioles (), surrounded by minimal amount of PCM.
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 (more ...)
De novo–assembled centrioles mature during second cell cycle
In control HeLa cells, the mother and daughter centrioles exhibit dramatically different mobility during early G1
. Whereas the mother centriole remains relatively stationary in the center of the cell, the daughter moves extensively in the cytoplasm making numerous excursions to the periphery of the cell and returning back to the center. These excursions cease at G1
/S transition (Piel et al., 2000
; Video 3, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1
Our recordings revealed that centrin aggregates also moved extensively in a random fashion throughout first interphase until the cells entered mitosis ( and Videos 1 and 2). During the first mitosis, approximately half of the of the mitotic cells (11/20) exhibited extra cleavage furrows and produced one mononucleated and one multinucleated daughter cell, which implies the assembly of multipolar spindles in cells with de novo–formed centrioles. Furthermore, we directly observed multipolar mitotic spindle in three cells fixed during first mitosis. Serial section EM reconstruction revealed that centrioles were associated with the spindle poles ().
After completion of first mitosis, which on average was longer in cells born without centrioles (2.9 ± 2.2 h; n = 13) than in their centrosomal sisters (2.0 ± 1.9 h; n = 12), the de novo–formed centrioles resumed movements that were similar to those exhibited by the centrin aggregates during the previous cell cycle. Then, at 2–10 h after mitosis, all centrioles suddenly coalesced into a relatively stationary complex (, E–F; and Videos 1 and 2). In all cases, the coalescence itself was relatively rapid, as all centrioles came together in <2 h. Thus, centriole coalescence corresponds to a pronounced change in behavior: from that normally exhibited by the daughter centrioles to the one typical for the mothers. The coalescence of the centrioles always occurred in the second cell cycle.
It has been previously shown that the motilities of the daughter and mother centrioles during G1
correlate with their ability to organize microtubule networks. Although both daughter and mother centrioles are capable of nucleating similar numbers of microtubules, only the mother can organize microtubules into a typical radial array (Piel et al., 2000
). As a result, mother centrioles always reside inside of microtubule foci, whereas the daughters, at least in some cell types (e.g., HeLa and L-929), are not associated with microtubule asters (Piel et al., 2000
). We investigated at which point the de novo–formed centrioles become associated with microtubule foci. Immunofluorescence analysis revealed that, not surprisingly, the moving centrin aggregates/centrioles of the first interphase were not associated with microtubules, even though some of them were associated with bona fide PCM components, such as γ-tubulin ( A). During the first cell cycle, the interphase microtubule array in cells born without centrioles did not converge on common focal points and were instead randomized, showing only loose concentration at the perinuclear region. Importantly, this difference in microtubule organization was observed even in those cells where the duration of first cell cycle was for some reason prolonged, something that occurs with equal frequency in cells born with and without centrosomes.
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 (more ...)
In contrast, after coalescence in the second cell cycle, all centrioles were found to be associated with prominent PCM and reside at the focus of microtubule array (). Although microtubules in HeLa cells are not prominently radial, the foci associated with the de novo–formed centrioles were very similar to those in the surrounding control cells. Even in those cells that by chance inherited only one centriole, this centriole was able to organize a microtubule focus ( B). The appearance of the centrosome in those cells, that by chance inherited two centrioles, was very similar to that in control cells during late G1 ( C). These observations revealed that, while formation of the morphologically complete centrioles occurs during the first cell cycle, they become competent microtubule-organizing centers only during G1 of the second cell cycle.
The difference between mature (mother) and immature (daughter) centrioles is not limited to their ability to organize microtubules. An important step in the maturation process is to gain the ability to give birth to a new daughter centriole (for conceptual review see Mazia, 1987
). Thus, if the de novo–assembled centrioles become mature during the second cell cycle, then they should begin to replicate in a normal fashion. We tested this prediction by following the progeny of cells born without centrioles with continuous time lapse microscopy over three consecutive cell cycles. We were able to obtain full three–cell cycle-long pedigrees for two cells born without centrioles. Analyses of these pedigrees revealed several important features of the centriole cycle. First, we found that centrioles formed de novo replicate in the second cell cycle (, cell a, which fortuitously inherited two de novo–formed centrioles, and its progeny aa and ab). Second, we found that the de novo pathway becomes active whenever the resident centrioles disappear from the cell. This was evident from those cases in which all the de novo–formed centrioles were distributed to only one of the progeny (, cell a; and Figs. S1 and S2). As a result, the sister cell was born without a centrosome (, cell b), not because of laser ablation, but because of centriole misdistribution. This cell exhibited de novo assembly of eight centrioles that were later distributed in a four-and-four fashion between the two progeny in the next mitosis (, cell b and its progeny ba and bb). It is noteworthy that this mitosis was multipolar; however, because of retraction of one of the cytokinesis furrows, it resulted in the formation of two cells—one mononucleated and one binucleated (not depicted). Thus, de novo–assembled centrioles become completely mature in the next cell cycle after their assembly.
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 (more ...)
De novo centriole assembly occurs during the S phase
Thus far, our data revealed that formation of a mature centriole takes place over two consecutive cell cycles in normally cycling cells. However, it remained unclear whether this was simply a time-dependent process or if progression through the cell cycle is required for centriole formation/maturation. To address this question, we conducted centriole ablations in cells arrested in G1 with lovastatin or S with hydroxyurea.
We found that formation of centrin aggregates did not occur after ablating resident centrioles in cells arrested during G1
( A; n
= 5). In contrast, cells arrested in S ( 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 ( and Video 4, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1
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 (more ...)
Figure 7. Intermediate stages of centriole formation in S-arrested cells. Similar procedure to 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 (more ...)
Fluorescence time lapse microscopy of centrin aggregates/centrioles formed in S-arrested cells revealed that they move continuously in the cytoplasm in the same fashion as centrin aggregates/immature centrioles do in cycling cells during the first interphase ( B and Video 5, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1
). This motion continued for as long as we were able to follow S-arrested cells (~50 h), and the aggregates never coalesced into a common complex. In light of our EM data, these observations indicate that the complete process of centriole biogenesis, but not maturation, can proceed to completion during the S phase.
De novo centriole formation is inhibited by the presence of a single resident centriole
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 (). 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 (; Piel et al., 2000
). 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.
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 (more ...)
Time lapse recordings of cells that inherited just one immature centriole revealed that all (n
= 3) entered the subsequent mitosis with only a single diplosome, which was formed as the result of replication of the resident centriole (). We did not detect any signs of the de novo pathway activity. The duration of the cell cycle appeared not affected in monocentriolar cells (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb200411126/DC1
), which was not surprising in light of our data on complete centrosome ablation. Importantly, the single diplosome present in these cells associated with only one of the two spindle poles (Fig. S2) and thus one of the two cells formed as the result of first mitosis was born with normal centriole complement, while its sister lacked centrioles completely. Whereas cells that inherited the diplosome proceeded with orderly centriole replication in the ensuing cell cycle, their sisters born without centrioles exhibited de novo assembly of a variable number of centrioles ( and Fig. S1).
De novo centriole formation is not a consequence of laser fragmentation of the resident centrosome
One formally possible complication of the laser ablation approach to centrosome inactivation is that the laser pulses may cause fragmentation of the centrioles and/or the PCM rather than their complete destruction. If so, the de novo assembly of centrosomes might simply reflect the seeding of centrosomes from tiny preexisting fragments of the original centrosome. To unequivocally test this possibility, we used glass needle microsurgery to remove centrosomes from HeLa cells and to determine whether centrioles would form de novo. Needle microsurgery is effective in completely removing the centrosome from interphase BSC-1 fibroblasts (Maniotis and Schliwa, 1991
; Hinchcliffe et al., 2001
), and this method cannot induce centrosome fragmentation.
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 (). 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.
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 (more ...)
The progeny of two karyoplasts were followed through the second cell cycle, second mitosis, and fixed at ~12 h into the third cell cycle. Each of these cells contained bright centrin aggregates clustered together in a common complex, as expected for de novo–formed centrioles after the maturation (unpublished data). Control amputations of large portions of cytoplasm, not containing centrioles, did not alter cell cycle progression, the normal pattern of centriole duplication, or induced the formation of supernumerary centrin foci (unpublished data).