Centrosome removal in G1: de novo centriole assembly
We initially worked with hTERT RPE1 and human mammary epithelial cells (HMECs) stably expressing human centrin-1/GFP to tag the centrioles. These normal human cells expressing centrin-1/GFP progress through the cell cycle at the same rate as the native cells, with a doubling time of 15–18 h. Centriole duplication and mitosis are normal. These cells have an intact p53 pathway, as indicated by cell cycle arrest with elevated levels of p21 in response to DNA damage (unpublished data).
We identified RPE1 cells that were in G1 by the presence of two bright focal centrin-1/GFP spots (centrioles); in phase contrast, we cut between the nucleus and the centrioles with a glass needle, as previously described (Hinchcliffe et al., 2001
; La Terra et al., 2005
), to form an acentrosomal cell and a centriole-containing cytoplasmic fragment called a cytoplast (). 15–30 min after the operation, cytoplasts were examined for ~1 s in fluorescence to confirm the presence of the centrioles (). Individual acentrosomal cells were followed with phase-contrast time-lapse video recordings after the coverslips were transferred from micromanipulation preparations to closed chambers, which allow cells to proliferate normally for at least 100 h, or until confluency is reached (Sluder et al., 2005
Figure 1. De novo centriole assembly in an acentrosomal RPE1 cell. (A, a) Acentrosomal cell and cytoplast 30 min after G1 microsurgery. (b) Fluorescence image of the GFP-tagged centriole pair (box) in the cytoplast. (c) The same acentrosomal cell at 1 h after microsurgery (more ...)
Before we fully describe the cell cycle progression of acentrosomal RPE1 cells (see next section), we first describe our observation that untransformed cells assemble centrioles de novo. Surprisingly, we found that acentrosomal RPE1 cells progressed through interphase and divided one or more times. During the first interphase and after the first or second mitosis, the cells contained 0–6 puncta of centrin-1 (, and ). The bright focal appearance and variable number of centrin foci is characteristic of de novo centriole assembly (Khodjakov et al., 2002
; La Terra et al., 2005
). Serial section electron microscopy of three acentrosomal cells previously followed in vivo revealed that the bright centrin foci assembled de novo corresponded to morphologically normal centrioles (). HMEC cells also formed bright centrin foci in the first interphase after G1 ablation of both centrioles. Serial section electron microscopy of two of these cells confirmed the de novo formation of centrioles (unpublished data).
Figure 2. Acentrosomal RPE1 cell progressing from G1 through two mitoses. (A) Acentrosomal cell 2 h after the microsurgery. (B) First mitosis. (C) Progeny of the first mitosis. The two daughters are indicated by arrows. (D and E and F and G) Second mitosis of both (more ...)
To determine when acentrosomal cells start to assemble centrioles de novo, we cut RPE1 cells in G1, added BrdU to the medium, and examined them at various times thereafter. We observed the formation of 2–7 centrin foci (precentrioles) starting ~9 h after the microsurgery (), which was temporally coincident with S phase as determined by BrdU incorporation. To test if the early, and perhaps invisible, formation of precentrioles occurs during G1 in RPE1 cells, we microsurgically removed the centrosome from 15 cells that were pretreated with 1 mM mimosine to arrest them in G1 (Krude, 1999
; Wang et al., 2000
). Because precentriole maturation into morphological centrioles is a time-dependent process in HeLa cells (La Terra et al., 2005
), there would be sufficient time for nascent precentrioles to mature and become readily visible in acentrosomal cells arrested in G1. 11 acentrosomal cells remained arrested in G1 for at least 24 h and, with one exception, none contained any visible centrin foci. The other four progressed into S phase, and all contained two or four centrin foci; these serve as internal controls, demonstrating that mimosine does not have an activity that shuts down the de novo centriole assembly pathway. Separately, we laser ablated the centrosome in five G1 RPE1 cells, and then hit the nucleus with the laser to induce DNA damage to hold the cells in G1. All arrested in interphase for at least 72 h, and none formed centrin foci. Together, these observations indicate that precentriole formation occurs in S phase, and thus, the G1 progression of acentrosomal cells reported in the next section is not supported by the presence of precentrioles.
Centrosome removal in G1: no cell cycle arrest
In 38 trials, all acentrosomal cells, even those produced 1 h after the completion of mitosis, progressed through interphase, entered mitosis (mean 15 h after cut), and completed cleavage into two daughters in a normal fashion (). However, instead of arresting in G1 after mitosis, as we would have expected from previous studies (Hinchcliffe et al., 2001
), out of 72 daughters that stayed in view, 2 arrested in G1, 5 progressed into S phase, and 65 divided at least one more time within 48 h (). To determine if G1 progression after centrosome removal is peculiar to microsurgery, we used laser ablation of the centrosome, which destroys only a small volume of the cell. Using a spinning disk confocal at low laser power setting (107 μW output at the objective lens) to visualize the GFP-tagged centrioles, we found that 7/8 RPE1 cells progressed through interphase to mitosis after G1 ablation of both centrioles. We also conducted G1 centrosome ablations on a p53-positive clone of HMECs expressing centrin-1/GFP to tag the centrioles. Using the same confocal power setting to visualize the GFP-tagged centrioles, we found that after ablation of both centrioles during G1, all 16 cells progressed through interphase into mitosis (, top, line A). In these experiments, we used pairs of sister G1 cells in which one received a cytoplasmic control ablation and the other a directed centrosome ablation; we found that the time from ablation to mitosis was the same (control irradiated cell mean = 26.9 h, n
= 15; experimental cell mean = 26.8 h, n
= 16). Together, these results demonstrate that G1 progression without a centrosome is not specific to the type of untransformed cell or the means used to remove the centrosome. Furthermore, when we induced physical damage to the centrosome by ablating one G1 centriole, three fourths of the cells progressed through interphase to mitosis (, top, line A).
Figure 3. Laser ablation of one or two centrioles during G1 predisposes HMEC cells to a p38-dependent interphase arrest. (top) The blue light level column shows the intensity of the confocal blue light power used to position cells at the coordinates of the laser (more ...)
Cells “born” without centrioles progress through G1
It is formally possible that we removed the centrosome from G1 cells after the point at which the centrosome becomes dispensable for cell cycle progression. To directly test whether or not RPE1 acentrosomal cells can progress through G1 in its entirety without a centrosome, we removed one of the two centrosomes from cells during late S–G2 (after centriole replication). The de novo pathway is inhibited as long as cells contain even a single centriole (La Terra et al., 2005
), and cells that enter mitosis with a single centrosome will divide into two daughters, one inheriting a centrosome and the other entering G1 without a centrosome (Khodjakov et al., 2000
; Khodjakov and Rieder, 2001
The 20 cells that entered mitosis within 8 h after the microsurgery all divided in a bipolar fashion. Shortly after mitosis, we added BrdU to the medium and later used an ~1-s fluorescence examination to identify which daughter did not contain centrioles. We followed the acentrosomal daughter cells for at least 36 h after mitosis to determine whether they progressed through interphase to mitosis. Those that did not were fixed at 36 h to assay for BrdU incorporation to determine if they progressed into S phase. All centrosome-containing daughters progressed to the next mitosis.
4 out of 20 acentrosomal daughter cells arrested in G1 after the first mitosis, as determined by lack of BrdU incorporation. Six acentrosomal daughters progressed into S phase, but did not enter mitosis within 36 h. The remaining 10 progressed through interphase and through the next mitosis (). An example of such an acentrosomal cell progressing from one mitosis to the next is shown in (an additional example is shown in Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200607073/DC1
). These observations reveal that 80% of the acentrosomal daughter cells progress through the entirety of G1 without a centrosome. Because precentrioles do not form during G1 (La Terra et al., 2005
; this study), the G1 progression we observed in this study is not supported by the assembly of precentrioles. This notion is further supported by one case of a G2 microsurgery in which we observed that the daughter cell “born” without centrioles progressed through the next mitosis, and no centrin foci were observed in the granddaughter cells.
Figure 4. RPE1 cells “born” without centrosomes progress through G1 in its entirety. (A) Summary of data. G2 cells were identified by the presence of four centrin dots (two centrosomes) and were cut to remove one centrosome. They were then followed (more ...)
These results are not peculiar to microsurgery or the cell type. We ablated one of the two centrosomes in G2 HMEC cells. In three experiments, all cells divided in a bipolar fashion between 1.5 and 7 h after the ablations, and the daughters born without centrioles progressed through interphase into the second mitosis. In two separate experiments on HMEC cells, we laser ablated one centrosome in metaphase cells, and then followed the daughters not inheriting centrioles. We found that both acentriolar daughters proceeded through interphase into mitosis within 24 h, as did their centriole-containing sisters (unpublished data).
Stress influences G1 progression
These observations are in clear contrast to previous reports that centrosome removal or the knockdown/displacement of a wide variety of centrosomal proteins lead to a G1 arrest in untransformed cells (for reviews see Sluder, 2005
; Doxsey et al., 2005a
). Insight into a possible reason for these fundamentally different observations was first suggested by our anecdotal observations that prolonged exposure of acentrosomal cells to 488-nm blue light, which is used to excite GFP, correlated with a G1 arrest, whereas control cells in the same microscope field continued through multiple cell cycles. This observation led us to ask if loss of the centrosome, by itself, is a stress for the cell, and if any additional stress (in this study, blue light) causes it to arrest in G1.
To test this notion, we used microsurgery of G1 cells to produce acentrosomal cells, and, in the same microscope field, performed control amputations of equivalent cell areas on other cells. The untouched cells in the same fields served as controls. 30 min after the cutting operations, we exposed the field of cells to various durations of 488-nm blue light (18 nW/μm2 at the field plane; 580 μW output at the objective lens) to controllably stress all the cells. The field was then followed by time-lapse microscopy to determine which cells progressed through interphase into mitosis and which did not. We used blue light as an exogenous stress, because it is deleterious to cells and dosages can be precisely controlled by computer control of the shutter on the epifluorescence pathway. Our results do not depend on knowing the details of how blue light stresses a cell; we use blue light only as an experimental tool. In this regard, other stressors, such as pH and composition of the media, could, in principle, be used in our application.
Our results, which are summarized in , reveal that the acentrosomal cells are most sensitive to blue light–induced stress, the control-amputated cells are sensitive, but less so, and the untouched control cells are not affected by blue light exposures within the range we used. The brevity of the blue light exposures that lead to a G1 arrest of acentrosomal cells and control-amputated cells reveals how sensitive they are to blue light, relative to the untouched controls. All untouched control cells exposed in G1 to 20–40 s exposures of blue light progress through interphase to mitosis (n = 34).
Figure 5. Microsurgery and centrosome removal predispose RPE1 cells to p38-dependent G1 arrest. For each experiment, a G1 cell was microsurgically cut to remove the centrosome and, in the same field, a cell was cut to amputate an equivalent portion of the cytoplasm (more ...)
Stresses such as UV light, heat, and osmotic shock result in activation of the MAP kinase p38, which in turn leads to G1 arrest by influencing cyclin D1 stability, as well as the phosphorylation of p53 and pRb (for reviews see Ambrosino and Nebreda, 2001
; Zarubin and Han, 2005
; Harris and Levine, 2005
). To assess the involvement of the p38 MAPK pathway in a G1 arrest of acentrosomal cells, we added SB 203580, which is an inhibitor of the p38 stress kinase (Kumar et al., 1999
), to the medium 30 min before the G1 cuts were made. 30 min after the microsurgery, the fields were exposed to blue light for either 2 or 4–5 s. We found that none of the acentrosomal cells or control amputees arrested in G1, even after 4–5 s exposures (, bottom).
These results are not peculiar to centrosome removal by microsurgery. When widefield excitation with blue light was used to position the centrosomes of RPE1 cells at the coordinates of the laser beam, and to later observe the cells, we found that laser ablation of centrosomes in G1 produced an interphase arrest in 14/16 cells, whereas the adjacent control cells progressed through interphase to mitosis. More recently, system upgrades, including the use of a spinning disk confocal (used at 107 μW output at the objective lens), allowed us to significantly reduce the intensity of blue light used to image the centrosomes. Under such conditions, we found that 7/8 RPE1 cells progressed to mitosis after complete centrosome ablation during early G1.
We also investigated how blue light exposure influences the G1 cell cycle progression of acentrosomal HMEC cells when two intensities of blue light were used to image centrosomes and monitor their ablation (the percentage of cells arresting in interphase under various conditions is summarized in ). At the lower blue light level (107 μW output at the objective lens), G1 laser irradiation of the cytoplasm adjacent to the centrosome (no damage to centrosome), damage to the centrosome in the form of ablation of one centriole, or the ablation of both centrioles did not give a substantial incidence of cell cycle arrest. Only one cell arrested (, top, line A). In contrast, at an approximately fourfold higher blue light level used to observe the cells (450 μW output at the objective lens for the same total amount of time), laser irradiation during G1 of the cytoplasm adjacent to the centrosome (control irradiation) led to an interphase arrest in approximately one third of the cells (, top, line B). Ablation of one or both centrioles during G1 arrested two thirds of the cells in interphase.
To determine if activation of the p38 stress-activated kinase plays a role in these observed interphase arrests at the higher blue light level, we repeated these ablations with cells continuously exposed to the p38 inhibitor SB 203580. Cytoplasmic laser irradiations, ablation of one centriole, or ablation of both centrioles did not lead to a G1 arrest in any of the cells (, top, line C). Together, these results indicate that physical damage to the centrosome, or its complete ablation, promotes a p38 stress-activated kinase–mediated interphase arrest when the HMEC cells are additionally stressed by blue light.
G1 progression of acentrosomal BSC-1 cells
We previously reported that after microsurgical removal of the centrosome during interphase, BSC-1 cells progressed through mitosis and 88% arrested in G1 after that mitosis (Hinchcliffe et al., 2001
). To test how our previous results fit with our current findings, we reinvestigated the consequences of microsurgical removal of the interphase centrosome from BSC-1 cells using our current methodology. Our current methods involve several system upgrades, such as the use of a mechanically more stable micromanipulator and more sensitive video cameras that allow ~64-fold lower green light (546 nm) intensities for time-lapse imaging (4.7 nW output from the condenser vs. 302 nW condenser output previously used). Also, after microsurgery, we now remount the cell bearing coverslips into sealed observation chambers (Sluder et al., 2005
) for time-lapse observations, rather than leaving them in oil-capped micromanipulation preparations. The sealed chambers contain an approximately threefold higher volume of medium (600 μl).
We cut a BSC-1 cell to remove the centrosome, and we performed a control amputation of cytoplasm from another in the same field of view. The untouched cells served as controls. For some experiments, the coverslips were transferred after the microsurgery to sealed observation chambers, as we have done after the microsurgery of RPE1 cells. For other experiments, we left the cells in the oil-capped micromanipulation preparations for time-lapse observations. Using our current observation conditions, we found that 14% of the acentrosomal cells arrested in interphase after mitosis, whereas none of the control amputation or untouched controls arrested in interphase (). When the cells were left in the micromanipulation chambers for time-lapse filming, 33% of the acentrosomal cells and 13% of the control-amputated cells arrested in interphase after mitosis; none of the untouched controls arrested.
Figure 6. G1 progression of acentrosomal and control cut BSC-1 cells under various experimental conditions. For each experiment, an interphase cell was microsurgically cut to remove the centrosome and, in the same field, a cell was cut to amputate an equivalent (more ...)
To test if acentrosomal BSC-1 cells are sensitive to the level of continuous green light used for time-lapse observations, we performed the same experiments, but raised the illumination intensity to 1,170-nW condenser output (3.8-fold higher than the Hinchcliffe et al. (2001)
study). Our results () show that for both the sealed and oil-capped micromanipulation chambers used for filming, a higher percentage of the acentrosomal cells and control cut cells arrest in interphase after mitosis under these higher green light conditions. Notably, none of the untouched control cells arrested under any of these conditions.