Here, we used a quantitative 4D imaging approach to analyze the processes of nucleolar breakdown and reassembly during mitosis in single live cells. HeLa cell lines were constructed that stably express either one or two FP-tagged markers for either different nucleolar subcompartments, nuclear lamina components, nuclear transport reporters and/or chromatin. By establishing double-transformed stable cell lines and parallel transient transfection, we could perform multi-wavelength 3D microscopy over time to correlate changes in the relative distributions and concentrations of multiple nuclear marker proteins in the same live cell during mitosis. The data for nucleolar disassembly and reassembly during mitosis () show that nucleolar breakdown begins with the loss of RNA polymerase I subunits from FCs, before the onset of NE breakdown. The subsequent disassembly of the DFC and GC subcompartments coincides with NE disassembly.
Figure 8. The dynamics of nucleolus during mitosis. The nucleolar disassembly and reassembly pathway are shown in a–c and d–i, respectively. During disassembly, (a and b) the RPA39 (blue) dissociates from the nucleoli ~2 min before the loss (more ...)
The RPA39 dissociates from the NORs during a brief period within metaphase, although the rRNA promoter binding factor, UBF, remains bound (). The pathway of nucleolar reformation in live cells after mitosis showed a strictly defined and reproducible temporal sequence of incorporation of markers in the order of FC, DFC, and finally GC. However, this did not correlate with the order in which the factors were imported into nuclei, as the DFC marker FIB accumulated early in nuclei but only later was incorporated into nucleolar foci, coincident with the maximal nuclear import of RNA polymerase I.
The advantages of live cell imaging have recently been applied to study several dynamic nuclear processes (Clute and Pines, 1999
; Gerlich et al., 2001
; Beaudouin et al., 2002
; Gerlich and Ellenberg, 2003a
; Prasanth et al., 2003
). A feature of the live cell approach used here is that we can conduct quantitative studies on nucleolar dynamics during mitosis at the single cell level. Moreover, we have directly correlated quantitatively the temporal changes in each of the separate nucleolar, NE and chromatin components within the nucleus. This has allowed us to detect small differences in the timing of events that would not be apparent by conventional immunofluorescence approaches, where temporal information is not available. Similarly, biochemical methods sample the mean properties of cell populations, rather than the behavior or sequence of events in individual cells. For example, we could reproducibly detect a timing difference as small as ~4 min in the loss of RPA39 from the FC, before loss of DFC or GC markers during nucleolar breakdown. Quantitative analyses of these time-lapse data obtained from multiple cells in each experiment also provided information regarding the kinetic behavior of nuclear proteins across the cell population and showed the degree of variation between nuclei.
Recent studies on the dynamics of nucleoli during mitosis have focused mainly on the process of postmitotic nucleolar reformation (Dundr et al., 2000
; Savino et al., 2001
). Two previous studies have included the use of live cell imaging to analyze events during the formation of nucleoli (Dundr et al., 2000
; Savino et al., 2001
). Both studies used a combination of immunofluorescence and cell lines expressing a single GFP-tagged marker to analyze the temporal order of formation of PNBs and nucleoli. In particular, both the composition of PNBs, which accumulate partially processed rRNA precursors and associated components, and how these components are subsequently transferred into NORs for nucleolar assembly have been studied (Jimenez-Garcia et al., 1994
; Dundr et al., 2000
; Savino et al., 2001
). Here, we have focused on quantitating the temporal pathway of nucleologenesis as well as studying the spatial organization of reforming NORs. In each of the live cells analyzed, multiple FP-tagged markers used for different nucleolar subcompartments allows us to distinguish reforming NORs and nucleoli from PNBs. For example, PNBs do not contain RNA polymerase I, which reassociates with the NOR early during nucleologenesis. Thus, we could perform a correlative as well as quantitative analysis of components from all three nucleolar subcompartments in parallel. More importantly, we have extended these analyses by comparing the processes of nucleolar breakdown as well as reassembly in the same cells. A novel conclusion from this work is that RNA polymerase I subunits RPA39 and RPA20 transiently leave the NORs during metaphase, whereas UBF remains associated with NORs throughout mitosis. Our findings are supported by the data from a recent independent study showing that other RNA polymerase I subunits, specifically, RPA194, RPA39, and RPA16, but not RPA43, also leave the NORs during metaphase (Dundr, M., and T. Misteli, personal communication). These results differ from the current view that RNA polymerase I, as well as UBF, remains associated with NORs throughout mitosis, based on immunofluorescence data using fixed cells, where it is difficult to detect a transient absence of RNA polymerase I. Our data in contrast indicate that multiple RNA polymerase I subunits either leave the chromosomes transiently, or else decrease in concentration below our detection limit, for a brief period at metaphase. We note that this can explain the previous observation based on high resolution in situ hybridization studies that metaphase chromosomes do not contain nascent rRNA (Weisenberger and Scheer, 1995
). Our data are also consistent with the findings from run-on assays that rRNA genes in metaphase chromosomes, whereas still in the same open configuration as interphase chromosomes, are less transcriptionally active (Conconi et al., 1989
). Our present data therefore indicate that the mitotic behavior of RNA polymerase I may be more similar to RNA polymerase II than was previously apparent.
We observe distinct kinetic behavior of individual proteins during nucleolar disassembly ( D and C). For example, although dissociation of B23 and RL27 from nucleoli initiates later than RPA39 or FIB, their dissociation rate is higher. Both GC components dissociate from nucleoli at similar rates, with comparable kinetics to cytoplasmic dispersal of IBB upon NE breakdown, which is most likely a diffusion-limited event. Although the molecular mechanism of nucleolar disassembly remains poorly understood, the present data raise the possibility that distinct processes could operate sequentially and/or independently during the disassembly of FC, DFC, and GC subcompartments. We observe that RPA39 leaves the nucleolus before breakdown of nuclear lamina components, whereas the DFC and GC markers are lost during the period of nuclear lamina breakdown. It is known from previous studies that lamina disassembly is triggered via cyclin B-CDK1 mediated phosphorylation of multiple components, including LB1 (Pines and Rieder, 2001
; Burke and Ellenberg, 2002
). Coincidently, the same cyclin complex is involved in repression of mitotic ribosomal transcription and nucleolar reformation. For example, phosphorylation of UBF and transcription factor SL1 by CDK1 causes shut-off of RNA polymerase I transcription (Heix et al., 1998
; Klein and Grummt, 1999
). The CDK1 inhibitor roscovitine also causes reactivation of RNA polymerase I transcription during mitosis but not the recruitment of rRNA processing factors to the rRNA gene repeats (Sirri et al., 2000
). Our data indicate that the loss of RNA polymerase I and hence transcription of rRNA genes is likely to be the initial event during mitotic disassembly of nucleoli. However, loss of rRNA gene transcription alone may not be sufficient to cause subsequent disassembly of the entire nucleolus. For example, although inhibition of ribosomal transcription during interphase causes RNA polymerase I subunits to leave nucleoli in vivo (unpublished data), the inhibition of ribosomal transcription by Actinomycin D in isolated nucleoli does not cause nucleoli to disintegrate in vitro (unpublished data). Therefore, we propose that the mitotic disassembly of the DFC and GC subcompartments is a result of an active mechanism rather than an indirect effect of the loss of transcriptional activity. This is consistent with a recent study that germ cell proteins FRGY2a and FRGY2b can reversibly disassemble somatic nucleoli in Xenopus
egg cytoplasm independent of rRNA transcription (Gonda et al., 2003
), suggesting that transcription activity and nucleolar integrity may not be obligatorily coupled. It will thus be interesting in future to test whether molecular mechanisms such as phosphorylation by the cyclin B–CDK1 complex may play a role in either RPA39 dissociation from FCs or in other steps in the nucleolar breakdown pathway.
In contrast with the precise temporal regulation in the order of events during both disassembly and reformation of nucleoli during mitosis, higher order aspects of nucleologenesis appear to be less strictly controlled. A statistical evaluation of the number of FC foci and functional nucleoli that appear after mitosis suggested that daughter nuclei are more similar to each other than to unrelated nuclei exiting mitosis elsewhere, consistent with the expected conservation in global chromosome positioning (Gerlich and Ellenberg, 2003b
; Walter et al., 2003
). Nonetheless, we observe statistically significant differences between the two daughter nuclei. At least from the point at which RNA polymerase I subunits reassociate with NORs, it appears that local effects, which can differ between daughter nuclei, influence the overall pathway of nucleologenesis. Effects acting at the level of the local chromosome environment, such as variations in chromosome orientation and decondensation (Thomson et al., 2004
), variable activation of rRNA genes within the repeat clusters and variation in protein concentration kinetics (Dundr et al., 2002a
), may all influence both the association of RNA polymerase I with NORs and the probability of NOR fusion and hence the number of active nucleoli formed. It is interesting to compare this view with a recent FISH analysis on the process of induced RNA polymerase II gene activation at the single cell level (Levsky et al., 2002
). Both this FISH analysis and our work suggest that the pattern and level of gene activation varies at the single cell level, which had not been apparent from previous biochemical studies from cell populations. Although it is increasingly appreciated that nuclear structure, including the relative 3D distribution of chromosomes (Parada and Misteli, 2002
; Bickmore and Chubb, 2003
), can influence gene expression by RNA polymerase II, we infer from this work that nuclear structure may also have an important effect on events connected with RNA polymerase I transcription.