Investigation of cell cycle-associated structural reorganization in nucleolar FC/DFCs from mouse MFC cells by electron microscopy

Investigation of cell cycle-associated structural reorganization in nucleolar FC/DFCs from mouse... Abstract Nucleolus structure alters as the cell cycle is progressing. It is established in telophase, maintained throughout the entire interphase and disassembled in metaphase. Fibrillar centers (FCs), dense fibrillar components (DFCs) and granular components (GCs) are essential nucleolar organizations where rRNA transcription and processing and ribosome assembly take place. Hitherto, little is known about the cell cycle-dependent reorganization of these structures. In this study, we followed the nucleolus structure during the cell cycle by electron microscopy (EM). We found the nucleolus experienced multiple rounds of structural reorganization within a single cell cycle: (1) when nucleoli are formed during the transition from late M to G1 phase, FCs, DFCs and GCs are constructed, leading to the establishment of tripartite nucleolus; (2) as FC/DFCs are disrupted at mid-G1, tripartite nucleolus is gradually changed into a bipartite organization; (3) at late G1, the reassembly of FC/DFCs results in a structural transition from bipartite nucleolus towards tripartite nucleolus; (4) as cells enter S phase, FC/DFCs are disassembled again and tripartite nucleolus is thus changed into a bipartite organization. Of note, FC/DFCs were not observed until late S phase; (5) FC/DFCs experience structural disruption and restoration during G2 and (6) when cells are at mitotic stage, FC/DFCs disappear before nucleolus structure is disassembled. These results also suggest that bipartite nucleolus can exist in higher eukaryotes at certain period of the cell cycle. As structures are the fundamental basis of diverse cell activities, unveiling the structural reorganization of nucleolar FCs and DFCs may bring insights into the spatial–temporal compartmentalization of relevant cellular functions. bipartite/tripartite, cell cycle, reorganization, FC/DFC unit, nucleolus, electron microscopy Introduction Nucleolus is a fundamentally important subcellular structure in most eukaryotes [1,2]. Since 1960s, this unique structure has been recognized as the compartment responsible for rRNA synthesis and ribosome assembly [3,4]. As proteins controlling diverse biological functions, such as cell proliferation and differentiation, are all synthesized in ribosomes, nucleolus is thus fundamentally important for the control of many cellular activities [5–7]. Increasing studies have demonstrated the important role of nucleolus in the regulation of cell cycle, cell senescence, tumorigenesis as well as the assembly of signal-recognition particles [7–14]. However, the connection between nucleolar structures and different biological functions still remains poorly understood. Nucleolus is an essential organelle dedicated to rRNA transcription and processing, products of which directly participate in the assembly of ribosome subunits with related proteins [15–17]. The nucleolus structure in eukaryote is characterized by fibrillar centers (FCs), dense fibrillar components (DFCs), granular components (GCs) as well as intranucleolar chromatin [18–28], among which FC and DFC are tightly associated with each other and often defined as a complex, namely the FC/DFC complex [29–31]. It has also been shown that FCs, DFCs and GCs are the nucleolar structures responsible for rDNA replication, rRNA transcription and RNA processing as well as the assembly of ribosome subunits [32–35]. However, the compartmentalization of these different events and the kinetics of their responsible nucleolar structure are still debatable [25,33,36–45]. Although previous studies indicated that FCs and DFCs are stable nucleolar structures [25,33,36–42,44], emerging evidence suggests that structures of FCs and DFCs change during the cell cycle [4,31,46,47]. This may explain, at least in part, the variable locations in the nucleolus for rDNA replication, rRNA transcription and processing as well as the assembly of ribosome subunits. We previously found that FC/DFCs of HeLa cells experience structural disruption and restoration during S phase [31]. However, it remains elusive whether FC/DFC structure is also reorganized in other stages of the cell cycle. If so, are these structural changes responsible for any cellular function? In this study, we used electron microscopy (EM) to follow the structural dynamics of nucleoli and revealed that FC/DFCs went through multiple rounds of reorganization during the cell cycle in MFC mouse cells. Material and method Cell culture and synchronization Mouse forestomach carcinoma (MFC) cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and incubated at 37°C in 5% CO2. As described previously [31], exponentially growing cells were incubated with thymidine (2 mM) at 37°C for 18 h. After washing with phosphate buffered saline (PBS, pH 7.0) for three times, cells were kept in fresh medium without thymidine for 5 h. Again, thymidine (2 mM) was added, and cells were synchronized for another 18 h. Following PBS washing, cells were released in fresh medium and collected every 30 min for an entire cell cycle. In the end, synchronized cells were analyzed with a Coulter EPICS XL-MCL flow cytometer (BECKMAN Coulter Corp., Hialeah, FL, USA). Ultrathin section preparation and electron microscopy As described previously [31], synchronized MFC cells were fixed with 2.5% glutaraldehyde (0.1 mol l−1 PBS, pH 7.0) for 2 h at 4°C. Subsequently, cells were rinsed with PBS for 30 min, fixed with 1% osmium tetraoxide for 2 h and dehydrated in ethanol with increasing concentrations at room temperature. Cell pellets were embedded with Epon812. Ultrathin sections were prepared with a Reichert Jung Ultracut and stained with uranyl acetate and lead citrate. Slides were imaged under a Hitachi-7500 transmission EM. Results Establishment of a synchronization system for MFC cell cycle analysis To systematically investigate the structural kinetics of MCF nucleolus during the cell cycle, we established a synchronization system. Cells were arrested at G1/S border by double-thymidine and collected at various time points (every 30 min) upon the release. According to the releasing time (from 0 to 13 h), samples were defined as T0, T0.5, T1, T1.5, T2, …… and T13 to indicate different stages of the cell cycle. Representative cell cycle profiles (Fig. 1) show that T0–T3 demonstrate S phase, T3–T7 represent G2 phase, T7–T8 indicate M phase and T8–T13 are G1 phase. In parallel, we confirmed these distinct cell cycle phases by nuclear morphology (Fig. 2) [48–51]. As anticipated, typical G1 cells (T10.5) possess condensed chromatin clumps (Fig. 2a, arrows) with irregular shape and distinct FC/DFCs (Fig. 2a, arrowheads) were observed in the nucleoli. When cells are in S phase (T0.5), thin chromatin fibers (Fig. 2b, arrows) are distributed sparsely and FC/DFCs are absent from the large nucleoli (Fig. 2b, arrowheads). In G2 (T3.5), chromatin fibers are condensed into thick ones (Fig. 2c, arrows) and FC/DFCs (Fig. 2c, arrowheads) in the nucleoli are under disruption. Given that the process of mitosis is typically divided into prophase, metaphase and telophase, we therefore further differentiated these stages in our system. In prophase (T7), chromosomes (Fig. 2d, arrows) are formed, and the nucleolus organization is clear. Moreover, FC/DFCs in nucleoli (Fig. 2d, arrowheads) are disassembled. As cells reach metaphase (T7.5), chromosomes are further compacted and nuclear membranes are collapsed (Fig. 2e). Meanwhile, nucleoli are not visible any more (Fig. 2e). Later, two sets of separated daughter chromosomes (Fig. 2f, arrows) become decondensed in telophase (T8). Fig. 1. View largeDownload slide Cell cycle analysis of synchronized MFC cells. S phase, T0–T3; G2 phase, T3–T7; M phase, T7–T8; G1 phase, T8–T13. Fig. 1. View largeDownload slide Cell cycle analysis of synchronized MFC cells. S phase, T0–T3; G2 phase, T3–T7; M phase, T7–T8; G1 phase, T8–T13. Fig. 2. View largeDownload slide Investigation of nuclei from different stages of the cell cycle. (a) Representative nucleus from G1 (T10.5) contains condensed chromatin lumps (a, arrows) with irregular size and nucleolus organization with FC/DFC structures (a, arrowheads). (b), In the nucleus from S (T0.5), thin chromatin fibers (b, arrows) distribute sparsely. Large nucleolus (b, arrowheads) does not contain FC/DFCs. (c) In nucleus from G2 (T3.5), thick chromatin fibers (c, arrows) are condensed and nucleolar FC/DFCs (c, arrowheads) are under disruption. (d–f) Nucleli in M (T7–T8). (d) When cells are in prophase, chromosomes (d, arrows) are formed and nucleolar FC/DFCs are disassembled. (e) In metaphase, chromosomes (e, arrows) are completely formed and nucleolus organizations disappear. (f) Sister chromatid separation occurs in telophase and associates with the establishment of two daughter nuclei (f, arrows). Scale bar, 1 μm. Fig. 2. View largeDownload slide Investigation of nuclei from different stages of the cell cycle. (a) Representative nucleus from G1 (T10.5) contains condensed chromatin lumps (a, arrows) with irregular size and nucleolus organization with FC/DFC structures (a, arrowheads). (b), In the nucleus from S (T0.5), thin chromatin fibers (b, arrows) distribute sparsely. Large nucleolus (b, arrowheads) does not contain FC/DFCs. (c) In nucleus from G2 (T3.5), thick chromatin fibers (c, arrows) are condensed and nucleolar FC/DFCs (c, arrowheads) are under disruption. (d–f) Nucleli in M (T7–T8). (d) When cells are in prophase, chromosomes (d, arrows) are formed and nucleolar FC/DFCs are disassembled. (e) In metaphase, chromosomes (e, arrows) are completely formed and nucleolus organizations disappear. (f) Sister chromatid separation occurs in telophase and associates with the establishment of two daughter nuclei (f, arrows). Scale bar, 1 μm. Structural changes of nucleolar FC/DFCs during the cell cycle To accurately characterize ultrastructural changes of the nucleolus, we carried out EM in synchronized MFC cells and followed their nucleolar organizations during the cell cycle. Notably, MFC nucleolus went through multiple rounds of structural reorganization within a single cell cycle: (1) When nucleoli are formed during the transition from M to G1, FCs, DFCs and GCs are constructed. This leads to the establishment of tripartite nucleolus; (2) As FC/DFCs are disrupted at mid-G1, tripartite nucleolus is gradually changed into a bipartite organization consisting of fibrillar and granular components; (3) At late G1, the reassembly of FC/DFCs results in the structural transition of bipartite nucleolus towards tripartite nucleolus; (4) As cells enter S phase, FC/DFCs are disassembled again and tripartite nucleolus is thus changed into a bipartite organization. Notably, FC/DFCs are not present until late S phase; (5) FC/DFCs experience structural disruption and restoration during G2 and (6) When cells are at mitosis, FC/DFCs disappear before nucleolus structure is disassembled. The construction of nucleolar FC/DFCs at early G1 We observed that, as cells were moving from M to G1, small nucleoli (Fig. 3a, arrows) with evenly distributed granular and fibrillar components started to appear. FC/DFCs were not formed yet in the nucleolus. When cells entered G1 (T8–T8.5), bipartite nucleolus consisting of fibrillar (Fig. 3b and c, arrows) and granular components (Fig. 3b and c, triangles) became larger. Regions of low electron density (Fig. 3b and c, arrowheads) surrounded by fibrillar components (Fig. 3b and c, arrows) were observed. FC/DFC-like compartments (Fig. 3d, arrows) and intermediate structures (Fig. 3d, hollow-arrows) formed in T8.5–T9. Distinct FC/DFCs started to appear in T9–T9.5 (Fig. 3e, arrows), suggesting the establishment of tripartite nucleolus in MFC cells. Till T9.5–T10, the construction of FC/DFCs (Fig. 3f, arrows) was completed. Fig. 3. View largeDownload slide Construction of nucleolar FC/DFCs from early to mid-G1. (a) Nucleolus (a, arrows) formed during the transition from M to G1. (b and c) Nucleolus in T8–T8.5 contains fibrillar components (b and c, arrows) mixed with granular components (b and c, triangles). Fibrillar-component-surrounded regions with low electron density (b and c, arrowheads) start to appear. (d) In T8.5–T9, fibrillar components accumulate around regions with low electron density to form FC/DFC-like compartments (d, arrows) and their intermediate structures (d, hollow-arrows). (e and f) Nucleolus in T9–T10 contains mature FC/DFC structures (e and f, arrows). Scale bar, 0.5 μm. Fig. 3. View largeDownload slide Construction of nucleolar FC/DFCs from early to mid-G1. (a) Nucleolus (a, arrows) formed during the transition from M to G1. (b and c) Nucleolus in T8–T8.5 contains fibrillar components (b and c, arrows) mixed with granular components (b and c, triangles). Fibrillar-component-surrounded regions with low electron density (b and c, arrowheads) start to appear. (d) In T8.5–T9, fibrillar components accumulate around regions with low electron density to form FC/DFC-like compartments (d, arrows) and their intermediate structures (d, hollow-arrows). (e and f) Nucleolus in T9–T10 contains mature FC/DFC structures (e and f, arrows). Scale bar, 0.5 μm. The disruption and restoration of nucleolar FC/DFCs in late G1 Compared to FC/DFCs in early G1 (T9–T9.5) (Fig. 3f), round-shaped FCs (Fig. 4a, arrowheads) in mid-G1 (T10–T10.5) were larger and DFCs (Fig. 4a, arrows) were disrupted and expanded into regions (Fig. 4a, triangles) filled with granular components. Gradually, FC/DFCs (Fig. 4b, hollow-arrows) were disrupted in T10.5–T11, and fibrillar components (Fig. 4b, arrows) derived from DFCs (T10–T10.5) were integrated into regions filled with granular components (Fig. 4b, triangles). As a consequence, a transition of nucleolus structure towards bipartite organization was initiated. In T11–T11.5, FC/DFCs were completely disassembled to give rise to the generation of fibrillar (Fig. 4c, arrows) and granular components (Fig. 4c, triangles), which mixed evenly and formed beehive-like structures (Fig. 4c, boxes). Thus, a bipartite nucleolus was established. In T11.5–T12, fibrillar components (Fig. 4d, arrows) surrounded the irregular regions of low electron density, resulting in FC/DFC-like structures (Fig. 4d, hollow-arrows). Of note, a transition from bipartite nucleolus to tripartite nucleolus with newly assembled FC/DFCs (Fig. 4d, arrows) was presented in T12–T12.5 (Fig. 4e). As cells exited G1 (T12.5–T13), the assembly of FC/DFCs (Fig. 4f, arrows) as well as the establishment of tripartite nucleolus were completed. Fig. 4. View largeDownload slide Disassembly and reassembly of nucleolar FC/DFCs from mid to late G1. (a) Nucleolar FCs (a, arrowheads) are relatively large in T10–T10.5. Their surrounding DFCs (a, arrows) are disrupted and expanded into regions (a, triangles) filled with granular components. (b) FC/DFCs (b, hollow-arrows) in T10.5–T11 appear to be unrecognizable and their surrounding DFCs are reorganized into fibrillar components (b, arrows) expanding to granular regions (b, triangles). (c) The structure of FC/DFCs in T11–T11.5 disappears, while fibrillar components (c, arrows) are mixed with granular components (c, triangles) to form beehive-like structures (c, boxes). (d) From T11.5–T12, fibrillar components (d, arrows) accumulate around regions of low electron density (d, arrowheads) to form FC/DFC-like structures (d, double-arrows). (e) In nucleolus from T12–T12.5, FC/DFCs start to appear (e, arrows). (f) In nucleolus from T12.5–T13, FC/DFCs (f, arrows) are completely constructed. Scale bar, 0.5 μm. Fig. 4. View largeDownload slide Disassembly and reassembly of nucleolar FC/DFCs from mid to late G1. (a) Nucleolar FCs (a, arrowheads) are relatively large in T10–T10.5. Their surrounding DFCs (a, arrows) are disrupted and expanded into regions (a, triangles) filled with granular components. (b) FC/DFCs (b, hollow-arrows) in T10.5–T11 appear to be unrecognizable and their surrounding DFCs are reorganized into fibrillar components (b, arrows) expanding to granular regions (b, triangles). (c) The structure of FC/DFCs in T11–T11.5 disappears, while fibrillar components (c, arrows) are mixed with granular components (c, triangles) to form beehive-like structures (c, boxes). (d) From T11.5–T12, fibrillar components (d, arrows) accumulate around regions of low electron density (d, arrowheads) to form FC/DFC-like structures (d, double-arrows). (e) In nucleolus from T12–T12.5, FC/DFCs start to appear (e, arrows). (f) In nucleolus from T12.5–T13, FC/DFCs (f, arrows) are completely constructed. Scale bar, 0.5 μm. The disruption and restoration of nucleolar FC/DFCs in S The second round of structural reorganization of FC/DFCs took place in S phase. FC/DFC disruption was demonstrated in the nucleoli from early S phase shown in T0–T1 (Fig. 5a–c). Compared to nucleolar structures in late G1 (Fig. 4f), DFCs (Fig. 5a, arrows) associated with large FCs (Fig. 5a, arrowheads) in T0–T0.5 expanded towards regions filled with homogeneously distributed granular components (Fig. 5a, triangles). In T0.5–T1, FC/DFCs were disrupted dramatically and most FC (Fig. 5b and c, arrowheads)-surrounding DFCs (Fig. 5b and c, arrows) expanded to granular regions. In mid-S (T1.5–T2), FC/DFCs were completely changed into fibrillar components (Fig. 5d, arrows), which in turn expanded and mingled with granular components (Fig. 5d, triangles). As fibrillar components were completely integrated into granular regions, FC/DFCs were no longer visible, indicating the re-establishment of bipartite nucleolus (Fig. 5d). In late S (T2–T2.5), regions with low electron density (Fig. 5e, arrowheads) surrounded by fibrillar components (Fig. 5e, arrows) reappeared and FC/DFCs (Fig. 5f, arrows) were constructed during the transition from S to G2 (T2.5–T3). Fig. 5. View largeDownload slide Structural reorganization of FC/DFCs in S phase. (a) FC /DFCs in early S phase (T0–T0.5) appear with clear outlines. Relatively large FCs (a, arrowheads) are surrounded by DFCs (a, arrows) and granular components (a, triangles) are distributed evenly. (b and c) In T0.5–T1, FC (b and c, arrowheads)-surrounding DFCs are disrupted into fibrillar components (b and c, arrows), which are present in granular regions (b and c, triangles). (d) In mid-S phase (T1.5–T2), FC/DFCs are completely disrupted into fibrillar (d, arrows) and granular components (d, triangles). (e) In late S phase (T2–T2.5), structures with low electron density (e, arrowheads) surrounded by fibrillar components (e, arrows) give rise to FC/DFCs. (f) FC/DFCs are completely constructed during the transition from late S to G2 (T2.5–T3). Scale bar, 0.5 μm. Fig. 5. View largeDownload slide Structural reorganization of FC/DFCs in S phase. (a) FC /DFCs in early S phase (T0–T0.5) appear with clear outlines. Relatively large FCs (a, arrowheads) are surrounded by DFCs (a, arrows) and granular components (a, triangles) are distributed evenly. (b and c) In T0.5–T1, FC (b and c, arrowheads)-surrounding DFCs are disrupted into fibrillar components (b and c, arrows), which are present in granular regions (b and c, triangles). (d) In mid-S phase (T1.5–T2), FC/DFCs are completely disrupted into fibrillar (d, arrows) and granular components (d, triangles). (e) In late S phase (T2–T2.5), structures with low electron density (e, arrowheads) surrounded by fibrillar components (e, arrows) give rise to FC/DFCs. (f) FC/DFCs are completely constructed during the transition from late S to G2 (T2.5–T3). Scale bar, 0.5 μm. The disruption and restoration of nucleolar FC/DFCs in G2 The third round of FC/DFCs reorganization occurred when cells progressed from S to G2. The nucleolus in early G2 (T3–T4) exhibited a distinct tripartite pattern with well-structured FC/DFCs (Fig. 6a, arrows). As the cell cycle was progressing, DFC structure (Fig. 6b and c, arrows) altered evidently. When DFCs (Fig. 6b, arrows) expanded into granular regions (Fig. 6b, triangles) in T4–T4.5, FCs (Fig. 6b, arrowheads) appeared as irregular-shaped nucleolar structures. In T4.5–T5, most FC/DFCs disappeared, while a few (Fig. 6c, arrows) were still under dramatic disruption. These observations suggest a switch from tripartite nucleolus to bipartite nucleolus. In T5–T6 (Fig. 6d), cells showed typical bipartite nucleoli with fibrillar (Fig. 6d, arrows) and granular components (Fig. 6d, triangles). In late G2 (T6–T6.5), new FC/DFCs (Fig. 6e, hollow-arrows) appeared, while most regions were still filled with fibrillar and granular components. Later (T6.5–T7.0), distinct FC/DFCs (Fig. 6f, arrows) and granular regions (Fig. 6f, triangles) were established. Fig. 6. View largeDownload slide Disassembly and reassembly of nucleolar FC/DFCs in G2. (a) Nucleolus in early G2 (T3–T4) is tripartite organization with distinct FC/DFCs (a, arrows). (b and c) In T4–T5, fibrillar components (b and c, arrows) around FCs (b and c, arrowheads) are reorganized and expanded into granular regions (b and c, triangles). (d) FC/DFCs are absent in T5–T6, but fibrillar components (d, arrows) distribute in granular regions (d, triangles), resulting in bipartite nucleolus. (e) In T6–T6.5, FC/DFCs (e, hollow-arrows) are gradually established, while some fibrillar components (e, arrows) are still mingled with granular components (e, triangles) in granular regions. (f) In late G2 (T6.5–T7), distinct structure of FC/DFC (f, arrows) and GC (f, triangles) is established. Scale bar, 0.5 μm. Fig. 6. View largeDownload slide Disassembly and reassembly of nucleolar FC/DFCs in G2. (a) Nucleolus in early G2 (T3–T4) is tripartite organization with distinct FC/DFCs (a, arrows). (b and c) In T4–T5, fibrillar components (b and c, arrows) around FCs (b and c, arrowheads) are reorganized and expanded into granular regions (b and c, triangles). (d) FC/DFCs are absent in T5–T6, but fibrillar components (d, arrows) distribute in granular regions (d, triangles), resulting in bipartite nucleolus. (e) In T6–T6.5, FC/DFCs (e, hollow-arrows) are gradually established, while some fibrillar components (e, arrows) are still mingled with granular components (e, triangles) in granular regions. (f) In late G2 (T6.5–T7), distinct structure of FC/DFC (f, arrows) and GC (f, triangles) is established. Scale bar, 0.5 μm. The disruption of nucleolar FC/DFCs in M When cells entered mitosis (T7–T8), FC/DFCs started to be disrupted. FCs formed in late G2 (T6.5–T7) (Fig. 6f) first became larger (Fig. 7a, arrowheads) and their surrounding fibrillar components (Fig. 7a, arrows) expanded into granular regions (Fig. 7a, triangles). Most FC/DFCs were disassembled in prophase, resulting in numerous fibrillar components (Fig. 7b and c, arrows) integrated into granular regions (Fig. 7b and c, triangles) in vicinity. Meanwhile, a few FC/DFCs (Fig. 7b and c, hollow-arrows) remained to be disassembled. In prometaphase, all FC/DFCs were disassembled into fibrillar components (Fig. 7d, arrows) mixing with granular components. Gradually, nucleolus matrix consisted of sparse granular components (Fig. 7e, triangles) and fibrillar components were missing from the nucleolus. When chromatin was compacted into chromosomes in metaphase (Fig. 7f), neither nuclear membrane nor nucleolus structure was visible. Fig. 7. View largeDownload slide Disruption of nucleolar FC/DFCs in M phase. (a) When cells enter M from G2, FCs (a, arrowheads) are relatively large and FC-surrounding DFCs (a, arrows) appear to be disrupted. (b and c) In prophase, most FC/DFCs were disrupted into fibrillar components (b and c, arrows) and few FC/DFCs (b and c, hollow-arrows) remained to be disassembled. (d) In prometaphase, fibrillar components (d, arrows) are mingled with granular components (d, triangles), while FC/DFCs are absent from the nucleolus. (e) Nucleolus matrix consists of sparse granular components (e, triangles). (f) Nucleolus structure and nuclear membrane are absent from the nucleus. Scale bar, 0.5 μm (a–e) and 1 μm (f). Fig. 7. View largeDownload slide Disruption of nucleolar FC/DFCs in M phase. (a) When cells enter M from G2, FCs (a, arrowheads) are relatively large and FC-surrounding DFCs (a, arrows) appear to be disrupted. (b and c) In prophase, most FC/DFCs were disrupted into fibrillar components (b and c, arrows) and few FC/DFCs (b and c, hollow-arrows) remained to be disassembled. (d) In prometaphase, fibrillar components (d, arrows) are mingled with granular components (d, triangles), while FC/DFCs are absent from the nucleolus. (e) Nucleolus matrix consists of sparse granular components (e, triangles). (f) Nucleolus structure and nuclear membrane are absent from the nucleus. Scale bar, 0.5 μm (a–e) and 1 μm (f). Discussion Dynamically regulated FC/DFC structures during the cell cycle Eukaryotic nucleolus is a fundamentally important structure with multiple cell cycle-dependent functions. It is established in telophase, maintained throughout the entire interphase and disassembled in metaphase [4,46,47,52]. FCs, DFCs and GCs are essential nucleolar organizations where rRNA transcription and processing and ribosome assembly occur [1,18,19,21–25,27]. Previous studies reported that the number and size of these nucleolar structures varied according to cell types, cellular activities and metabolic fluctuations [38,44,53–59]. However, less is known regarding the cell cycle-associated reorganization of FC, DFC and GC structure. We previously showed that FC/DFCs from G1 in human HeLa cells experienced structural disruption and restoration during S phase [31,60]. It still remains unclear whether FC/DFCs are also structurally reorganized in other stages of the cell cycle. By using EM, we systematically investigated nucleolar structures during the cell cycle. Notably, the nucleolus went through multiple rounds of structural reorganization within a single cell cycle. (1) FC/DFCs were established in early G1. (2) FC/DFCs were disrupted in mid-G1, giving rise to fibrillar and granular components. (3) In late G1, fibrillar and granular components were assembled into FC/DFCs. (4) FC/DFCs were disassembled as cells entered S and reassembled once again in late S. (5) In G2, FC/DFCs experienced structural disruption and restoration for the third time. (6) When cells were at mitotic stage, FC/DFCs disappeared before nucleolus structure was disassembled. Identification of bipartite nucleolus in higher eukaryotic cells Nucleolus is mainly comprised of fibrillar and granular components [59]. These two basic building components of nucleolar organizations appeared to have variable distribution in different species [22,59,61], resulting in distinct nucleolus patterns, namely bipartite and tripartite organization [59,62,63]. In tripartite nucleolus, fibrillar and granular components give rise to FCs, DFCs and GCs. In contrast, only fibrillar and granular structures have been observed in bipartite nucleolus [59,62,63]. Many studies support the view that tripartite organization is a typical feature for higher eukaryotic nucleoli and bipartite nucleolus only exists in lower eukaryotic cells, such as yeast [59,62,63]. For instance, nucleoli from human HeLa cells were previously characterized as a tripartite organization [61,62]. However, recent studies found bipartite nucleolus in plant and anamniotic vertebrates, and thereby suggested a transition from bipartite to tripartite organization as a part of the evolution process [59,62]. In addition, we have recently demonstrated a structural transition from tripartite organization to bipartite organization in HeLa nucleoli due to the disruption and restoration of FC/DFCs in S phase [31]. To address whether distinct nucleolus patterns are tightly associated with different stages of the eukaryotic cell cycle, we dissected the kinetics of important nucleolar structures in MFC cells. Interestingly, we found that nucleolar FC/DFCs went through multiple rounds of structural reorganization, leading to the switch between bipartite nucleolus and tripartite nucleolus. Notably, bipartite nucleolus can also exist in higher eukaryotes at certain period of the cell cycle. Functional influences from FC/DFCs reorganization FCs, DFCs and GCs are prominent nucleolar structures in nuclei. Accumulating evidence based on EM, such as immunoelectron microscopy and EM autoradiography, has shown that FCs, DFCs and GCs are the organizations responsible for rDNA replication and transcription, RNA processing and ribosome assembly [36–38,40,64,65]. However, the precise localization in these nucleolar compartments for variable cellular functions still remains debatable. For instance, Koberna et al. [44] found that rDNA transcription occurred in DFCs, while others reported that this event took place in either FCs or the junction between FCs and DFCs [66,67]. Given that FCs and DFCs were considered as stable structures maintained throughout the entire interphase in those studies, our present findings on dynamically regulated FC/DFC structures may provide useful evidence to reconcile the variable observations. We found that the structure of FCs and DFCs was disassembled and reassembled during the cell cycle. Structural reorganization of FCs and DFCs contributes to the regulation of their distribution in the nucleolus and thus influence the compartmentalization of specific functions, such as rDNA transcription. As structural changes often reflect alterations in cellar functions, dissecting the cell cycle-regulated dynamics of important structure units may help us understand the coordination and compartmentalization of key cellular functions in eukaryotes. Funding This work was supported by the National Natural Science Foundation of China (No. 31271425 and No. 31600645). Abbreviations FC, fibrillar center; DFC, dense fibrillar component; GC, granular component; MFC, mouse forestomach carcinoma; EM, electron microscopy. References 1 Jordan E G ( 1984 ) Nucleolar nomenclature . J. Cell Sci. 67 ( 1 ): 217 – 220 . Google Scholar PubMed 2 Lam Y W , Trinkle-Mulcahy L , and Lamond A I ( 2005 ) The nucleolus . J. Cell Sci. 118 ( 7 ): 1335 – 1337 . 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Investigation of cell cycle-associated structural reorganization in nucleolar FC/DFCs from mouse MFC cells by electron microscopy

Microscopy , Volume 67 (4) – Aug 1, 2018

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Abstract

Abstract Nucleolus structure alters as the cell cycle is progressing. It is established in telophase, maintained throughout the entire interphase and disassembled in metaphase. Fibrillar centers (FCs), dense fibrillar components (DFCs) and granular components (GCs) are essential nucleolar organizations where rRNA transcription and processing and ribosome assembly take place. Hitherto, little is known about the cell cycle-dependent reorganization of these structures. In this study, we followed the nucleolus structure during the cell cycle by electron microscopy (EM). We found the nucleolus experienced multiple rounds of structural reorganization within a single cell cycle: (1) when nucleoli are formed during the transition from late M to G1 phase, FCs, DFCs and GCs are constructed, leading to the establishment of tripartite nucleolus; (2) as FC/DFCs are disrupted at mid-G1, tripartite nucleolus is gradually changed into a bipartite organization; (3) at late G1, the reassembly of FC/DFCs results in a structural transition from bipartite nucleolus towards tripartite nucleolus; (4) as cells enter S phase, FC/DFCs are disassembled again and tripartite nucleolus is thus changed into a bipartite organization. Of note, FC/DFCs were not observed until late S phase; (5) FC/DFCs experience structural disruption and restoration during G2 and (6) when cells are at mitotic stage, FC/DFCs disappear before nucleolus structure is disassembled. These results also suggest that bipartite nucleolus can exist in higher eukaryotes at certain period of the cell cycle. As structures are the fundamental basis of diverse cell activities, unveiling the structural reorganization of nucleolar FCs and DFCs may bring insights into the spatial–temporal compartmentalization of relevant cellular functions. bipartite/tripartite, cell cycle, reorganization, FC/DFC unit, nucleolus, electron microscopy Introduction Nucleolus is a fundamentally important subcellular structure in most eukaryotes [1,2]. Since 1960s, this unique structure has been recognized as the compartment responsible for rRNA synthesis and ribosome assembly [3,4]. As proteins controlling diverse biological functions, such as cell proliferation and differentiation, are all synthesized in ribosomes, nucleolus is thus fundamentally important for the control of many cellular activities [5–7]. Increasing studies have demonstrated the important role of nucleolus in the regulation of cell cycle, cell senescence, tumorigenesis as well as the assembly of signal-recognition particles [7–14]. However, the connection between nucleolar structures and different biological functions still remains poorly understood. Nucleolus is an essential organelle dedicated to rRNA transcription and processing, products of which directly participate in the assembly of ribosome subunits with related proteins [15–17]. The nucleolus structure in eukaryote is characterized by fibrillar centers (FCs), dense fibrillar components (DFCs), granular components (GCs) as well as intranucleolar chromatin [18–28], among which FC and DFC are tightly associated with each other and often defined as a complex, namely the FC/DFC complex [29–31]. It has also been shown that FCs, DFCs and GCs are the nucleolar structures responsible for rDNA replication, rRNA transcription and RNA processing as well as the assembly of ribosome subunits [32–35]. However, the compartmentalization of these different events and the kinetics of their responsible nucleolar structure are still debatable [25,33,36–45]. Although previous studies indicated that FCs and DFCs are stable nucleolar structures [25,33,36–42,44], emerging evidence suggests that structures of FCs and DFCs change during the cell cycle [4,31,46,47]. This may explain, at least in part, the variable locations in the nucleolus for rDNA replication, rRNA transcription and processing as well as the assembly of ribosome subunits. We previously found that FC/DFCs of HeLa cells experience structural disruption and restoration during S phase [31]. However, it remains elusive whether FC/DFC structure is also reorganized in other stages of the cell cycle. If so, are these structural changes responsible for any cellular function? In this study, we used electron microscopy (EM) to follow the structural dynamics of nucleoli and revealed that FC/DFCs went through multiple rounds of reorganization during the cell cycle in MFC mouse cells. Material and method Cell culture and synchronization Mouse forestomach carcinoma (MFC) cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and incubated at 37°C in 5% CO2. As described previously [31], exponentially growing cells were incubated with thymidine (2 mM) at 37°C for 18 h. After washing with phosphate buffered saline (PBS, pH 7.0) for three times, cells were kept in fresh medium without thymidine for 5 h. Again, thymidine (2 mM) was added, and cells were synchronized for another 18 h. Following PBS washing, cells were released in fresh medium and collected every 30 min for an entire cell cycle. In the end, synchronized cells were analyzed with a Coulter EPICS XL-MCL flow cytometer (BECKMAN Coulter Corp., Hialeah, FL, USA). Ultrathin section preparation and electron microscopy As described previously [31], synchronized MFC cells were fixed with 2.5% glutaraldehyde (0.1 mol l−1 PBS, pH 7.0) for 2 h at 4°C. Subsequently, cells were rinsed with PBS for 30 min, fixed with 1% osmium tetraoxide for 2 h and dehydrated in ethanol with increasing concentrations at room temperature. Cell pellets were embedded with Epon812. Ultrathin sections were prepared with a Reichert Jung Ultracut and stained with uranyl acetate and lead citrate. Slides were imaged under a Hitachi-7500 transmission EM. Results Establishment of a synchronization system for MFC cell cycle analysis To systematically investigate the structural kinetics of MCF nucleolus during the cell cycle, we established a synchronization system. Cells were arrested at G1/S border by double-thymidine and collected at various time points (every 30 min) upon the release. According to the releasing time (from 0 to 13 h), samples were defined as T0, T0.5, T1, T1.5, T2, …… and T13 to indicate different stages of the cell cycle. Representative cell cycle profiles (Fig. 1) show that T0–T3 demonstrate S phase, T3–T7 represent G2 phase, T7–T8 indicate M phase and T8–T13 are G1 phase. In parallel, we confirmed these distinct cell cycle phases by nuclear morphology (Fig. 2) [48–51]. As anticipated, typical G1 cells (T10.5) possess condensed chromatin clumps (Fig. 2a, arrows) with irregular shape and distinct FC/DFCs (Fig. 2a, arrowheads) were observed in the nucleoli. When cells are in S phase (T0.5), thin chromatin fibers (Fig. 2b, arrows) are distributed sparsely and FC/DFCs are absent from the large nucleoli (Fig. 2b, arrowheads). In G2 (T3.5), chromatin fibers are condensed into thick ones (Fig. 2c, arrows) and FC/DFCs (Fig. 2c, arrowheads) in the nucleoli are under disruption. Given that the process of mitosis is typically divided into prophase, metaphase and telophase, we therefore further differentiated these stages in our system. In prophase (T7), chromosomes (Fig. 2d, arrows) are formed, and the nucleolus organization is clear. Moreover, FC/DFCs in nucleoli (Fig. 2d, arrowheads) are disassembled. As cells reach metaphase (T7.5), chromosomes are further compacted and nuclear membranes are collapsed (Fig. 2e). Meanwhile, nucleoli are not visible any more (Fig. 2e). Later, two sets of separated daughter chromosomes (Fig. 2f, arrows) become decondensed in telophase (T8). Fig. 1. View largeDownload slide Cell cycle analysis of synchronized MFC cells. S phase, T0–T3; G2 phase, T3–T7; M phase, T7–T8; G1 phase, T8–T13. Fig. 1. View largeDownload slide Cell cycle analysis of synchronized MFC cells. S phase, T0–T3; G2 phase, T3–T7; M phase, T7–T8; G1 phase, T8–T13. Fig. 2. View largeDownload slide Investigation of nuclei from different stages of the cell cycle. (a) Representative nucleus from G1 (T10.5) contains condensed chromatin lumps (a, arrows) with irregular size and nucleolus organization with FC/DFC structures (a, arrowheads). (b), In the nucleus from S (T0.5), thin chromatin fibers (b, arrows) distribute sparsely. Large nucleolus (b, arrowheads) does not contain FC/DFCs. (c) In nucleus from G2 (T3.5), thick chromatin fibers (c, arrows) are condensed and nucleolar FC/DFCs (c, arrowheads) are under disruption. (d–f) Nucleli in M (T7–T8). (d) When cells are in prophase, chromosomes (d, arrows) are formed and nucleolar FC/DFCs are disassembled. (e) In metaphase, chromosomes (e, arrows) are completely formed and nucleolus organizations disappear. (f) Sister chromatid separation occurs in telophase and associates with the establishment of two daughter nuclei (f, arrows). Scale bar, 1 μm. Fig. 2. View largeDownload slide Investigation of nuclei from different stages of the cell cycle. (a) Representative nucleus from G1 (T10.5) contains condensed chromatin lumps (a, arrows) with irregular size and nucleolus organization with FC/DFC structures (a, arrowheads). (b), In the nucleus from S (T0.5), thin chromatin fibers (b, arrows) distribute sparsely. Large nucleolus (b, arrowheads) does not contain FC/DFCs. (c) In nucleus from G2 (T3.5), thick chromatin fibers (c, arrows) are condensed and nucleolar FC/DFCs (c, arrowheads) are under disruption. (d–f) Nucleli in M (T7–T8). (d) When cells are in prophase, chromosomes (d, arrows) are formed and nucleolar FC/DFCs are disassembled. (e) In metaphase, chromosomes (e, arrows) are completely formed and nucleolus organizations disappear. (f) Sister chromatid separation occurs in telophase and associates with the establishment of two daughter nuclei (f, arrows). Scale bar, 1 μm. Structural changes of nucleolar FC/DFCs during the cell cycle To accurately characterize ultrastructural changes of the nucleolus, we carried out EM in synchronized MFC cells and followed their nucleolar organizations during the cell cycle. Notably, MFC nucleolus went through multiple rounds of structural reorganization within a single cell cycle: (1) When nucleoli are formed during the transition from M to G1, FCs, DFCs and GCs are constructed. This leads to the establishment of tripartite nucleolus; (2) As FC/DFCs are disrupted at mid-G1, tripartite nucleolus is gradually changed into a bipartite organization consisting of fibrillar and granular components; (3) At late G1, the reassembly of FC/DFCs results in the structural transition of bipartite nucleolus towards tripartite nucleolus; (4) As cells enter S phase, FC/DFCs are disassembled again and tripartite nucleolus is thus changed into a bipartite organization. Notably, FC/DFCs are not present until late S phase; (5) FC/DFCs experience structural disruption and restoration during G2 and (6) When cells are at mitosis, FC/DFCs disappear before nucleolus structure is disassembled. The construction of nucleolar FC/DFCs at early G1 We observed that, as cells were moving from M to G1, small nucleoli (Fig. 3a, arrows) with evenly distributed granular and fibrillar components started to appear. FC/DFCs were not formed yet in the nucleolus. When cells entered G1 (T8–T8.5), bipartite nucleolus consisting of fibrillar (Fig. 3b and c, arrows) and granular components (Fig. 3b and c, triangles) became larger. Regions of low electron density (Fig. 3b and c, arrowheads) surrounded by fibrillar components (Fig. 3b and c, arrows) were observed. FC/DFC-like compartments (Fig. 3d, arrows) and intermediate structures (Fig. 3d, hollow-arrows) formed in T8.5–T9. Distinct FC/DFCs started to appear in T9–T9.5 (Fig. 3e, arrows), suggesting the establishment of tripartite nucleolus in MFC cells. Till T9.5–T10, the construction of FC/DFCs (Fig. 3f, arrows) was completed. Fig. 3. View largeDownload slide Construction of nucleolar FC/DFCs from early to mid-G1. (a) Nucleolus (a, arrows) formed during the transition from M to G1. (b and c) Nucleolus in T8–T8.5 contains fibrillar components (b and c, arrows) mixed with granular components (b and c, triangles). Fibrillar-component-surrounded regions with low electron density (b and c, arrowheads) start to appear. (d) In T8.5–T9, fibrillar components accumulate around regions with low electron density to form FC/DFC-like compartments (d, arrows) and their intermediate structures (d, hollow-arrows). (e and f) Nucleolus in T9–T10 contains mature FC/DFC structures (e and f, arrows). Scale bar, 0.5 μm. Fig. 3. View largeDownload slide Construction of nucleolar FC/DFCs from early to mid-G1. (a) Nucleolus (a, arrows) formed during the transition from M to G1. (b and c) Nucleolus in T8–T8.5 contains fibrillar components (b and c, arrows) mixed with granular components (b and c, triangles). Fibrillar-component-surrounded regions with low electron density (b and c, arrowheads) start to appear. (d) In T8.5–T9, fibrillar components accumulate around regions with low electron density to form FC/DFC-like compartments (d, arrows) and their intermediate structures (d, hollow-arrows). (e and f) Nucleolus in T9–T10 contains mature FC/DFC structures (e and f, arrows). Scale bar, 0.5 μm. The disruption and restoration of nucleolar FC/DFCs in late G1 Compared to FC/DFCs in early G1 (T9–T9.5) (Fig. 3f), round-shaped FCs (Fig. 4a, arrowheads) in mid-G1 (T10–T10.5) were larger and DFCs (Fig. 4a, arrows) were disrupted and expanded into regions (Fig. 4a, triangles) filled with granular components. Gradually, FC/DFCs (Fig. 4b, hollow-arrows) were disrupted in T10.5–T11, and fibrillar components (Fig. 4b, arrows) derived from DFCs (T10–T10.5) were integrated into regions filled with granular components (Fig. 4b, triangles). As a consequence, a transition of nucleolus structure towards bipartite organization was initiated. In T11–T11.5, FC/DFCs were completely disassembled to give rise to the generation of fibrillar (Fig. 4c, arrows) and granular components (Fig. 4c, triangles), which mixed evenly and formed beehive-like structures (Fig. 4c, boxes). Thus, a bipartite nucleolus was established. In T11.5–T12, fibrillar components (Fig. 4d, arrows) surrounded the irregular regions of low electron density, resulting in FC/DFC-like structures (Fig. 4d, hollow-arrows). Of note, a transition from bipartite nucleolus to tripartite nucleolus with newly assembled FC/DFCs (Fig. 4d, arrows) was presented in T12–T12.5 (Fig. 4e). As cells exited G1 (T12.5–T13), the assembly of FC/DFCs (Fig. 4f, arrows) as well as the establishment of tripartite nucleolus were completed. Fig. 4. View largeDownload slide Disassembly and reassembly of nucleolar FC/DFCs from mid to late G1. (a) Nucleolar FCs (a, arrowheads) are relatively large in T10–T10.5. Their surrounding DFCs (a, arrows) are disrupted and expanded into regions (a, triangles) filled with granular components. (b) FC/DFCs (b, hollow-arrows) in T10.5–T11 appear to be unrecognizable and their surrounding DFCs are reorganized into fibrillar components (b, arrows) expanding to granular regions (b, triangles). (c) The structure of FC/DFCs in T11–T11.5 disappears, while fibrillar components (c, arrows) are mixed with granular components (c, triangles) to form beehive-like structures (c, boxes). (d) From T11.5–T12, fibrillar components (d, arrows) accumulate around regions of low electron density (d, arrowheads) to form FC/DFC-like structures (d, double-arrows). (e) In nucleolus from T12–T12.5, FC/DFCs start to appear (e, arrows). (f) In nucleolus from T12.5–T13, FC/DFCs (f, arrows) are completely constructed. Scale bar, 0.5 μm. Fig. 4. View largeDownload slide Disassembly and reassembly of nucleolar FC/DFCs from mid to late G1. (a) Nucleolar FCs (a, arrowheads) are relatively large in T10–T10.5. Their surrounding DFCs (a, arrows) are disrupted and expanded into regions (a, triangles) filled with granular components. (b) FC/DFCs (b, hollow-arrows) in T10.5–T11 appear to be unrecognizable and their surrounding DFCs are reorganized into fibrillar components (b, arrows) expanding to granular regions (b, triangles). (c) The structure of FC/DFCs in T11–T11.5 disappears, while fibrillar components (c, arrows) are mixed with granular components (c, triangles) to form beehive-like structures (c, boxes). (d) From T11.5–T12, fibrillar components (d, arrows) accumulate around regions of low electron density (d, arrowheads) to form FC/DFC-like structures (d, double-arrows). (e) In nucleolus from T12–T12.5, FC/DFCs start to appear (e, arrows). (f) In nucleolus from T12.5–T13, FC/DFCs (f, arrows) are completely constructed. Scale bar, 0.5 μm. The disruption and restoration of nucleolar FC/DFCs in S The second round of structural reorganization of FC/DFCs took place in S phase. FC/DFC disruption was demonstrated in the nucleoli from early S phase shown in T0–T1 (Fig. 5a–c). Compared to nucleolar structures in late G1 (Fig. 4f), DFCs (Fig. 5a, arrows) associated with large FCs (Fig. 5a, arrowheads) in T0–T0.5 expanded towards regions filled with homogeneously distributed granular components (Fig. 5a, triangles). In T0.5–T1, FC/DFCs were disrupted dramatically and most FC (Fig. 5b and c, arrowheads)-surrounding DFCs (Fig. 5b and c, arrows) expanded to granular regions. In mid-S (T1.5–T2), FC/DFCs were completely changed into fibrillar components (Fig. 5d, arrows), which in turn expanded and mingled with granular components (Fig. 5d, triangles). As fibrillar components were completely integrated into granular regions, FC/DFCs were no longer visible, indicating the re-establishment of bipartite nucleolus (Fig. 5d). In late S (T2–T2.5), regions with low electron density (Fig. 5e, arrowheads) surrounded by fibrillar components (Fig. 5e, arrows) reappeared and FC/DFCs (Fig. 5f, arrows) were constructed during the transition from S to G2 (T2.5–T3). Fig. 5. View largeDownload slide Structural reorganization of FC/DFCs in S phase. (a) FC /DFCs in early S phase (T0–T0.5) appear with clear outlines. Relatively large FCs (a, arrowheads) are surrounded by DFCs (a, arrows) and granular components (a, triangles) are distributed evenly. (b and c) In T0.5–T1, FC (b and c, arrowheads)-surrounding DFCs are disrupted into fibrillar components (b and c, arrows), which are present in granular regions (b and c, triangles). (d) In mid-S phase (T1.5–T2), FC/DFCs are completely disrupted into fibrillar (d, arrows) and granular components (d, triangles). (e) In late S phase (T2–T2.5), structures with low electron density (e, arrowheads) surrounded by fibrillar components (e, arrows) give rise to FC/DFCs. (f) FC/DFCs are completely constructed during the transition from late S to G2 (T2.5–T3). Scale bar, 0.5 μm. Fig. 5. View largeDownload slide Structural reorganization of FC/DFCs in S phase. (a) FC /DFCs in early S phase (T0–T0.5) appear with clear outlines. Relatively large FCs (a, arrowheads) are surrounded by DFCs (a, arrows) and granular components (a, triangles) are distributed evenly. (b and c) In T0.5–T1, FC (b and c, arrowheads)-surrounding DFCs are disrupted into fibrillar components (b and c, arrows), which are present in granular regions (b and c, triangles). (d) In mid-S phase (T1.5–T2), FC/DFCs are completely disrupted into fibrillar (d, arrows) and granular components (d, triangles). (e) In late S phase (T2–T2.5), structures with low electron density (e, arrowheads) surrounded by fibrillar components (e, arrows) give rise to FC/DFCs. (f) FC/DFCs are completely constructed during the transition from late S to G2 (T2.5–T3). Scale bar, 0.5 μm. The disruption and restoration of nucleolar FC/DFCs in G2 The third round of FC/DFCs reorganization occurred when cells progressed from S to G2. The nucleolus in early G2 (T3–T4) exhibited a distinct tripartite pattern with well-structured FC/DFCs (Fig. 6a, arrows). As the cell cycle was progressing, DFC structure (Fig. 6b and c, arrows) altered evidently. When DFCs (Fig. 6b, arrows) expanded into granular regions (Fig. 6b, triangles) in T4–T4.5, FCs (Fig. 6b, arrowheads) appeared as irregular-shaped nucleolar structures. In T4.5–T5, most FC/DFCs disappeared, while a few (Fig. 6c, arrows) were still under dramatic disruption. These observations suggest a switch from tripartite nucleolus to bipartite nucleolus. In T5–T6 (Fig. 6d), cells showed typical bipartite nucleoli with fibrillar (Fig. 6d, arrows) and granular components (Fig. 6d, triangles). In late G2 (T6–T6.5), new FC/DFCs (Fig. 6e, hollow-arrows) appeared, while most regions were still filled with fibrillar and granular components. Later (T6.5–T7.0), distinct FC/DFCs (Fig. 6f, arrows) and granular regions (Fig. 6f, triangles) were established. Fig. 6. View largeDownload slide Disassembly and reassembly of nucleolar FC/DFCs in G2. (a) Nucleolus in early G2 (T3–T4) is tripartite organization with distinct FC/DFCs (a, arrows). (b and c) In T4–T5, fibrillar components (b and c, arrows) around FCs (b and c, arrowheads) are reorganized and expanded into granular regions (b and c, triangles). (d) FC/DFCs are absent in T5–T6, but fibrillar components (d, arrows) distribute in granular regions (d, triangles), resulting in bipartite nucleolus. (e) In T6–T6.5, FC/DFCs (e, hollow-arrows) are gradually established, while some fibrillar components (e, arrows) are still mingled with granular components (e, triangles) in granular regions. (f) In late G2 (T6.5–T7), distinct structure of FC/DFC (f, arrows) and GC (f, triangles) is established. Scale bar, 0.5 μm. Fig. 6. View largeDownload slide Disassembly and reassembly of nucleolar FC/DFCs in G2. (a) Nucleolus in early G2 (T3–T4) is tripartite organization with distinct FC/DFCs (a, arrows). (b and c) In T4–T5, fibrillar components (b and c, arrows) around FCs (b and c, arrowheads) are reorganized and expanded into granular regions (b and c, triangles). (d) FC/DFCs are absent in T5–T6, but fibrillar components (d, arrows) distribute in granular regions (d, triangles), resulting in bipartite nucleolus. (e) In T6–T6.5, FC/DFCs (e, hollow-arrows) are gradually established, while some fibrillar components (e, arrows) are still mingled with granular components (e, triangles) in granular regions. (f) In late G2 (T6.5–T7), distinct structure of FC/DFC (f, arrows) and GC (f, triangles) is established. Scale bar, 0.5 μm. The disruption of nucleolar FC/DFCs in M When cells entered mitosis (T7–T8), FC/DFCs started to be disrupted. FCs formed in late G2 (T6.5–T7) (Fig. 6f) first became larger (Fig. 7a, arrowheads) and their surrounding fibrillar components (Fig. 7a, arrows) expanded into granular regions (Fig. 7a, triangles). Most FC/DFCs were disassembled in prophase, resulting in numerous fibrillar components (Fig. 7b and c, arrows) integrated into granular regions (Fig. 7b and c, triangles) in vicinity. Meanwhile, a few FC/DFCs (Fig. 7b and c, hollow-arrows) remained to be disassembled. In prometaphase, all FC/DFCs were disassembled into fibrillar components (Fig. 7d, arrows) mixing with granular components. Gradually, nucleolus matrix consisted of sparse granular components (Fig. 7e, triangles) and fibrillar components were missing from the nucleolus. When chromatin was compacted into chromosomes in metaphase (Fig. 7f), neither nuclear membrane nor nucleolus structure was visible. Fig. 7. View largeDownload slide Disruption of nucleolar FC/DFCs in M phase. (a) When cells enter M from G2, FCs (a, arrowheads) are relatively large and FC-surrounding DFCs (a, arrows) appear to be disrupted. (b and c) In prophase, most FC/DFCs were disrupted into fibrillar components (b and c, arrows) and few FC/DFCs (b and c, hollow-arrows) remained to be disassembled. (d) In prometaphase, fibrillar components (d, arrows) are mingled with granular components (d, triangles), while FC/DFCs are absent from the nucleolus. (e) Nucleolus matrix consists of sparse granular components (e, triangles). (f) Nucleolus structure and nuclear membrane are absent from the nucleus. Scale bar, 0.5 μm (a–e) and 1 μm (f). Fig. 7. View largeDownload slide Disruption of nucleolar FC/DFCs in M phase. (a) When cells enter M from G2, FCs (a, arrowheads) are relatively large and FC-surrounding DFCs (a, arrows) appear to be disrupted. (b and c) In prophase, most FC/DFCs were disrupted into fibrillar components (b and c, arrows) and few FC/DFCs (b and c, hollow-arrows) remained to be disassembled. (d) In prometaphase, fibrillar components (d, arrows) are mingled with granular components (d, triangles), while FC/DFCs are absent from the nucleolus. (e) Nucleolus matrix consists of sparse granular components (e, triangles). (f) Nucleolus structure and nuclear membrane are absent from the nucleus. Scale bar, 0.5 μm (a–e) and 1 μm (f). Discussion Dynamically regulated FC/DFC structures during the cell cycle Eukaryotic nucleolus is a fundamentally important structure with multiple cell cycle-dependent functions. It is established in telophase, maintained throughout the entire interphase and disassembled in metaphase [4,46,47,52]. FCs, DFCs and GCs are essential nucleolar organizations where rRNA transcription and processing and ribosome assembly occur [1,18,19,21–25,27]. Previous studies reported that the number and size of these nucleolar structures varied according to cell types, cellular activities and metabolic fluctuations [38,44,53–59]. However, less is known regarding the cell cycle-associated reorganization of FC, DFC and GC structure. We previously showed that FC/DFCs from G1 in human HeLa cells experienced structural disruption and restoration during S phase [31,60]. It still remains unclear whether FC/DFCs are also structurally reorganized in other stages of the cell cycle. By using EM, we systematically investigated nucleolar structures during the cell cycle. Notably, the nucleolus went through multiple rounds of structural reorganization within a single cell cycle. (1) FC/DFCs were established in early G1. (2) FC/DFCs were disrupted in mid-G1, giving rise to fibrillar and granular components. (3) In late G1, fibrillar and granular components were assembled into FC/DFCs. (4) FC/DFCs were disassembled as cells entered S and reassembled once again in late S. (5) In G2, FC/DFCs experienced structural disruption and restoration for the third time. (6) When cells were at mitotic stage, FC/DFCs disappeared before nucleolus structure was disassembled. Identification of bipartite nucleolus in higher eukaryotic cells Nucleolus is mainly comprised of fibrillar and granular components [59]. These two basic building components of nucleolar organizations appeared to have variable distribution in different species [22,59,61], resulting in distinct nucleolus patterns, namely bipartite and tripartite organization [59,62,63]. In tripartite nucleolus, fibrillar and granular components give rise to FCs, DFCs and GCs. In contrast, only fibrillar and granular structures have been observed in bipartite nucleolus [59,62,63]. Many studies support the view that tripartite organization is a typical feature for higher eukaryotic nucleoli and bipartite nucleolus only exists in lower eukaryotic cells, such as yeast [59,62,63]. For instance, nucleoli from human HeLa cells were previously characterized as a tripartite organization [61,62]. However, recent studies found bipartite nucleolus in plant and anamniotic vertebrates, and thereby suggested a transition from bipartite to tripartite organization as a part of the evolution process [59,62]. In addition, we have recently demonstrated a structural transition from tripartite organization to bipartite organization in HeLa nucleoli due to the disruption and restoration of FC/DFCs in S phase [31]. To address whether distinct nucleolus patterns are tightly associated with different stages of the eukaryotic cell cycle, we dissected the kinetics of important nucleolar structures in MFC cells. Interestingly, we found that nucleolar FC/DFCs went through multiple rounds of structural reorganization, leading to the switch between bipartite nucleolus and tripartite nucleolus. Notably, bipartite nucleolus can also exist in higher eukaryotes at certain period of the cell cycle. Functional influences from FC/DFCs reorganization FCs, DFCs and GCs are prominent nucleolar structures in nuclei. Accumulating evidence based on EM, such as immunoelectron microscopy and EM autoradiography, has shown that FCs, DFCs and GCs are the organizations responsible for rDNA replication and transcription, RNA processing and ribosome assembly [36–38,40,64,65]. However, the precise localization in these nucleolar compartments for variable cellular functions still remains debatable. For instance, Koberna et al. [44] found that rDNA transcription occurred in DFCs, while others reported that this event took place in either FCs or the junction between FCs and DFCs [66,67]. Given that FCs and DFCs were considered as stable structures maintained throughout the entire interphase in those studies, our present findings on dynamically regulated FC/DFC structures may provide useful evidence to reconcile the variable observations. We found that the structure of FCs and DFCs was disassembled and reassembled during the cell cycle. 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MicroscopyOxford University Press

Published: Aug 1, 2018

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