| Nucleolus,which contains ribosomal genes and ribosome biogenesis factors,is the most significant and important structure in the eukaryotic cell.Within the nucleolus,three basic structural compartments,including the fibrillar center(FC),the dense fibrillar component(DFC),and the granular component(GC)can be visualized under electron microscope.As FCs are typically surrounded or partially surrounded by DFCs,the complex is therefore often referred to as FC/DFCs.Previously,we and others have shown that nucleolar structures,especially FC/DFCs,in human cancer and plant garlic meristem cells experience dynamic changes during the cell cycle.However,the mechanism responsible for the cell-cycle-dependent reorganization of FC/DFCs still remains unclear.Considering one of the essential components of FC/DFCs is DNA,we tracked rDNA and replication protein,PCNA(proliferating cell nuclear antigen),during the cell cycle to understand the structural changes.Over past decades,studies based on variable research approaches and subjects have led to debatable views on functional sites in nucleolus,where the biogenesis of ribosomal subunits as well as transcription of rDNA and the processing of pre-rRNA take place.Besides,most previous studies substantially focus on the snapshots of nucleolar structures,instead of dynamic changes during the cell cycle.These may contribute to the different observations as well.Therefore,dissecting the structural dynamics of nucleolar structures and their related functions during the cell cycle appears to be pivotal.In this work,we performed conventional electron microscopy,NAMA-Ur DNA-selective staining and Lowicryl K4 M embedding combined with immunoelectron microscopy of DNA/RNA hybrids and nucleolin.We synchronized mouse forestomach carcinoma cells(MFC)by double-thymidine block and followed the structural dynamics of the nucleolar organization during the cell cycle by monitoring PCNA and nucleolin.Based on our data,the main conclusions are as follows:1.MFC cells blocked by double-thymidine at the G1/S boundary are indicated as T0.T1-T2 are samples at S phase and T3-T6 are G2 phase cells.Moreover,T7 is M phase sample and T8-T14 are G1 phase samples.2.Chromatin-bound PCNA appeared in nucleus in early-S phase,enriched in the nucleolus in mid-S phase and gradually decreased in late-S phase.In early-G2 phase,PCNA were undetectable.3.Conventional electron microscopy of MFC cells showed that the nucleolus structure went through dynamically changes during the cell cycle.The nucleolus was disrupted in prophase and reconstructed in telophase.During interphase,the disruption and reconstruction of nucleolar FC/DFCs were repeated a number of times.Of note,transient bipartite organization in nucleolus,which has not been detected in amnion vertebrates other than turtles,was observed in S phase.4.Combining NAMA-Ur DNA-selective staining,conventional electron microscopy and immunofluorescence labeling of PCNA,we found that DNA metabolism and its associated protein contributed to the dynamic changes of nucleolar structure during the cell cycle.5.Using immunoelectron microscopy,we successfully showed that nucleolin in nucleolus distributed differently within the cell cycle.In G1 phase,early-S phase and late-G2 phase,little nucleolin appeared at the periphery of FCs,whereas a large amount of nucleolin accumulated in DFCs and GCs.However,nucleolin was also detected in the central region of FC in mid,late-S as well as early-G2 phase.6.Co-staining of DNA/RNA hybrids and nucleolin showed that the silent rDNA distributed inside FC.Transcriptionally active rDNA partially existed at the periphery of FC and mainly stayed in DFC.The transcription sites of rDNA located at the periphery of FC as well as DFC,where the transcription intensity in DFC was higher than that in FC.In addition,the pre-rRNA processing sites located all over GCs.DFC and the periphery of FC might also contain pre-rRNA processing sites. |
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