Nucleolus

About: Nucleolus is a research topic. Over the lifetime, 5873 publications have been published within this topic receiving 232435 citations. The topic is also known as: GO:0005730 & cell nucleolus.

The Ultrastructure of Synchronized Hela Cells

Abstract: Synchronous populations of mitotic HeLa cells were obtained by the controlled agitation method, and a detailed morphological study of the cells in all phases of the cell cycle was undertaken to correlate variations in cell structures to known coexisting biochemical events. Autoradiographic techniques using tritiated thymidine ( 3 H-TdR) were used to detect S cells, and colcemid was added to some G 2 samples to prevent the cells from going into the next cycle, thus preventing contamination with G 1 cells. The approximate duration (in hours) of the 4 phases were as follows: M = 1.4, G 1 = 8-9, S = 7, G 2 = 4, and the generation time ( T ) = 21 ± 2 h. Randomization of the cell populations became apparent in the G 2 phase (contaminated with S and M cells) and was most likely a result of the genetic make-up of the individual (mixoploid) HeLa cells, nutritional factors (serum lots used), temperature shock, and handling effects. Polyribosomes shifted to monomeric ribosomes during late prophase, at which time nucleoli also break down. These changes are correlated with the drop in protein and RNA synthesis reported for mitotic mammalian cells. The Golgi apparatus persisted in a modified form throughout mitosis. The mid-body forms from the anaphase stem-body and may interfere with the separation of daughter cells. Our studies suggest that the mid-body goes to one of the daughter cells where remnants of this structure were seen in early G 1 cells. Large numbers of polyribosomes and the presence of well-developed nucleoli (many attached to the nuclear envelope) characterized G 1 . These structures, which play a major role in protein and RNA synthesis, persist with slight variations throughout interphase. Dense fibrillar nuclear bodies were prominent in late G 1 cells. Centrioles separate during G 1 , and replicate by orthogonal budding during the S phase. Reproducible labelling patterns which reflect the asynchronous multireplicon nature of DNA synthesis in mammalian cells were characteristic of the various stages of the S phase. Granular nuclear bodies, which were prominent in S and G 2 cells, may correspond to the larger species of heterogeneous nuclear RNA found in HeLa cells. G 2 cells were similar in appearance to S cells. As heterochromatin areas increased in late G 2 and prophase, perichromatin granules (of unknown significance) became prominent. Mitochondria behaved as independent cell organelles throughout the cell cycle. Hypertrophied RER, SER, and annulate lamellae, characterized the cytoplasm of colcemidtreated cells. The above changes are indicative of increased metabolic activity, and these structures may function in the production of colcemid-detoxify enzymes in a manner analogous to that of drug-treated hepatocytes, such as those treated with phenobarbital.

Interdependence of Pes1, Bop1, and WDR12 Controls Nucleolar Localization and Assembly of the PeBoW Complex Required for Maturation of the 60S Ribosomal Subunit

Abstract: The PeBoW complex is essential for cell proliferation and maturation of the large ribosomal subunit in mammalian cells. Here we examined the role of PeBoW-specific proteins Pes1, Bop1, and WDR12 in complex assembly and stability, nucleolar transport, and pre-ribosome association. Recombinant expression of the three subunits is sufficient for complex formation. The stability of all three subunits strongly increases upon incorporation into the complex. Only overexpression of Bop1 inhibits cell proliferation and rRNA processing, and its negative effects could be rescued by coexpression of WDR12, but not Pes1. Elevated levels of Bop1 induce Bop1/WDR12 and Bop1/Pes1 subcomplexes. Knockdown of Bop1 abolishes the copurification of Pes1 with WDR12, demonstrating Bop1 as the integral component of the complex. Overexpressed Bop1 substitutes for endogenous Bop1 in PeBoW complex assembly, leading to the instability of endogenous Bop1. Finally, indirect immunofluorescence, cell fractionation, and sucrose gradient centrifugation experiments indicate that transport of Bop1 from the cytoplasm to the nucleolus is Pes1 dependent, while Pes1 can migrate to the nucleolus and bind to preribosomal particles independently of Bop1. We conclude that the assembly and integrity of the PeBoW complex are highly sensitive to changes in Bop1 protein levels.

Stabilization of PML nuclear localization by conjugation and oligomerization of SUMO-3.

Abstract: The PML gene of acute promyelocytic leukemia (APL) encodes a cell-growth and tumor suppressor. PML localizes to discrete nuclear bodies (NBs) that are disrupted in APL cells, resulting from a reciprocal chromosome translocation t (15;17). Here we show that the nuclear localization of PML is also regulated by SUMO-3, one of the three recently identified SUMO isoforms in human cells. SUMO-3 bears similar subcellular distribution to those of SUMO-1 and -2 in the interphase nuclear body, which is colocalized with PML protein. However, both SUMO-2 and -3 are also localized to nucleoli, a region lacking SUMO-1. Immunoprecipitated PML protein bears SUMO-3 moiety in a covalently modified form, supporting the notion that PML is conjugated by SUMO-3. To determine the functional relevance of SUMO-3 conjugation on PML molecular dynamics, we suppressed SUMO-3 protein expression using a siRNA-mediated approach. Depletion of SUMO-3 markedly reduced the number of PML-containing NBa and their integrity, which is rescued by exogenous expression of SUMO-3 but not SUMO-1 or SUMO-2. The specific requirement of SUMO-3 for PML nuclear localization is validated by expression of SUMO-3 conjugation defective mutant. Moreover, we demonstrate that oligomerization of SUMO-3 is required for PML retention in the nucleus. Taken together, our studies provide first line of evidence showing that SUMO-3 is essential for PML localization and offer novel insight into the pathobiochemistry of APL.

The NBS1–Treacle complex controls ribosomal RNA transcription in response to DNA damage

Abstract: Chromosome breakage elicits transient silencing of ribosomal RNA synthesis, but the mechanisms involved remained elusive. Here we discover an in trans signalling mechanism that triggers pan-nuclear silencing of rRNA transcription in response to DNA damage. This is associated with transient recruitment of the Nijmegen breakage syndrome protein 1 (NBS1), a central regulator of DNA damage responses, into the nucleoli. We further identify TCOF1 (also known as Treacle), a nucleolar factor implicated in ribosome biogenesis and mutated in Treacher Collins syndrome, as an interaction partner of NBS1, and demonstrate that NBS1 translocation and accumulation in the nucleoli is Treacle dependent. Finally, we provide evidence that Treacle-mediated NBS1 recruitment into the nucleoli regulates rRNA silencing in trans in the presence of distant chromosome breaks.