Cajal Bodies, Nucleoli, and Speckles in the Xenopus Oocyte Nucleus Have a Low-Density, Sponge-like Structure

Nuclear organelles, unlike many cytoplasmic organelles, lack investing membranes and are thus in direct contact with the surrounding nucleoplasm. Because the properties of the nucleoplasm and nuclear organelles influence the exchange of molecules from one compartment to another, it is important to understand their physical structure. We studied the density of the nucleoplasm and the density and permeability of nucleoli, Cajal bodies (CBs), and speckles in the Xenopus oocyte nucleus or germinal vesicle (GV). Refractive indices were measured by interferometry within intact GVs isolated in oil. The refractive indices were used to estimate protein concentrations for nucleoplasm (0.106 g/cm3), CBs (0.136 g/cm3), speckles (0.162 g/cm3), and the dense fibrillar region of nucleoli (0.215 g/cm3). We determined similar protein concentrations for nuclear organelles isolated in aqueous media, where they are no longer surrounded by nucleoplasm. To examine the permeability of nuclear organelles, we injected fluorescent dextrans of various molecular masses (3–2000 kDa) into the cytoplasm or directly into the GV and measured the extent to which they penetrated the organelles. Together, the interferometry and dextran penetration data show that organelles in the Xenopus GV have a low-density, sponge-like structure that provides access to macromolecules from the nucleoplasm.

Organization of the double-stranded RNA-activated protein kinase DAI and virus-associated VA RNAI in adenovirus-2-infected HeLa cells

We have examined the cellular distribution of the double-stranded RNA-activated protein kinase DAI in adenovirus 2 (Ad2)-infected and uninfected HeLa cells. In uninfected cells DAI was found to be concentrated in the cytoplasm. In addition, DAI was localized in the nucleoli and diffusely distributed throughout the nucleoplasm. Cells treated with alpha-interferon displayed a similar pattern of distribution for DAI. When RNA polymerase I activity was inhibited by the drug actinomycin D, nucleoli segregated and DAI was found to colocalize with the dense fibrillar region of the nucleoli. During mitosis, the distribution of DAI paralleled that of rRNA. In adenovirus-infected cells the localization of DAI was similar to that in uninfected interphase cells. VA RNAI was detected in Ad2-infected cells by 10–14 hours post-infection as fine dots in the nucleoplasm. By 18–24 hours post-infection, VA RNAI appeared in bigger and more abundant dots in the nucleoplasm and the cytoplasm was intensively labeled. Transient expression of the VA RNAI gene in uninfected cells resulted in a similar localization of the RNA. Our results are consistent with a role for DAI and VA RNAI in protein synthesis and suggest that DAI may play an early role in ribosome biogenesis in the nucleolus in addition to its cytoplasmic role in translation.

SSB-1 of the yeast Saccharomyces cerevisiae is a nucleolar-specific, silver-binding protein that is associated with the snR10 and snR11 small nuclear RNAs.

SSB-1, the yeast single-strand RNA-binding protein, is demonstrated to be a yeast nucleolar-specific, silver-binding protein. In double-label immunofluorescence microscopy experiments antibodies to two other nucleolar proteins, RNA Pol I 190-kD and fibrillarin, were used to reveal the site of rRNA transcription; i.e., the fibrillar region of the nucleolus. SSB-1 colocalized with fibrillarin in a double-label immunofluorescence mapping experiment to the yeast nucleolus. SSB-1 is located, though, over a wider region of the nucleolus than the transcription site marker. Immunoprecipitations of yeast cell extracts with the SSB-1 antibody reveal that in 150 mM NaCl SSB-1 is bound to two small nuclear RNAs (snRNAs). These yeast snRNAs are snR10 and snR11, with snR10 being predominant. Since snR10 has been implicated in pre-rRNA processing, the association of SSB-1 and snR10 into a nucleolar snRNP particle indicates SSB-1 involvement in rRNA processing as well. Also, another yeast protein, SSB-36-kD, isolated by single-strand DNA chromatography, is shown to bind silver under the conditions used for nucleolar-specific staining. It is, most likely, another yeast nucleolar protein.

The nucleolus and meiosis during microsporogenesis in Endymion non-scriptus (L.)

Stages of meiosis from the bluebell Endymion non-scriptus (L.) were studied by electron microscopy. The segregated components of the nucleolus at meiotic prophase underwent fragmentation and dissolution at pachytene-diplotene. Nucleoli were absent during both meiotic divisions and reformed on the nucleolus organizer into a fibrillar mass from scattered fibrillar components at the dyad and tetrad stages. Ti is argued that the fibrillar region shows continuity through nuclear division though undergoing structural transformations in the process. Nucleolar reformation occurs on condensed nucleolus organizers. Processing of the ribosomal precursors and the resumption of RNA synthesis is discussed in relation to the dispersal of the nucleolus organizer into the fibrillar region of the reformed nucleolus.

RNA synthesis in the ultrastructural and biochemical components of the nucleolus of Chinese hamster ovary cells.

A correlated autoradiographic and biochemical study of RNA synthesis in the nucleoli of chinese hamster ovary cells has been made. Quantitative analysis of the labeling indicates that the fibrillar ribonucleoprotein (RNP) component is labeled faster than 80S RNP and 45S RNA molecules, but approaches simultaneously a steady-state 3H to 14C ratio or grains/mum2 after 30 min of [3H]uridine incorporation. On the other hand, the 55S RNP, the 36S + 32S RNA, and the granular RNP components have the same kinetic of labeling with [3H]uridine. These results suggest that the fibrillar and granular RNP components of the nucleolus are the ultrastructural substratum of, respectively, the 80S RNP (45S RNA) and 55S RNP (36S + 32S RNA). The possibility that precursors to 80S RNP exist also in the fibrillar region of the nucleolus is strongly suggested by the rapid labeling of the fibrils on the autoradiographs.

The arrangement and ultrastructure of the musculature, nerves and epidermis, in the tail of the cercaria of Cryptocotyle lingua (Creplin) from Littorina littorea (L.)

The ultrastructure of the caudal muscles of the cercaria of Cryptocotyle lingua is related to the rapid swimming movements. Dorsal and ventral striated longitudinal muscle cells extend in a straight line along the tail length each containing a single myofibril, U-shaped in transverse section and divided into sarcomeres. A-, I- and H-bands are recognizable and the fragmented Z-band consists of a row of dense bars to which the thin myofilaments are attached. A pair of proximal ventro-lateral striated muscles may be responsible for the figure of eight pattern of movement. Large mitochondria are abundant in the non-fibrillar region of the muscle cells. Tubular sarcoplasmic reticulum envelops the myofibril giving off branches at the Z-bands which extend into and across the myofibril alternating with the dense bars. T-tubules are absent but cisternae of the sarcoplasmic reticulum form numerous dyadic junctions with the sarcolemma. Dorsal and ventral motor caudal nerves arise from a nerve plexus behind the tail root; axo-axonal and neuromuscular synapses are frequent. Clear and dense synaptic vesicles occur in the presynaptic terminals.