Localization of rRNA transcribed spacer domains in the nucleolinus and maternal procentrosomes of surf clam (Spisula) oocytes.

The nucleolinus is a nuclear subcompartment long ago posited to play a role in cell division. In a recent study using surf clam oocytes, cytoplasmic foci containing a nucleolinar protein were shown to later recruit γ-tubulin, identifying them as centrosomal precursors. (1) We now demonstrate the presence of structural RNAs from the nucleolinus in these procentrosomes. They include the well-known but poorly understood rRNA-transcribed spacer regions. In situ hybridization revealed a specific and dynamic association of these structural RNAs with the cell division apparatus that extends through the early stages of meiosis. In addition to their bearing on the debate over the nature of centrosome- and spindle-associated RNAs, the observations also suggest that rRNA spacer regions are not simply waste products to be discarded immediately, but may be functional byproducts that play a role in formation of the cell division apparatus.

Composition and dynamics of the nucleolinus, a link between the nucleolus and cell division apparatus in surf clam (Spisula) oocytes.

The nucleolinus is a little-known cellular structure, discovered over 150 years ago (Agassiz, L. (1857) Contributions to the Natural History of the United States of America, First Monograph, Part IIL, Little, Brown and Co., Boston) and thought by some investigators in the late 19th to mid-20th century to function in the formation of the centrosomes or spindle. A role for the nucleolinus in formation of the cell division apparatus has recently been confirmed in oocytes of the surf clam, Spisula solidissima (Alliegro, M. A., Henry, J. J., and Alliegro, M. C. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 13718-13723). However, we know so little about the composition and dynamics of this compartment, it is difficult to construct mechanistic hypotheses or even to be sure that prior reports were describing analogous structures in the cells of mammals, amphibians, plants, and other organisms where it was observed. Surf clam oocytes are an attractive model to approach this problem because the nucleolinus is easily visible by light microscopy, making it accessible by laser microsurgery as well as isolation by common cell fractionation techniques. In this report, we analyze the macromolecular composition of isolated Spisula nucleolini and examine the relationship of this structure to the nucleolus and cell division apparatus. Analysis of nucleolinar RNA and protein revealed a set of molecules that overlaps with but is nevertheless distinct from the nucleolus. The proteins identified were primarily ones involved in nucleic acid metabolism and cell cycle regulation. Monoclonal antibodies generated against isolated nucleolini revealed centrosomal forerunners in the oocyte cytoplasm. Finally, induction of damage to the nucleolinus by laser microsurgery altered the trafficking of α- and γ-tubulin after fertilization. These observations strongly support a role for the nucleolinus in cell division and represent our first clues regarding mechanism.

Rediscovery of the nucleolinus, a dynamic RNA-rich organelle associated with the nucleolus, spindle, and centrosomes.

The nucleolinus is an RNA-rich compartment, closely apposed to or embedded within the nucleolus. Discovered over 150 y ago, fewer than two dozen articles have been published on the nucleolinus, probably because complex histochemical stains are required for its visualization in the great majority of cells. The nucleolinus has been reported in invertebrate oocytes, mammalian and amphibian epithelial cells, neurons, and several transformed cell lines. A prominent nucleolinus, clearly visible with transmitted light microscopes at 10x magnification, is present in each oocyte of the surf clam, Spisula solidissima. We observed a consistent relationship between the nucleolinus and the developing meiotic apparatus following Spisula oocyte activation. Through sonication and sucrose gradient fractionation of purified oocyte nuclei, we isolated nucleolini, extracted their RNA, and prepared an in situ riboprobe (NLi-1), which is associated specifically with the nucleolinus, confirming its unique composition. Other in situ observations revealed a NLi-1 and nucleolinar association with the developing spindle and centrosomes. Laser microsurgery that targeted the nucleolinus resulted in failed meiotic cell division in parthenogenetically activated oocytes and failed mitosis in fertilized oocytes. Although the nucleolinus may be a forgotten organelle, its demonstrated role in spindle formation suggests it deserves renewed attention.

Centrosomal RNA correlates with intron-poor nuclear genes in Spisula oocytes.

The evolutionary origin of centriole/kinetosomes, centrosomes, and other microtubule organizing centers (MTOCs), whether by direct filiation or symbiogenesis, has been controversial for >50 years. Centrioles, like mitochondria and chloroplasts, duplicate independently of the nucleus and constitute a heritable system independent of chromosomal DNA. Nucleic acids endogenous to the MTOC would support evolutionary origin by symbiogenesis. To date, most reports of centrosome-associated nucleic acids have used generalized reagents such as RNases and nucleic acid dyes. Here, from a library of RNAs extracted from isolated surf clam (Spisula solidissima) centrosomes, we describe a group of centrosome-associated transcripts representing a structurally unique intron-poor collection of nuclear genes skewed toward nucleic acid metabolism. Thus, we resolve the debate over the existence of centrosome-associated RNA (cnRNA). A subset of cnRNAs contain functional domains that are highly conserved across distant taxa, such as nucleotide polymerase motifs. In situ localization of cnRNA65, a molecule with an RNA polymerase domain, showed it is present in the intact oocyte nucleus (germinal vesicle). Its expression, therefore, precedes the appearance of gamma-tubulin-containing centrosomes. At this stage, the in situ signal resembles the nucleolinus, a poorly understood organelle proposed to play a role in spindle formation. After oocyte activation and germinal vesicle breakdown, cnRNA65 persists as a cytoplasmic patch within which gamma-tubulin-stained centrosomes can be seen. These observations provoke the question of whether cnRNAs and the nucleolinus serve as cytological progenitors of the centrosome and may support a symbiogenetic model for its evolution.

Echinonectin is a Del-1-like molecule with regulated expression in sea urchin embryos.

Echinonectin (EN) is a dimeric galactosyl-binding protein found in sea urchin eggs and embryos. It had been postulated in earlier studies that EN is secreted into the hyaline layer, a stratified matrix deposited on the apical surface of cells, and serves as an attachment substrate for cells of the blastoderm. However, the dynamics of EN expression have rendered past observations difficult to interpret on this point and others. Radioiodination experiments in this study indicate that the bulk of EN is, at any one time, maintained in its vesicular compartment beneath the plasma membrane, but that a portion of the protein is secreted onto the cell surface during early development. The primary structure of EN was determined. The protein consists of a series of coagulation factor 5/8 repeats and discoidin-like lectin domains, and bears similarity to the secreted proteins DEL-1 and lactadherin from angiogenic endothelial cells. In situ hybridization analysis indicates that EN mRNA levels are regulated to coincide with periods of reduced motility in embryonic cells, supporting the postulate that the protein is involved in cell anchoring.

Centrosome-associated RNA in surf clam oocytes.

Centrosomes are the major microtubule-organizing center in animal cells. They are composed of a pair of [9(3) + 0] centrioles surrounded by a relatively ill-defined pericentriolar matrix, provide the ciliary centriole-kinetosome (basal body) progenitor, and organize the assembly of microtubules into the mitotic spindle during cell division. Despite >100 years of microscopic observation and their obvious significance, our understanding of centrosome composition, dynamic organization, and mechanism of action is limited when compared with that of other cellular organelles. Centrosomes duplicate only once per cell cycle to ensure development of a normal bipolar spindle. The initial event in centrosome duplication is centriole replication, which is generative, semiconservative, and independent of the nucleus. Such observations led to the proposal that centrosomes contain their own complement of nucleic acids, possibly representative of an organellar genome comparable with those described for mitochondria and chloroplasts. The consensus in the field is that centrosomes lack DNA but may contain RNA. We isolated centrosomes from oocytes of the surf clam, Spisula solidissima, and purified from them a unique set of RNAs. We show here by biochemical means and subcellular in situ hybridization that the first transcript we analyzed is intimately associated with centrosomes. Sequence analysis reveals that this centrosome-associated RNA encodes a conserved RNA-directed polymerase domain. The hypothesis that centrosomes contain an intrinsic complement of specific RNAs suggests new opportunities to address the century-old problem of centrosome function, heredity, and evolution.

Differential expression of tyrosinated tubulin in Spisula solidissima polar bodies.

The C-terminus of alpha-tubulin can be reversibly modified by a specific tyrosine ligase to yield an isoform known as Tyr-tubulin. Tyr-tubulin is typically found in more dynamic microtubule arrays such as the mitotic spindle, as opposed to stable structures like centrioles and flagella. In developing systems, it is expressed in relatively undifferentiated, proliferative cell types but is replaced by detyrosinated (Glu-) tubulin during differentiation. We found Tyr-tubulin highly enriched in a single polar body of Spisula solidissima embryos. Quantitation of DNA content by Hoechst staining indicates that polar body 1 (with twice the DNA content of polar body 2) is the Tyr-tubulin-positive cell. Other than the apoptosis marker caspase, this is, to our knowledge, the first distinguishing marker antigen for polar bodies, particularly for one polar body vs. another. This localization of Tyr-tubulin is unlikely to be a byproduct of the meiotic process itself, because it arises after ejection of both polar bodies is complete. Although polar bodies are typically thought of as a terminally differentiated vestige of meiosis, the localization of this more dynamic tubulin isoform suggests an active role in early development.

Mouse pigpen encodes a nuclear protein whose expression is developmentally regulated during craniofacial morphogenesis.

Pigpen, a nuclear protein with RNA-binding motifs and a putative transcriptional activation domain (TAD), is expressed at high levels in proliferating endothelial cells and expression is down-regulated when cells adopt a quiescent or differentiated phenotype. We cloned the mouse homolog of pigpen and investigated the regulation of its expression during embryogenesis. In situ hybridization demonstrated that a broad pattern of pigpen expression became restricted during tooth formation in the mandible. In the eye, pigpen showed a spatial restriction to the more proliferating and less differentiated regions of the lens and neural retina. Expression was also restricted in the developing vibrissae, lung, and kidney, all sites where epithelial-mesenchymal interactions are vital for morphogenesis. In vitro assays, that focused on the mandible and tooth development, indicated that epithelial signals, mediated by fibroblast growth factor-8, were required to maintain pigpen expression in the mandibular mesenchyme, whereas bone morphogenetic protein-4 negatively regulated expression in that tissue during early odontogenesis. At the protein level, immunocytochemistry demonstrated that Pigpen was expressed diffusely in the cytoplasm and more concentratedly in focal granules within the nuclei of mouse embryonic cells. Lastly, CAT reporter assays showed that the N-terminus of mouse pigpen encodes an active TAD. These data suggest that mouse Pigpen may activate transcription in vivo in response to specific growth factor signals and regulate proliferation and/or differentiation events during mouse organogenesis.

Receptor-bound uPA is reversibly protected from inhibition by low molecular weight inhibitors.

Urokinase-type plasminogen activator (uPA) plays a ubiquitous role in cell migration and invasiveness. Amiloride, a competitive inhibitor of uPA, can inhibit endothelial cell (EC) outgrowth during angiogenesis. To address the question of whether amiloride blocked angiogenesis by inhibiting uPA, we undertook a study of uPA expression in sprouting EC in vitro and the effects of amiloride on both enzymatic and morphogenetic activity. As expected, amiloride inhibited soluble uPA (suPA) with an IC(50) of 45-85 microm, however, receptor-bound uPA (rbuPA) from the sprouting EC was insensitive to amiloride. Removal of uPA from its receptors confers sensitivity to inhibition by amiloride suggesting that a reversible conformational change may mediate the insensitivity of rbuPA to amiloride and its analogs. In summary, we found no evidence to support the hypothesis that amiloride blocks capillary outgrowth by inhibition of uPA, but were able to successfully demonstrate a functional difference between two physiological forms of this important matrix-degrading enzyme.

Nuclear injection of anti-pigpen antibodies inhibits endothelial cell division.

Endothelial cell proliferation is required for angiogenesis in both embryonic and adult tissues. In rat brain tumors, it has recently been shown that the nuclear protein pigpen is expressed selectively in endothelial cells of developing microvasculature but not in the established peritumoral vessels (Blank, M., Weinschenk, T., Priemer, M., and Schluesener, H. (2001) J. Biol. Chem. 276, 16464-16468). This finding suggests that pigpen may be important for promoting the undifferentiated, or "angiogenic" endothelial cell phenotype. Our studies show that pigpen protein and mRNA are expressed in actively dividing endothelial cells and down-regulated as they become confluent. Protein distribution is regulated in a cell cycle-dependent manner. We conclude that this expression pattern is important for and not simply ancillary to proliferation because nuclear microinjection of anti-pigpen Fab fragments inhibited endothelial cell division. Moreover, expression of the proliferating cell marker Ki67 was inhibited in antibody-injected cells. The absence of Ki67 suggests exit from rather than arrest within (for example, at the G(1)/S interface) the cell cycle. Together with earlier observations on the structure and expression of this molecule, our data support the hypothesis that pigpen helps regulate endothelial cell differentiation state.