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.

High-throughput optofluidic system for the laser microsurgery of oocytes.

This study combines microfluidics with optical microablation in a microscopy system that allows for high-throughput manipulation of oocytes, automated media exchange, and long-term oocyte observation. The microfluidic component of the system transports oocytes from an inlet port into multiple flow channels. Within each channel, oocytes are confined against a microfluidic barrier using a steady fluid flow provided by an external computer-controlled syringe pump. This allows for easy media replacement without disturbing the oocyte location. The microfluidic and optical-laser microbeam ablation capabilities of the system were validated using surf clam (Spisula solidissima) oocytes that were immobilized in order to permit ablation of the 5 µm diameter nucleolinus within the oocyte nucleolus. Oocytes were the followed and assayed for polar body ejection.

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.

The karyomastigont as an evolutionary seme.

The problem of eukaryogenesis--the evolutionary mechanism whereby eukaryotic cells evolved from prokaryotes--remains one of the great unsolved mysteries of cell biology, possibly due to the reductionist tendency of most scientists to work only within their subdisciplines. Communication between biologists who conduct research on the nucleus and those working on the cytoskeleton or endomembrane system are sometimes wanting, and yet, all of these quintessentially eukaryotic elements of the cell are interdependent, and are physically associated in many protists as the karyomastigont organellar system: nucleus, one or more basal bodies and flagella, nuclear connector, and Golgi apparatus. Here we suggest a more holistic view of the karyomastigont as not simply an organellar system, but an evolutionary seme, the archaic state of the eukaryotic cell. We also present a scheme whereby the karyomastigont may have dissociated, giving rise in more derived cells to one or more free nuclei and discrete flagellar apparati (akaryomastigonts).

The Nucleolinus: A disappearing, forgotten and (maybe) misnamed organelle.

It is common knowledge that many of the cell components we study today were discovered more than a century ago. Some have been renamed due to a newer understanding of their physiology or composition, and in some cases the old terminology is abandoned. However, it is unusual to find a structure that has not been renamed but simply forgotten. This appears to be the case for the nucleolinus, discovered at least 150 years ago and studied by Agassiz, Haekel, Montgomery and others until it virtually dropped from the literature in the early 1970s. The nucleolinus was thought to have a role in cell division, but with little knowledge of its composition and no molecular markers (until recently) available for its study, we do not know if the nucleolinus is a ubiquitous structure or an antiquated descriptor. This brief article relates most of what we know about the nucleolinus and where to find more information. Our growing knowledge concerning the role of the closely allied nucleolus in cell cycle regulation suggests that renewed study of the nucleolinus will yield important information about the biogenesis and evolution of the cell division apparatus.

The centrosome and spindle as a ribonucleoprotein complex.

The notion of nucleic acids in the spindle, and particularly, the centrosome has a long history of inquiry, doubt, and debate. However, the association of specific RNAs with these structures is now confirmed by several investigators. What their presence means for the assembly, function, and evolution of the cell division apparatus is not known; but with newly available information and probes, these are questions that can finally be addressed. The present article summarizes the history of this field, what we know about the molecules in question, and in light of these findings, emphasizes the need to view the cell division apparatus for what it is by definition, a ribonucleoprotein complex.

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.

Origin of the cilium: novel approaches to examine a centriolar evolution hypothesis.

Recently, a new hypothesis was proposed regarding the evolution of the cilium from an enveloped RNA virus (Satir et al., 2007, Cell Motil. Cytoskeleton 64, 906). The hypothesis predicts that there may be specific centriolar or basal body RNAs with sequences reminiscent of retroviruses, and/or that the nuclear genes for certain centriole-specific proteins would have viral origins. Four independent laboratories have reported the existence of centrosomal RNA (cnRNA). Methods for studying cnRNA are described. We analyzed evidence of relatedness of known full-length cnRNAs to extant viral molecules. Out of 14 cnRNAs studied, 12 have similarity to entries in viral databases, all but one of these with E-values of < or = 1e(-4). Some centrosomal, and possibly uniquely centriolar, proteins also have relatives in viral databases that meet the criteria accepted to indicate a relationship by descent. Nine general cytoskeleton proteins exhibited no significant similarity to viral proteins. The speculation that centrioles are invaders of RNA viral origin in the evolving eukaryotic cell is strengthened by these findings.

The implications of centrosomal RNA.

The existence of nucleic acids associated with the centrosome has been controversial for many years. Founded in the 1950s and the subject of active inquiry in the 1970s and 1980s, the topic entered a period of hiatus for over a decade that was essentially capped by a comprehensive and authoritative review in 2000.(1) The consensus at the time was that there was no evidence supporting the presence of DNA in the centrosome and related structures, although the existence of centrosomal RNA remained a possibility. The question of centrosomal DNA remains unresolved. To this day the evidence-with no recent additions-is inconclusive and sometimes contradictory. The existence of RNA at the centrosome, however, has been conclusively demonstrated. Consequently, we may now revisit the lines of inquiry established decades ago and ask, what are the functions and origins of centrosome-associated RNA (cnRNA)? Is it important for replication of the mitotic center? Does it play a role in spindle assembly? Does it represent the remnant of a once-independent genome, derived by symbiogenesis? The questions are fundamental, interdependent and now answerable.

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.

Myosin II in retinal pigmented epithelial cells: evidence for an association with membranous vesicles.

The goal of this study was to further characterize and identify possible functions for a cytoplasmic myosin II protein which we have isolated from retinal pigmented epithelial (RPE) cells. The nucleotide and deduced amino acid sequences are highly identical to non-muscle myosin heavy chain II-A (NMMHC II-A). However, this RPE myosin displays characteristics that are atypical of other myosins, including an affinity for carbohydrate and a C-terminal sequence extension, suggesting it may have a specialized function. In this study, reverse transcriptase-PCR using isoform-specific primers demonstrated that the RPE myosin and conventional NMMHC II-A have overlapping but distinguishable tissue expression profiles. To gain clues to function, subcellular distribution was determined in motile RPE cells using indirect immunofluorescence. In addition to subtle differences in localization that appeared to further distinguish this molecule from NMMHC II-A, these studies revealed a colocalization with phagocytosed intracellular vesicles. In vitro experiments suggest that the association in situ was not simply coincidental, because isolated vesicles interacted with the protein in cosedimentation assays. Taken together, our observations suggest the RPE myosin exhibits characteristics different from conventional myosin II-A and may function in intracellular vesicle transport.

Coiled body heterogeneity induced by G(1) arrest with amiloride + bumetanide.

Ki67 is a nuclear protein expressed in proliferating cells, but not in quiescent or G(0)-arrested cells. Similar to the proliferating cell nuclear antigen and several other well-characterized molecules, Ki67 exhibits a repeating pattern of regulated expression and redistribution during the cell cycle, making it a useful marker for cell cycle phase. In addition to other structures labeled, concentrated foci may be observed in the nucleus and sometimes the cytoplasm. We observed that these Ki67 foci can be found at any stage of the endothelial cell cycle. They are not coincident with coiled bodies (CB), as determined in double-label immunofluorescence experiments with anti-Ki67 and antibodies to the CB marker protein pigpen. However, arrest of BPA47 endothelial cells in G(1) with amiloride + bumetanide induces colocalization of pigpen and Ki67 in 40% of cells exhibiting Ki67 foci. We conducted a series of experiments to examine the possibilities that pigpen was exported from CB and into unique, Ki67-containing foci or that Ki67 was imported into pigpen-containing CB. Our results showed us that although CB typically contain both coilin and pigpen, amiloride + bumetanide-induced G(1) arrest reconfigured the CB compartment into three populations of foci: one containing pigpen without coilin, the second containing coilin without pigpen, and a third containing both pigpen and coilin together. Furthermore, G(1) arrest resulted in Ki67 redistribution into both coilin- and pigpen-containing foci. The results suggest that under certain conditions, "resident" CB proteins can be differentially redistributed, and proteins not previously recognized as resident in CB can be driven into that compartment. Our observations underscore the fluid nature of CB. They demonstrate that previously reported heterogeneity in the CB compartment can be amplified by a specific experimental manipulation. This may be useful in future analyses of protein trafficking within the CB compartment and between CB and other cellular compartments. Finally, the redistribution of Ki67 into CB represents a new finding for a widely expressed but poorly understood molecule, one that may be useful in elucidating function.

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.