Mark C. Alliegro

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 γ-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 γ-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.

Intima-like smooth muscle cells: developmental link between endothelium and media?

 The presence of non-contractile smooth muscle cells within the arterial wall raises questions as to their origin and function. These cells abound within the aortae of murine and porcine neonates, but are also present within the intimal and medial layers of adult arteries. They are largely devoid of smooth muscle-associated proteins and manifest an epithelioid form. Their morphological resemblance to endothelial cells prompted us to explore this potential relationship and to investigate their angiogenic properties in three-dimensional collagen gels. Using well-characterized smooth muscle cell lines, displaying either the intima-like (epithelioid) or media-like (spindle-shaped) morphology, we were able to show that intima-like cells share several features in common with endothelial ones and can transform into a media-like phenotype, whereby they irreversibly lose their characteristic pattern of protein expression. Intima-like, but not media-like, vascular smooth muscle cells are capable of forming capillary tubes, and, in co-cultures, can induce media-like ones to participate in this process. Such capillaries consist of a randomly-organized, mixed population of endothelial cells with intima-like or media-like smooth muscle ones. The functional significance of this diversity in smooth muscle cell type is not well understood, but phenotypic plasticity could conceivably figure as an important adaptive response to changes in the local environment.

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 10× 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.

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.

PIGPEN AND ENDOTHELIAL CELL DIFFERENTIATION

Endothelial cells can toggle back and forth between differentiated and relatively undifferentiated states with comparative ease. This is an important characteristic, particularly in adult tissues where the constitutive endothelial cell phenotype is quiescent. It enables rapid repair of wounds, renewal of the vascular intima in parts of the circulatory system with high flow and turbulence, and is essential to the cyclic function of reproductive organs. However, the ability to dedifferentiate can be a severe disadvantage when it is subverted to the support of disease processes such as tumor growth and metastasis. The control of endothelial cell differentiation state is, therefore, a matter of significance to investigators of basic developmental mechanism, as well as those studying an array of neovascular disorders. Recently, studies have advanced beyond the identification of extracellular triggers and overt cellular responses to the analysis of signal transduction pathways and nuclear events. This review focuses on the nuclear protein pigpen that is found in the right place at the right time, and with the necessary equipment, to modulate endothelial cell differentiation. We project that when we better understand the relationship of pigpen to its upstream regulators and downstream effectors, we will also have a better understanding of the mechanisms underlying capillary morphogenesis.

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 (Marshall and Rosenbaum, 2000). 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.

Novel Characteristics of a Myosin Isolated from Mammalian Retinal Pigment Epithelial and Endothelial Cells

We have isolated a novel, high Mr protein from human retinal pigment epithelial cells and endothelial cells by affinity chromatography on Sepharose 4B. Two polypeptides are present on SDS-gels of the 8 M urea eluent with apparent molecular mass of ~210 and 47 kDa. In the absence of dithiothreitol, the two polypeptides migrate as one protein band with an apparent molecular mass of ~550 kDa. “Piglet,” as this molecule is tentatively named, is present in retinal pigment epithelial and endothelial cells of several species, but could not be detected in the nonepithelial cells we examined. Immunofluorescent localization using an antibody to the 210-kDa polypeptide revealed a filamentous network in the cytoplasm of cultured cells. This antibody was used to identify a cDNA for piglet in a bovine aortic endothelial cell expression library. Sequence data indicate a high degree of identity with non-muscle myosin II heavy chain. We subsequently found that piglet had an actin-activated ATPase activity, colocalized with actin in cells, and reacted on Western blots with a pan-non-muscle myosin II heavy chain antiserum. The protein was also recognized by antibodies specific for myosin heavy chain isoform A, but did not react with anti-isoform B antibodies. Although piglet has several features in common with known forms of non-muscle myosin II, the distinctly unconventional features it displays suggest that it is a novel myosin.

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.

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. Developmental Dynamics 228:59–71, 2003.

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.