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

Abstract

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.cell division | mitosis | meiosis T he nucleolinus was described by Montgomery in eggs of the nudibranch mollusk, Montagua, in his 1898 monograph, "Comparative Cytological Studies, with Especial Regard to the Morphology of the Nucleolus" (1). As the title indicates, the study focuses on the appearance and behavior of the nucleolus. In it, Montgomery presents his own thoughts on the significance of the nucleolinus as well as the observations of other investigators, including Louis Agassiz, over the previous 40 y (2-4). These range from the attachment of no morphological significance to the structure to suggestions that the nucleolinus is a microorganism enclosed in the nucleus and a direct morphological progenitor of the centrosome. Twenty-two years later, in his "Observations on an Intra-Nucleolar Body," Carleton experimented with Cajal's formol-silver nitrate technique to visualize the nucleolinus in cat and frog intestinal epithelial cells (5). In some cases, he reported, the nucleolinus was the only silverimpregnated structure within the entire nucleus. As of 90 y ago, then, the nucleolinus was clearly distinguished from the surrounding or adjacent nucleolus, but only via histochemistry. Carleton, on the basis of morphological observations of fixed, sectioned tissues, suggested that the nucleolinus divided into two during prophase. Allen (6, 7) subsequently proposed a "spindle forming role" for the nucleolinus, on the basis of his observations of live Spisula oocytes, but no photomicrographs or other data were presented to directly support this hypothesis.The most comprehensive analysis of the nucle...

The centrosome and spindle as a ribonucleoprotein complex

“…At least fifteen distinct molecules for which the full length sequences are known have been localized to the centrosome by in situ hybridization in four laboratories (Lambert and Nagy, 2004;Alliegro et al, 2006;Alliegro and Alliegro, 2008;Kingsley et al, 2007;Lecuyer et al, 2007). Others have been localized to the spindle (Groismann et al, 2000;Lecuyer et al, 2007;Alliegro et al, 2010) and dozens more are either presumed (by biochemical enrichment) or known (by in situ localization based on partial sequences) to be present in these structures (Lecuyer et al, 2007;Alliegro and Alliegro, 2008). We may now return our attention to the function and origin of spindle and centrosomal RNAs.…”

“…Three other studies have found specific RNAs associated with the centrosomes and/or spindle in surf clam (Spisula solidissima) oocytes (Alliegro et al, 2006;Alliegro and Alliegro, 2008;Alliegro et al, 2010). The first two reports described a set of RNAs that are significantly enriched in isolated centrosomes and includes confirmatory in situ hybridizations for four of these.…”

The Karyomastigont as an Evolutionary Seme

“…These include cilia and flagella, the basal bodies from which they arise (themselves derived from centrioles), and the mitotic spindle. There is evidence to suggest that certain intranuclear structures may be considered part of this system as well (Allen 1951(Allen , 1953Tanaka 1973;Laane and Haugli 1974;Alliegro et al 2010Alliegro et al , 2012.…”

“…From this perspective, the observation that a component of a unitary microtubule-based motility organelle could nucleate microtubules is no revelation. The karyomastigont perspective also addresses reports of MTOC components in the nucleus of some cells (Tanaka 1973;Laane and Haugli 1974;Alliegro et al 2010Alliegro et al , 2012. Viewed in light of the unitary karyomastigont, the relationship between the microtubule-based motility system and the nucleus is not simply one devised to segregate genetic material, but a much more integrated, reciprocal relationship with shared and exchanged elements.…”

Composition and Dynamics of the Nucleolinus, a Link between the Nucleolus and Cell Division Apparatus in Surf Clam (Spisula) Oocytes

“…The nucleolinus (NLi) 2 is a compartment or organelle described in a wide variety of cells and with a long although virtually forgotten history (2,3). In cells where it is clearly visible, it is closely apposed to the nucleolus, and in other cells where it has been described it appears to be integrated within the nucleolus.…”