Patrick J. DiMario

Nucleolar stress with and without p53.

A veritable explosion of primary research papers within the past 10 years focuses on nucleolar and ribosomal stress, and for good reason: with ribosome biosynthesis consuming ~80% of a cell’s energy, nearly all metabolic and signaling pathways lead ultimately to or from the nucleolus. We begin by describing p53 activation upon nucleolar stress resulting in cell cycle arrest or apoptosis. The significance of this mechanism cannot be understated, as oncologists are now inducing nucleolar stress strategically in cancer cells as a potential anti-cancer therapy. We also summarize the human ribosomopathies, syndromes in which ribosome biogenesis or function are impaired leading to birth defects or bone narrow failures; the perplexing problem in the ribosomopathies is why only certain cells are affected despite the fact that the causative mutation is systemic. We then describe p53-independent nucleolar stress, first in yeast which lacks p53, and then in other model metazoans that lack MDM2, the critical E3 ubiquitin ligase that normally inactivates p53. Do these presumably ancient p53-independent nucleolar stress pathways remain latent in human cells? If they still exist, can we use them to target >50% of known human cancers that lack functional p53?

Cell and Molecular Biology of Nucleolar Assembly and Disassembly

Nucleoli disassemble in prophase of the metazoan mitotic cycle, and they begin their reassembly (nucleologenesis) in late anaphase?early telophase. Nucleolar disassembly and reassembly were obvious to the early cytologists of the eighteenth and nineteenth centuries, and although this has lead to a plethora of literature describing these events, our understanding of the molecular mechanisms regulating nucleolar assembly and disassembly has expanded immensely just within the last 10-15 years. We briefly survey the findings of nineteenth-century cytologists on nucleolar assembly and disassembly, followed by the work of Heitz and McClintock on nucleolar organizers. A primer review of nucleolar structure and functions precedes detailed descriptions of modern molecular and microscopic studies of nucleolar assembly and disassembly. Nucleologenesis is concurrent with the reinitiation of rDNA transcription in telophase. The perichromosomal sheath, prenucleolar bodies, and nucleolar-derived foci serve as repositories for nucleolar processing components used in the previous interphase. Disassembly of the perichromosomal sheath along with the dynamic movements and compositional changes of the prenucleolar bodies and nucleolus-derived foci coincide with reactivation of rDNA synthesis within the chromosomal nucleolar organizers during telophase. Nucleologenesis is considered in various model organisms to provide breadth to our understanding. Nucleolar disassembly occurs at the onset of mitosis primarily as a result of the mitosis-specific phosphorylation of Pol I transcription factors and processing components. Although we have learned much regarding nucleolar assembly and disassembly, many questions still remain, and these questions are as vibrant for us today as early questions were for nineteenth- and early twentieth-century cytologists.

Knockdown of the Drosophila GTPase nucleostemin 1 impairs large ribosomal subunit biogenesis, cell growth, and midgut precursor cell maintenance.

Mammalian nucleostemin (NS) is a nucleolar guanosine triphosphate-binding protein implicated in cell cycle progression, stem cell proliferation, and ribosome assembly. Drosophila melanogaster contains a four-member nucleostemin family (NS1-4). NS1 is the closest orthologue to human NS; it shares 33% identity and 67% similarity with human NS. We show that NS1 has intrinsic GTPase and ATPase activity and that it is present within nucleoli of most larval and adult cells. Endogenous NS1 and lightly expressed green fluorescent protein (GFP)-NS1 enrich within the nucleolar granular regions as expected, whereas overexpressed GFP-NS1 localized throughout the nucleolus and nucleoplasm, and to several transcriptionally active interbands of polytene chromosomes. Severe overexpression correlated with the appearance of melanotic tumors and larval/pupal lethality. Depletion of 60% of NS1 transcripts also lead to larval and pupal lethality. NS1 protein depletion>95 correlated with the loss of imaginal island (precursor) cells in the larval midgut and to an apparent block in the nucleolar release of large ribosomal subunits in terminally differentiated larval midgut polyploid cells. Ultrastructural examination of larval Malpighian tubule cells depleted for NS1 showed a loss of cytoplasmic ribosomes and a concomitant appearance of cytoplasmic preautophagosomes and lysosomes. We interpret the appearance of these structures as indicators of cell stress response.

Deletion and site-specific mutagenesis of nucleolin's carboxy GAR domain

Vertebrate nucleolin is an abundant RNA-binding protein in the dense fibrillar component of active nucleoli. Nucleolin is modular in composition. Its amino-terminal third contains alternating acidic and basic domains, its middle section contains four consensus RNA-binding domains (cRBDs), and its carboxy-terminus contains a distinctive glycine/arginine-rich (GAR) domain with several RGG motifs. The arginines within these motifs are asymmetrically dimethylated. Several laboratories have shown that the GAR domain is necessary but not sufficient for the efficient localization of nucleolin to nucleoli. We examined the distribution of endogenous fibrillarin, Nopp140, and B23 when full-length and ΔGAR nucleolin were expressed exogenously as enhanced green fluorescent protein (EGFP)-tagged fusions. Only B23 redistributed when ΔGAR-EGFP was expressed at moderate to high levels, suggesting an in vivo interaction between nucleolin and B23. Next we substituted all ten arginines within the GAR domain of Chinese hamster ovary (CHO) nucleolin with lysines to test the hypothesis that methylation of the carboxy GAR domain is necessary for the nucleolar association of nucleolin. The lysine-substituted mutant was not an in vitro substrate for the yeast protein methyltransferase, Hmt1p/Rmt1. It was, however, able to associate properly with interphase nucleoli and with interphase pre-nucleolar bodies upon recovery from hypotonic shock. We conclude, therefore, that although the GAR domain is necessary for the efficient localization of nucleolin to nucleoli, methylation of this domain is not required for proper nucleolar localization.

Mycobacterium smegmatis histone-like protein Hlp is nucleoid associated

Eubacteria encode proteins that are required for nucleoid organization and for regulation of DNA-dependent processes. Of these histone-like proteins (Hlps), Escherichia coli HU has been shown to associate with the nucleoid and to regulate processes such as DNA repair and recombination. In contrast, the divergent HU homologs encoded by mycobacteria have been variously identified as involved in the physiology of dormancy, in the response to cold shock, or as laminin-binding proteins associated with the cell envelope. We show here, contrary to previous reports that the HU-related Hlp from Mycobacterium smegmatis associates with the nucleoid in vivo. Using indirect fluorescent antibody microscopy we show that cold shock causes Hlp to accumulate in the cytoplasm of M. smegmatis. No evidence of surface-associated Hlp was found in M. smegmatis cells treated for cell wall permeabilization. Quantitative Western blots indicate that exponentially growing cells contain c. 120 molecules per cell, with upregulation of Hlp after cold shock estimated to be c. 10-fold. That Hlp associates with the nucleoid in vivo suggests functions in DNA metabolism, perhaps in adaptation to environmental stress.

Structural changes in oocyte nucleoli ofXenopus laevis during oogenesis and meiotic maturation

Immunoelectron microscopy with anti-nucleolin defined substructures within the multiple nucleoli of biosynthetically active stage II–III oocytes and within the nucleoli of relatively quiescent stage VI oocytes ofXenopus laevis. Dense fibrillar components (DFCs) of nucleoli from stage II–III oocytes consisted of nucleolonemas that radiated from a continuous DFC sheath surrounding fibrillar centers (FCs). Discernible granular regions (GRs) were absent in these same nucleoli. Conversely, stage VI oocyte nucleoli displayed compacted DFCs and prominent GRs. Immunofluorescence microscopy then tracked fibrillarin, nucleolin, and condensed DNA through oogenesis and into progesterone-induced meiotic maturation and nuclear breakdown. In stage II–III oocyte nucleoli, fibrillarin was enriched near the FC-DFC boundaries, while nucleolin was distributed throughout these same DFCs. Both proteins were enriched within the compacted DFCs of stage VI oocyte nucleoli. Staining with (DAPI) 4′,6-diamidino-2-phenyl-indole showed condensed DNA within nucleolar FCs of both stage II–III and stage VI oocyte. Upon nuclear breakdown, we found fibrillarin and nucleolin in small particles and in the surrounding cytoplasm. Although we saw no trace of fibrillarin or nucleolin in nuclear remnants prepared just minutes later, DAPI-stained particles remained within these preparations, thus suggesting that FCs were at least slow to disassemble.

A complete nucleolin cDNA sequence from Xenopus laevis

Nucleolin is a nucleolar specific protein that is thought to play a role in the processing of pre-ribosomal RNA as ribosomes are assembled (1). A full length Xenopus nucleolin cDNA was constructed from overlapping sequences recovered from an ovary cDNA library. The intact cDNA is 2443 bp in length with the ATG start codon at ops 64-66 and the TGA stop codon at bps 2017-2019. A stop codon at bps 19-21 is in frame with the ATG start codon suggesting that the cDNA is complete. The deduced protein is 651 amino acid residues in length (Fig. 1). Like other vertebrate forms of nucleolin, the amino terminal third of Xenopus nucleolin contains alternating basic and acidic domains, while the carboxy two-thirds consists of four RNAbinding domains and a glycine/arginine rich domain (2). The largest basic domain within the amino terminal third of chicken (3), CHO (4), and Xenopus nucleolin (underlined in Fig. 1) are compared in Fig. 2. Both nucleolin TPA/GKK motifs (underlined in Fig. 2) and histone HI SPKK motifs are substrates for p34°* kinase (5,6), and we have previously shown with a monoclonal antibody, that various vertebrate nucleolin proteins share an antigenic epitope with histone HI (7). The largest basic domain from the amino terminal third of Xenopus nucleolin compares well with sea urchin gonadal histone HI (Fig. 3A), specifically, the spacing of proline residues (asterisks). Similar periodicities of proline residues are evident when the largest basic domain within the amino terminal region of CHO nucleolin is compared to Xenopus histone HI A (Fig. 3B). The similar periodicity of proline residues in nucleolin and histone HI suggests similar structures, perhaps reverse /3-turns (8, 9) or kinking alpha helices (10, 11).

Drosophila delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDh) is required for proline breakdown and mitochondrial integrity-Establishing a fly model for human type II hyperprolinemia.

Delta-1-pyrroline-5-carboxylate dehydrogenase (P5CDh) is a nuclear-encoded mitochondrial enzyme that catalyzes the second step in proline degradation. Mutations in human P5CDh cause type II hyperprolinemia, a complex syndrome displaying increased serum proline and mental disabilities. Conceptual gene CG7145 in Drosophila melanogaster encodes the orthologous DmP5CDh1. The mutant allele CG7145(f04633) contains a piggyBac transposon that truncates the enzyme by 83 residues. Heterozygous (CG7145(f04633)/TM3) individuals developed normally, while homozygous (CG7145(f04633)/CG7145(f04633)) individuals displayed proline levels twice that of normal, swollen mitochondria, and ultimately larval and pupal lethality. We believe this is the first correlation between the loss of P5CDh and morphological defects in mitochondria.

Tracking nucleolar dynamics with GFP-Nopp140 during Drosophila oogenesis and embryogenesis

We expressed two green fluorescent protein (GFP)-tagged Nopp140 isoforms in transgenic Drosophila melanogaster to study nucleolar dynamics during oogenesis and early embryogenesis. Specifically, we wanted to test whether the quiescent oocyte nucleus stored maternal Nopp140 and then to determine precisely when nucleoli formed during embryogenesis. During oogenesis nurse cell nucleoli accumulated GFP-Nopp140 gradually such that posterior nurse cell nucleoli in egg chambers at stage 10 were usually brighter than the more anterior nurse cell nucleoli. Nucleoli within apoptotic nurse cells disassembled in stages 12 and 13, but not all GFP-Nopp140 entered the oocyte through inter-connecting cytoplasmic bridges. Oocytes, on the other hand, lost their nucleoli by stage 3, but GFP-Nopp140 gradually accumulated in oocyte nuclei during stages 8–13. Most oocyte nuclei at stage 10 stored GFP-Nopp140 uniformly, but many stage 10 oocytes accumulated GFP-Nopp140 in presumed endobodies or in multiple smaller spheres. All oocyte nuclei at stages 11-12 were uniformly labeled, and GFP-Nopp140 diffused to the cytoplasm upon nuclear disassembly in stage 13. GFP-Nopp140 reappeared during embryogenesis; initial nucleologenesis occurred in peripheral somatic nuclei during embryonic stage 13, one stage earlier than reported previously. These GFP-Nopp140-containing foci disassembled at the 13th syncytial mitosis, and a second nucleologenesis occurred in early stage 14. The resulting nucleoli occupied nuclear regions closest to the periphery of the embryos. Pole cells contained GFP-Nopp140 during the syncytial embryonic stages, but their nucleologenesis started at gastrulation.

Nucleolin is a calcium‐binding protein

We have purified a prominent 110‐kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with ‘Stains‐All’ in sodium dodecyl sulfate–polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin‐dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half‐maximal binding observed at 105 μM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained “Stains‐All”, ruthenium red, and 45Ca2+ binding. N‐terminal sequencing of intact p110 and a 70‐kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two‐dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pIs from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti‐nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin‐like Ca2+‐binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N‐terminus with autolysis apparently resulting in largely selective removal of its basic C‐terminal domain. Although the Ca2+‐dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG‐1, its Ca2+‐dependent actions may regulate chromatin structure, possibly during apoptosis. J. Cell. Biochem. 85: 268–278, 2002.