Cytoplasmic Particles Research Articles

Identification of the Junctional Plaque Protein Plakophilin 3 in Cytoplasmic Particles Containing RNA-binding Proteins and the Recruitment of Plakophilins 1 and 3 to Stress Granules

Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell-cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25-35 S and 45-55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in "stress granules" known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism.

Desmoplakin assembly dynamics in four dimensions: multiple phases differentially regulated by intermediate filaments and actin.

The intermediate filament (IF)–binding protein desmoplakin (DP) is essential for desmosome function and tissue integrity, but its role in junction assembly is poorly understood. Using time-lapse imaging, we show that cell–cell contact triggers three temporally overlapping phases of DP-GFP dynamics: (1) the de novo appearance of punctate fluorescence at new contact zones after as little as 3 min; (2) the coalescence of DP and the armadillo protein plakophilin 2 into discrete cytoplasmic particles after as little as 15 min; and (3) the cytochalasin-sensitive translocation of cytoplasmic particles to maturing borders, with kinetics ranging from 0.002 to 0.04 μm/s. DP mutants that abrogate or enhance association with IFs exhibit delayed incorporation into junctions, altering particle trajectory or increasing particle pause times, respectively. Our data are consistent with the idea that DP assembles into nascent junctions from both diffusible and particulate pools in a temporally overlapping series of events triggered by cell–cell contact and regulated by actin and DP–IF interactions.

Particles in Practice: How Ultrafines Disseminate in the Body

Ultrafine particles (UFPs), those less than 100 nanometers in diameter, have existed for millennia in natural settings. But with the significant increase in UFPs resulting from human activities in the past few centuries (largely through combustion processes) and the potential for a deluge of nanoparticles as that industry gears up, are ancient human bodily defenses up to the substantial new hazards they now face? Findings by a team of Swiss, German, and Canadian researchers suggest that animals may be largely defenseless against the rapid dissemination of UFPs into cells throughout the body [EHP 113:1555–1560]. Their findings, which include the first evidence of how individual particles are distributed within the lung, raise some concerns, especially since UFPs often end up in locations within cells where the tiny particles can impair many cellular functions. General knowledge about the rapid penetration of UFPs into various body organs has surfaced in the past few years, but the specific distribution and mechanisms remain largely unknown. To explore the distribution, the research team performed two parallel sets of experiments. In the first set of experiments, they investigated the spread of titanium dioxide UFPs in rats after a 1-hour inhalation of an aerosol containing the material. The team then evaluated lung tissue taken from the rats either 1 or 24 hours after inhalation. They found that on average, 24% of the inhaled titanium dioxide they detected had penetrated cells throughout the lung and the bloodstream just 1 hour after inhalation. Within cells in different lung compartments, there was no difference in the 1-hour and 24-hour samples, suggesting that UFPs can easily move between compartments. The team continues to investigate what happens with the remaining 76% of the particles and with those that enter the bloodstream. There is evidence the particles spread throughout the body. Of the particles they did find, 79.3% lodged in cells on the inner surface of airways and alveoli, 11.3% were within capillaries, 4.8% were within connective tissue, and 4.6% were within epithelial or endothelial cells. The researchers were surprised to find that most of the particles in the cellular cytoplasm were not attached to the membrane, as would have been expected if the particles had been encapsulated through endocytosis or phagocytosis. Floating in the cytoplasm, the particles can access many of the structures within the cell, such as the nucleus and mitochondria, increasing the potential toxicity of the particles. In the second set of experiments, the researchers explored the movement of three sizes of fluorescent polystyrene UFPs and of gold UFPs after the particles were introduced to cultures of swine macrophages and human red blood cells. They found all three particle sizes (1.0, 0.2, and 0.078 micrometer) penetrated the swine macrophages, though in perplexingly different proportions—only 21% of the macrophages contained the medium size, while 77% contained the smallest and 56% contained the largest. In human red blood cells, they found the smallest and medium sizes, but not the largest. The experiments did not offer evidence about exactly how the tiny, insoluble particles disseminate so extensively and rapidly into so many different cells, but the researchers note that other experiments have demonstrated a number of possible mechanisms. The researchers also note their findings are specific to just the few substances they studied, and differ in some ways from those for iridium, one of the few other materials evaluated in some detail.

Efficient Protein Trafficking Requires Trailer Hitch, a Component of a Ribonucleoprotein Complex Localized to the ER in Drosophila

Translational control of localized messenger mRNAs (mRNAs) is critical for cell polarity, synaptic plasticity, and embryonic patterning. While progress has been made in identifying localization factors and translational regulators, it is unclear how broad a role they play in regulating basic cellular processes. We have identified Drosophila trailer hitch (tral) as a gene that is required for the proper secretion of the dorsal-ventral patterning factor Gurken, as well as the vitellogenin receptor Yolkless. Surprisingly, biochemical purification of Tral revealed that it is part of a large RNA-protein complex that includes the translation/localization factors Me31B and Cup as well as the mRNAs for endoplasmic reticulum (ER) exit site components. This complex is localized to subdomains of the ER that border ER exit sites. Furthermore, tral is required for normal ER exit site formation. These findings raise exciting new possibilities for how the mRNA localization machinery could interface with the classical secretory pathway to promote efficient protein trafficking in the cell.

A conserved RNA-protein complex component involved in physiological germline apoptosis regulation inC. elegans

Two conserved features of oogenesis are the accumulation of translationally quiescent mRNA, and a high rate of stage-specific apoptosis. Little is understood about the function of this cell death. In C. elegans, apoptosis occurring through a specific ;physiological' pathway normally claims about half of all developing oocytes. The frequency of this germ cell death is dramatically increased by a lack of the RNA helicase CGH-1, orthologs of which are involved in translational control in oocytes and decapping-dependent mRNA degradation in yeast processing (P) bodies. Here, we describe a predicted RNA-binding protein, CAR-1, that associates with CGH-1 and Y-box proteins within a conserved germline RNA-protein (RNP) complex, and in cytoplasmic particles in the gonad and early embryo. The CGH-1/CAR-1 interaction is conserved in Drosophila oocytes. When car-1 expression is depleted by RNA interference (RNAi), physiological apoptosis is increased, brood size is modestly reduced, and early embryonic cytokinesis is abnormal. Surprisingly, if apoptosis is prevented car-1(RNAi) animals are characterized by a progressive oogenesis defect that leads rapidly to gonad failure. Elevated germ cell death similarly compensates for lack of the translational regulator CPB-3 (CPEB), orthologs of which function together with CGH-1 in diverse organisms. We conclude that CAR-1 is of critical importance for oogenesis, that the association between CAR-1 and CGH-1 has been conserved, and that the regulation of physiological germ cell apoptosis is specifically influenced by certain functions of the CGH-1/CAR-1 RNP complex. We propose that this cell death pathway facilitates the formation of functional oocytes, possibly by monitoring specific cytoplasmic events during oogenesis.

Lung pathology and pathogenesis of severe acute respiratory syndrome: a report of six full autopsies

Severe acute respiratory syndrome (SARS) is an emerging infectious disease that first manifested in humans in November 2002. The SARS-associated coronavirus (SARS-CoV) has been identified as the causal agent, but the pathology and pathogenesis are still not quite clear. Post-mortem lung samples from six patients who died from SARS from April to July 2003 were studied by light and electron microscopy, Masson trichromal staining and immunohistochemistry. Evidence of infection with the SARS-CoV was determined by reverse-transcription PCR (RT-PCR) , serological examination and electron microscopy. Four of six patients had serological and RT-PCR evidence of recent infection of SARS-CoV. Morphologic changes are summarized as follows: (1) Diffuse and bilateral lung consolidation was seen in all patients (6/6) with increasing lung weight. (2) Diffuse alveolar damage was universal (6/6) with hyaline membrane formation (6/6), intra-alveolar edema/hemorrhage (6/6), fibrin deposition (6/6), pneumocyte desquamation (6/6). A marked disruption in the integrity of the alveolar epithelium was confirmed by immunostaining for the epithelial marker AE1/AE3 (6/6). (3) Type II pneumocytes, with mild hyperplasia, atypia, cytomegaly with granular amphophilic cytoplasm and intracytoplasmic lipid accumulation (5/6). (4) Giant cells in the alveoli were seen in five of 6 patients (5/6) , most of which were positive for the epithelial marker AE1/AE3 (5/6), but some cells were positive for the macrophage marker CD68(2/6). (5) A pronounced increase of macrophages were seen in the alveoli and the interstitium of the lung (6/6), which was confirmed by histological study and immunohistochemistry. (6) Haemophagocytosis was present in five of the 6 patients(5/6). (7) Lung fibrosis was seen in five patients(5/6), with alveolar septa and interstitium thickening(5/6), intraalveolar organizing exudates (6/6) and pleura thickening (4/6). Proliferation of collagen was confirmed by Masson trichromal staining, most of which was type III collagen by immunostaining. The formation of distinctive fibroblast/myofibroblast foci was seen in five patients (5/6) by light microscopy and immunochemistry. (8) Squamous metaplasia of bronchial mucosa was seen in five patients(5/6). (9) Thrombi was seen in all patients(6/6). (10) Accompanying infection was present in two patients, one was bacteria, the other was fungus. In addition, electron microscopy revealed viral particles in the cytoplasm of alveolar epithelial cells and endothelial cells corresponding to coronavirus. Direct injury of SARS-CoV on alveolar epithelium, prominent macrophage infiltration and distinctive fibroblast/myofibroblast proliferation may play major roles in the pathogenesis of SARS.

Mass flow and pressure-driven hyphal extension in Neurospora crassa

Mass flow of cytoplasm in Neurospora crassa trunk hyphae was directly confirmed by injecting oil droplets into the hyphae. The droplets move in a manner similar to cytoplasmic particles and vacuoles within the hyphae. The direction of mass flow is towards the growing hyphal tips at the colony edge. Based on flow velocities (about 5 microm s(-1)), hyphal radius and estimates of cytoplasm viscosity, the Reynolds number is about 10(-4), indicating that mass flow is laminar. Therefore, the Poiseulle equation can be used to calculate the pressure gradient required for mass flow: 0.0005-0.1 bar cm(-1) (depending on the values used for septal pore radius and cytoplasmic viscosity). These values are very small compared to the normal hydrostatic pressure of the hyphae (4-5 bar). Mass flow stops after respiratory inhibition with cyanide, or creation of an extracellular osmotic gradient. The flow is probably caused by internal osmotic gradients created by differential ion transport along the hyphae. Apical cytoplasm migrates at the same rate as tip extension, as do oil droplets injected near the tip. Thus, in addition to organelle positioning mediated by molecular motors, pressure-driven mass flow may be an integral part of hyphal extension.

The Haloferax volcanii FtsY Homolog Is Critical for Haloarchaeal Growth but Does Not Require the A Domain

The targeting of many Sec substrates to the membrane-associated translocation pore requires the cytoplasmic signal recognition particle (SRP). In Eukarya and Bacteria it has been shown that membrane docking of the SRP-substrate complex occurs via the universally conserved SRP receptor (Sralpha/beta and FtsY, respectively). While much has been learned about the archaeal SRP in recent years, few studies have examined archaeal Sralpha/FtsY homologs. In the present study the FtsY homolog of Haloferax volcanii was characterized in its native host. Disruption of the sole chromosomal copy of ftsY in H. volcanii was possible only under conditions where either the full-length haloarchaeal FtsY or an amino-terminally truncated version of this protein lacking the A domain, was expressed in trans. Subcellular fractionation analysis of H. volcanii ftsY deletion strains expressing either one of the complementing proteins revealed that in addition to a cytoplasmic pool, both proteins cofractionate with the haloarchaeal cytoplasmic membrane. Moreover, membrane localization of the universally conserved SRP subunit SRP54, the key binding partner of FtsY, was detected in both H. volcanii strains. These analyses suggest that the H. volcanii FtsY homolog plays a crucial role but does not require its A domain for haloarchaeal growth.

Group 13 Allergens as Environmental and Immunological Markers for Grass Pollen Allergy: Studies by Immunogold Field Emission Scanning and Transmission Electron Microscopy

Background: Polygalacturonases were recently identified as important grass pollen allergens and designated group 13 allergens. The objective of the present study was to investigate the presence of group 13 grass pollen allergens in different grass species, their release and ultrastructural location in dry and hydrated grass pollen. Methods: Nitrocellulose-blotted allergen extracts from 12 wild and cultivated grass genera were probed with a rabbit antiserum raised against purified recombinant timothy grass pollen allergen, Phl p 13. The release kinetics of Phl p 13 from timothy grass pollen hydrated for 0.5 min to 3 h were analyzed by immunoblotting. Phl p 13 was localized in dry and hydrated grass pollen grains by immunogold field emission scanning and transmission electron microscopy. Results: Group 13 allergens were detected in all 12 wild and cultivated grass genera representing the major subfamilies of the Poaceae. Ultrastructurally, the allergen was located in the wall and in the cytoplasm of timothy grass pollen grains. In the cytoplasm, Phl p 13 was associated with polysaccharide particles and as yet undescribed stacks of microtubule-like structures. After hydration in rain water, pollen grains expel cytoplasmic particles of respirable size containing Phl p 13, which becomes detectable in aqueous supernatants already after 0.5 min. Conclusions: Group 13 allergens represent one set of marker allergens which specifically occur in pollen of the major grass subfamilies and are rapidly released in association with respirable particles after pollen hydration. They may be considered as environmental markers for grass pollen exposure and group 13-specific IgE antibodies as immunological markers for genuine grass pollen sensitization.

Characterization of 20-S and 40-S non-polysomal cytoplasmic messenger ribonucleoprotein particles from rat liver.

Two populations of free messenger ribonucleoprotein (mRNP) particles, sedimenting at 20 S and 40 S respectively, were isolated from a rat liver postpolysomal supernatant. After treatment with 0.5 M KCl and recentrifugation through a sucrose layer, the mRNP particles were characterized with respect to their low-molecular-weight RNA and protein components. 40-S and 20-S particles show very different RNA patterns. Four distinct low-molecular-weight RNA species of approximately 105, 139, 187 and 256 nucleotides were found as components of the 40-S mRNPs. The 20-S mRNP particles contain one major low-Mr RNA species of approximately 243 nucleotides and a characteristic pattern of low-Mr RNAs similar to the one found in nuclear ribonucleoprotein particles. In contrast to the low-Mr RNAs found in nuclear RNP particles most of the low-Mr RNA species present in 20-S and 40-S mRNP particles are rapidly labeled after [3H]orotate administration. Whereas the low-Mr RNA composition of 20-S and 40-S mRNP particles is very different, the protein patterns of both mRNP complexes are very similar. Six major polypeptides with the following molecular weights of 117000, 79800, 76700, 53800, 43900, 36300 and several minor ones were found in both 20-S and 40-S mRNPs. In a cell-free system from wheat germs neither 20-S nor 40-S mRNP particles stimulated the incorporation of [3H]leucine into proteins. However, phenol-extracted RNA from 20-S and 40-S mRNPs stimulated total protein synthesis 16-fold and 3-fold, respectively. Furthermore, the RNA from both mRNP pools directed the synthesis of albumin in vitro.

Synthesis in vitro of novel nucleic-acid-like polymers of Ehrlich ascites tumor cells.

1 When cytoplasmic particles isolated from Ehrlich ascites tumor cells were incubated with [14C]ribose, es and inorganic phosphate in the presence of MgCl2 and Tris/HCl (pH 7.0), radioactivity was incorporated into trichloroacetic-acid-insoluble materials. 2 Radioactive products, extracted and purified by DEAE-cellulose column chromatography, were hydrolyzed in i M HCl at 100°C for 1 h and the resultant radioactive neutral sugar components were purified and identified as ribose, glucose and rhamnose by gas chromatography and mass spectrometry. 3 When [14C]glucose was incubated in the same reaction system, radioactivity was also incorporated into trichloroacetic-acid-insoluble materials. Purified radioactive products were hydrolyzed in 1 M HCl at 100°C for 1 h and the resultant radioactive neutral sugar components were also shown to be ribose, glucose and a little rhamnose, as was the case with the products synthesized from [14C]ribose. 4 When [14C]deoxyribose was incubated in the same reaction system, little incorporation was observed. 5 The radioactive products were incubated with venom phosphodiesterase and the digests were analyzed by Dowex lX4 column chromatography. The results suggest that the radioactive products contained ribo-nuclcotides and ‘nucleotides’ containing glucose (glucose ‘nucleotides’). 6 When the radioactive products were incubated with both venom phosphodiesterase and alkaline phosphatase, ribonucleosides and glucose ‘nucleosides’ were detected. In Dowex 1X4 (borate form) chromatography, tibonuclcosides were eluted coincidentalhy with ribonucleoside markers (Cyd Ado Urd and Guo), whereas the elution profiles of glucose ‘nucleosides’ were totallydifferent fromfacto of ribonucleosides.

Isolation and characterization of nucleic-acid-like polymers containing glucose from Ehrlich ascites tumor cells.

1. Novel nucleic-acid-like polymers containing glucose, glucose 'nucleic acid', were isolated from cytoplasmic particles of Ehrlich ascites tumor cells. It is assumed that the basic structure of the polymers is similar to those of known nucleic acids except that their sugar constituents are glucose derivatives instead of ribose or deoxyribose. 2. When the glucose 'nucleic acid' was incubated with venom phosphodiesterase, four kinds of glucose 'nucleotides' containing bases of cytosine, adenine, guanine and thymine were obtained. 3. When the polymers were incubated with both venom phosphodiesterase and alkaline phosphatase, inorganic phosphates of glucose 'nucleotides' were removed and each nucleotide was converted to a glucose 'nucleoside'.

Dynamic Recruitment of Nek2 Kinase to the Centrosome Involves Microtubules, PCM-1, and Localized Proteasomal Degradation

Centrosomes undergo dramatic changes in composition and activity during cell cycle progression. Yet mechanisms involved in recruiting centrosomal proteins are poorly understood. Nek2 is a cell cycle-regulated protein kinase required for regulation of centrosome structure at the G2/M transition. Here, we have addressed the processes involved in trafficking of Nek2 to the centrosome of human adult cells. We find that Nek2 exists in small, highly dynamic cytoplasmic particles that move to and from the centrosome. Many of these particles align along microtubules and a motif was identified in the Nek2 C-terminal noncatalytic domain that allows both microtubule binding and centrosome localization. FRAP experiments reveal that 70% of centrosomal Nek2 is rapidly turned over (t(1/2) approximately 3 s). Microtubules facilitate Nek2 trafficking to the centrosome but only over long distances. Cytoplasmic Nek2 particles colocalize in part with PCM-1 containing centriolar satellites and depletion of PCM-1 interferes with centrosomal recruitment of Nek2 and its substrate C-Nap1. Finally, we show that proteasomal degradation is necessary to allow rapid recruitment of new Nek2 molecules to the centrosome. Together, these data highlight multiple processes involved in regulating the abundance of Nek2 kinase at the centrosome including microtubule binding, the centriolar satellite component PCM-1, and localized protein degradation.

Requirement for P granules and meiosis for accumulation of the germline RNA helicase CGH-1

In Caenorhabditis elegans, lack of the conserved germline RNA helicase CGH-1 causes infertility and excessive levels of physiological germline apoptosis, a process that normally claims about half of all developing oocytes. In yeast the CGH-1 ortholog is a key component of degradative "processing (P) bodies," which may share some properties with germline protein-RNA complexes such as P granules. During oogenesis CGH-1 associates with P granules, but also accumulates to high levels in additional cytoplasmic particles. Here we show that appropriate levels and localization of CGH-1 depends on some P granule components and on mechanisms that establish meiotic development. At the same time, germ cell death is not increased by various abnormalities in P granules or meiosis. We conclude that in developing oocytes CGH-1 particles accumulate specifically in response to meiotic development and have distinct functions from P granules, and may be dynamic protein-mRNA structures.

Characterization of MVP and VPARP assembly into vault ribonucleoprotein complexes

Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins: the major vault protein (MVP), the vault poly(ADP-ribose)polymerase (VPARP), and the telomerase-associated protein 1, together with one or more small untranslated RNAs. To date, little is known about the process of vault assembly or about the stability of vault components. In this study, we analyzed the biosynthesis of MVP and VPARP, and their half-lives within the vault particle in human ACHN renal carcinoma cells. Using an immunoprecipitation assay, we found that it took more than 4 h for newly synthesized MVPs to be incorporated into vault particles but that biosynthesized VPARPs were completely incorporated into vaults within 1.5 h. Once incorporated into the vault complex, both MVP and VPARP were very stable. Expression of human MVP alone in Escherichia coli resulted in the formation of particles that had a distinct vault morphology. The C-terminal region of VPARP that lacks poly(ADP-ribose)polymerase activity co-sedimented with MVP particles. This suggests that the activity of VPARP is not essential for interaction with MVP-self-assembled vault-like particles. In conclusion, our findings provide an insight into potential mechanisms of physiological vault assembly.

A Tribute to the Xenopus laevis Oocyte and Egg

When I was asked to reflect as part of the celebration of the 100-year anniversary of the Journal of Biological Chemistry I recalled a talk by Seymour Cohen at the Federation meetings in Atlantic City about 1960. He began by thanking bacteriophage for providing him with so many wonderful research problems. I decided in the same spirit to pay homage to the Xenopus laevis egg and oocyte, two states of a cell that has played and continues to play a central role in most of the disciplines of modern biology including biochemistry and molecular biology. Many of us owe a debt of gratitude to this cell. The egg is the most important and interesting cell in the repertoire of any organism. In X. laevis this single cell has a diameter of about 1.3 mm. The oocyte is permeable to small molecules, but after it undergoes meiosis and becomes an unfertilized egg it is impermeable to these same molecules. The oocyte is an active site of RNA and protein synthesis but not DNA replication, whereas the unfertilized egg is poised to replicate every 10 min after it is fertilized. During the cleavage period the embryo is transcriptionally silent. An oocyte can be cultured for days, but once ovulated the egg degenerates rapidly if it is not fertilized. The oocyte is the largest single cell in the body yet it has many of the same structures as somatic cells. These compartments are so exaggerated in size that they are accessible to cell biologists for manipulation and visualization. The huge oocyte nucleus is called a germinal vesicle (GV). The premeiotic tetraploid chromosomes are expanded into a “lampbrush” configuration so that they can be studied with a light microscope. The X. laevis egg and its possibilities for experimental manipulation were introduced to modern biology in the late 1950s by John Gurdon (Fig. 1), then a graduate student in the laboratory of Michail Fischberg in the Department of Zoology at Oxford University. Gurdon’s thesis was to reproduce with the South African “clawed toad” X. laevis the famous nuclear transplantation experiments that Briggs and King had perfected with the American leopard frog, Rana pipiens (1). Embryonic nuclei were injected into eggs whose own nuclei had been removed or destroyed. These early cloning experiments addressed the question of whether a differentiated and specialized somatic cell nucleus was still capable of expressing its full genetic repertoire. Briggs and King found that nuclei from R. pipiens embryos older than gastrula never promoted normal development, suggesting that the genetic material might change in cells as they specialize during embryonic development. This result was challenged by John Gurdon’s demonstration that nuclei from X. laevis embryos are more permissive. Even a small fraction of nuclei derived from differentiated intestinal epithelial cells supported normal development (2). This crucial scientific question of whether irreversible changes occur in the genome of highly specialized cells was addressed more precisely by experimental manipulation than by biochemistry or genetics. The concepts behind these experiments have influenced the stem cell field. The topic of whether the genome is altered in specialized somatic cells was revisited just this year by the same strategy of nuclear transplantation. Postmitotic nuclei from olfactory neurons support the development of normal fertile mice when transplanted into enucleated mouse eggs (3). THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 44, Issue of October 29, pp. 45291–45299, 2004 © 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Glycogen-rich clear cell mammary malignant myoepithelioma

Primary clear cell tumors of the breast are uncommon and often present a diagnostic challenge. We describe an extremely rare case of glycogen-rich clear cell malignant myoepithelioma in a 43-year-old woman. Histologically, this tumor is composed of clear cells with abundant cytoplasmic glycogen particles. Immunohistochemically, these tumor cells show co-expression of vimentin, smooth muscle actin, epithelial membrane antigen, S-100 protein, and cytokeratin as evidence of myoepithelial cell tumor. The pathological staging of the patient is IIB (pT3N0M0) and the nuclear grading is 2. The patient demonstrated no evidence of recurrence or metastasis over a period of 42 months. We suggest that glycogen-rich clear cell malignant myoepithelioma be included in the histological differential diagnosis of clear cell tumors of the breast.

Involvement of the nucleolus in plant virus systemic infection.

The nucleolus is a prominent subnuclear domain and is classically regarded as the site of transcription of rRNA, processing of the precursor rRNAs and biogenesis of pre-ribosomal particles. In addition to these traditionally recognized activities, the nucleolus also participates in many other aspects of cell function. The umbravirus-encoded ORF3 protein is a multifunctional RNA-binding protein involved in long-distance RNA movement, and protection of viral RNA from RNase attack, including possibly small interfering RNA-guided RNA silencing. In addition to its presence in cytoplasmic ribonucleoprotein particles containing viral RNA, the umbraviral ORF3 protein accumulates in nuclei, preferentially targeting nucleoli. The ORF3 protein domains involved in the localization of the protein to the nucleolus were identified. Functional analysis of the mutants revealed the correlation between the ORF3 protein nucleolar localization and its ability to form the cytoplasmic ribonucleoprotein particles and transport viral RNA long distances via the phloem. Possible mechanisms of the nucleolar involvement in systemic virus infection are discussed.

Vault poly(ADP-ribose) polymerase is associated with mammalian telomerase and is dispensable for telomerase function and vault structure in vivo.

Vault poly(ADP-ribose) polymerase (VPARP) was originally identified as a minor protein component of the vault ribonucleoprotein particle, which may be involved in molecular assembly or subcellular transport. In addition to the association of VPARP with the cytoplasmic vault particle, subpopulations of VPARP localize to the nucleus and the mitotic spindle, indicating that VPARP may have other cellular functions. We found that VPARP was associated with telomerase activity and interacted with exogenously expressed telomerase-associated protein 1 (TEP1) in human cells. To study the possible role of VPARP in telomerase and vault complexes in vivo, mVparp-deficient mice were generated. Mice deficient in mVparp were viable and fertile for up to five generations, with no apparent changes in telomerase activity or telomere length. Vaults purified from mVparp-deficient mouse liver appeared intact, and no defect in association with other vault components was observed. Mice deficient in mTep1, whose disruption alone does not affect telomere function but does affect the stability of vault RNA, showed no additional telomerase or telomere-related phenotypes when the mTep1 deficiency was combined with an mVparp deficiency. These data suggest that murine mTep1 and mVparp, alone or in combination, are dispensable for normal development, telomerase catalysis, telomere length maintenance, and vault structure in vivo.

Live imaging of nuage and polar granules: evidence against a precursor-product relationship and a novel role for Oskar in stabilization of polar granule components.

Nuage, a germ line specific organelle, is remarkably conserved between species, suggesting that it has an important germline cell function. Very little is known about the specific role of this organelle, but in Drosophila three nuage components have been identified, the Vasa, Tudor and Aubergine proteins. Each of these components is also present in polar granules, structures that are assembled in the oocyte and specify the formation of embryonic germ cells. We used GFP-tagged versions of Vasa and Aubergine to characterize and track nuage particles and polar granules in live preparations of ovaries and embryos. We found that perinuclear nuage is a stable structure that maintains size, seldom detaches from the nuclear envelope and exchanges protein components with the cytoplasm. Cytoplasmic nuage particles move rapidly in nurse cell cytoplasm and passage into the oocyte where their movements parallel that of the bulk cytoplasm. These particles do not appear to be anchored at the posterior or incorporated into polar granules, which argues for a model where nuage particles do not serve as the precursors of polar granules. Instead, Oskar protein nucleates the formation of polar granules from cytoplasmic pools of the components shared with nuage. Surprisingly, Oskar also appears to stabilize at least one shared component, Aubergine, and this property probably contributes to the Oskar-dependent formation of polar granules. We also find that Bruno, a translational control protein, is associated with nuage, which is consistent with a model in which nuage facilitates post transcriptional regulation by promoting the formation or reorganization of RNA-protein complexes.