C-terminal moiety of Tudor contains its in vivo activity in Drosophila

Background

In early Drosophila embryos, the germ plasm is localized to the posterior pole region and is partitioned into the germline progenitors, known as pole cells. Germ plasm, or pole plasm, contains the polar granules which form during oogenesis and are required for germline development. Components of these granules are also present in the perinuclear region of the nurse cells, the nuage. One such component is Tudor (Tud) which is a large protein containing multiple Tudor domains. It was previously reported that specific Tudor domains are required for germ cell formation and Tud localization.

Methodology/Principal Findings

In order to better understand the function of Tud the distribution and functional activity of fragments of Tud were analyzed. These fragments were fused to GFP and the fusion proteins were synthesized during oogenesis. Non-overlapping fragments of Tud were found to be able to localize to both the nuage and pole plasm. By introducing these fragments into a tud mutant background and testing their ability to rescue the tud phenotype, I determined that the C-terminal moiety contains the functional activity of Tud. Dividing this fragment into two parts reduces its localization in pole plasm and abolishes its activity.

Conclusions/Significance

I conclude that the C-terminal moiety of Tud contains all the information necessary for its localization in the nuage and pole plasm and its pole cell-forming activity. The present results challenge published data and may help refining the functional features of Tud.     

Valois, a component of the nuage and pole plasm, is involved in assembly of these structures, and binds to Tudor and the methyltransferase Capsuléen

Using the Capsuleen (Csul) methyltransferase as bait in the yeast two-hybrid system, we have identified a novel Drosophila protein containing multiple WD repeats and encoded by the valois (vsl) gene, which acts in pole plasm function. Vls is homologous to human MEP50, which forms a complex with the PRMT5 methyltransferase--the human homologue of Csul. We found that Vls localizes to the nuage in the nurse cells and to the pole plasm in the oocyte. Moreover vls is required for the synthesis and/or stability of Oskar and the localization of Tudor (Tud) in both the nuage and at the posterior pole of the oocyte. Furthermore, we show that Vls and a fragment of Tud interact directly in binding assay. As the PMRT5/MEP50 complex is involved in ribonucleoprotein complex assembly, we hypothesize that the Vls complex may play a similar function in assembling the nuage in nurse cells and the polar granules in the oocyte.     

Nuage morphogenesis becomes more complex: two translocation pathways and two forms of nuage coexist in Drosophila germline syncytia

We have developed a simple and reliable method of preserving antigen immunoreactivity with concomitant excellent retention of the cell ultrastructure. Using this method, we have been able to follow the origin and developmental stages of nuage accumulations within the nurse cell/oocyte syncytium in the ovary of the fruit fly, Drosophila melanogaster, at the ultrastructural level. We have found two morphologically and biochemically distinct forms of nuage material in the nurse cell cytoplasm: translocating accumulations of nuage containing the Vasa protein, termed sponge bodies and stationary polymorphic accumulations of nuage enriched in Argonaute and Survival of motor neuron proteins. Immunogold labeling combined with confocal fluorescent and ultrastructural analyses have revealed that the Vasa-containing nuage accumulations remain closely associated with the cisternae of the endoplasmic reticulum throughout their lifetimes. The migration mechanism of the Vasa-positive nuage appears distinct from the microtubule-dependent translocation of oskar ribonucleoprotein complexes. We postulate that these two distinct nuage translocation pathways converge in the formation of the polar granules within the polar/germ plasm of the oocyte posterior pole. We also provide morphological and immunocytochemical evidence that these polymorphic nuage accumulations correspond to the recently described cytoplasmic domains termed U body-P body complexes.     

Arginine methyltransferase Capsuleen is essential for methylation of spliceosomal Sm proteins and germ cell formation in Drosophila

Although arginine modification has been implicated in a number of cellular processes, the in vivo requirement of protein arginine methyltransferases (PRMTs) in specific biological processes remain to be clarified. In this study we characterize the Drosophila PRMT Capsuléen, homologous to human PRMT5. During Drosophila oogenesis, catalytic activity of Capsuléen is necessary for both the assembly of the nuage surrounding nurse cell nuclei and the formation of the pole plasm at the posterior end of the oocyte. In particular, we show that the nuage and pole plasm localization of Tudor, an essential component for germ cell formation, are abolished in csul mutant germ cells. We identify the spliceosomal Sm proteins as in vivo substrates of Capsuléen and demonstrate that Capsuléen, together with its associated protein Valois, is essential for the synthesis of symmetric di-methylated arginyl residues in Sm proteins. Finally, we show that Tudor can be targeted to the nuage in the absence of Sm methylation by Capsuléen, indicating that Tudor localization and Sm methylation are separate processes. Our results thus reveal the role of a PRMT in protein localization in germ cells.     

Arginine Methylation of Vasa Protein Is Conserved across Phyla

Recent studies have uncovered an unexpected relationship between factors that are essential for germline development in Drosophila melanogaster: the arginine protein methyltransferase 5 (dPRMT5/Csul/Dart5) and its cofactor Valois, methylate the Piwi family protein Aub, enabling it to bind Tudor. The RNA helicase Vasa is another essential protein in germline development. Here, we report that mouse (mouse Vasa homolog), Xenopus laevis, and D. melanogaster Vasa proteins contain both symmetrical and asymmetrical dimethylarginines. We find that dPRMT5 is required for the production of sDMAs of Vasa in vivo. Furthermore, we find that the mouse Vasa homolog associates with Tudor domain-containing proteins, Tdrd1 and Tdrd6, as well as the Piwi proteins, Mili and Miwi. Arginine methylation is thus emerging as a conserved and pivotal post-translational modification of proteins that is essential for germline development.     

Doubly uniparental inheritance of mitochondria as a model system for studying germ line formation

Background

Doubly Uniparental Inheritance (DUI) of mitochondria occurs when both mothers and fathers are capable of transmitting mitochondria to their offspring, in contrast to the typical Strictly Maternal Inheritance (SMI). DUI was found in some bivalve molluscs, in which two mitochondrial genomes are inherited, one through eggs, the other through sperm. During male embryo development, spermatozoon mitochondria aggregate in proximity of the first cleavage furrow and end up in the primordial germ cells, while they are dispersed in female embryos.

Methodology/Principal Findings

We used MitoTracker, microtubule staining and transmission electron microscopy to examine the mechanisms of this unusual distribution of sperm mitochondria in the DUI species Ruditapes philippinarum. Our results suggest that in male embryos the midbody deriving from the mitotic spindle of the first division concurs in positioning the aggregate of sperm mitochondria. Furthermore, an immunocytochemical analysis showed that the germ line determinant Vasa segregates close to the first cleavage furrow.

Conclusions/Significance

In DUI male embryos, spermatozoon mitochondria aggregate in a stable area on the animal-vegetal axis: in organisms with spiral segmentation this zone is not involved in cleavage, so the aggregation is maintained. Moreover, sperm mitochondria reach the same embryonic area in which also germ plasm is transferred. In 2-blastomere embryos, the segregation of sperm mitochondria in the same region with Vasa suggests their contribution in male germ line formation. In DUI male embryos, M-type mitochondria must be recognized by egg factors to be actively transferred in the germ line, where they become dominant replacing the Balbiani body mitochondria. The typical features of germ line assembly point to a common biological mechanism shared by DUI and SMI organisms. Although the molecular dynamics of the segregation of sperm mitochondria in DUI species are unknown, they could be a variation of the mechanism regulating the mitochondrial bottleneck in all metazoans.     

OST-HTH: a novel predicted RNA-binding domain

Background  

The mechanism by which the arthropod Oskar and vertebrate TDRD5/TDRD7 proteins nucleate or organize structurally related ribonucleoprotein (RNP) complexes, the polar granule and nuage, is poorly understood. Using sequence profile searches we identify a novel domain in these proteins that is widely conserved across eukaryotes and bacteria.     

Fat facets interacts with vasa in the Drosophila pole plasm and protects it from degradation

Anterior-posterior patterning and germ cell specification in Drosophila requires the establishment, during oogenesis, of a specialized cytoplasmic region termed the pole plasm. Numerous RNAs and proteins accumulate to the pole plasm and assemble in polar granules. Translation of some of these RNAs is generally repressed and active only in pole plasm. Vasa (VAS) protein, an RNA helicase and a component of polar granules, is essential maternally for posterior patterning and germ cell specification, and VAS is a candidate translational activator in the pole plasm. VAS is stabilized within the pole plasm in that it is initially present throughout the entire embryo but strictly limited to the pole cells by the cellular blastoderm stage. hsp83 mRNA, which accumulates in the pole plasm through a stabilization-degradation mechanism, is another example. Here, we used a biochemical approach to identify proteins that copurify with VAS in crosslinked extracts. Prominent among these proteins was the ubiquitin-specific protease Fat facets (FAF), a pole plasm component [7], but one whose roles in posterior patterning and germ line specification have remained unclear. We present evidence that FAF interacts with VAS physically and reverses VAS ubiquitination, thereby stabilizing VAS in the pole plasm.     

Projected Polar Bear Sea Ice Habitat in the Canadian Arctic Archipelago

Background

Sea ice across the Arctic is declining and altering physical characteristics of marine ecosystems. Polar bears (Ursus maritimus) have been identified as vulnerable to changes in sea ice conditions. We use sea ice projections for the Canadian Arctic Archipelago from 2006 – 2100 to gain insight into the conservation challenges for polar bears with respect to habitat loss using metrics developed from polar bear energetics modeling.

Principal Findings

Shifts away from multiyear ice to annual ice cover throughout the region, as well as lengthening ice-free periods, may become critical for polar bears before the end of the 21^st century with projected warming. Each polar bear population in the Archipelago may undergo 2–5 months of ice-free conditions, where no such conditions exist presently. We identify spatially and temporally explicit ice-free periods that extend beyond what polar bears require for nutritional and reproductive demands.

Conclusions/Significance

Under business-as-usual climate projections, polar bears may face starvation and reproductive failure across the entire Archipelago by the year 2100.     

The QKI-6 RNA Binding Protein Localizes with the MBP mRNAs in Stress Granules of Glial Cells

Background

The quaking viable (qk^v) mouse has several developmental defects that result in rapid tremors in the hind limbs. The qkI gene expresses three major alternatively spliced mRNAs (5, 6 and 7 kb) that encode the QKI-5, QKI-6 and QKI-7 RNA binding proteins that differ in their C-terminal 30 amino acids. The QKI isoforms are known to regulate RNA metabolism within oligodendrocytes, however, little is known about their roles during cellular stress.

Methodology/Principal Findings

In this study, we report an interaction between the QKI-6 isoform and a component of the RNA induced silencing complex (RISC), argonaute 2 (Ago2). We show in glial cells that QKI-6 co-localizes with Ago2 and the myelin basic protein mRNA in cytoplasmic stress granules.

Conclusions

Our findings define the QKI isoforms as Ago2-interacting proteins. We also identify the QKI-6 isoform as a new component of stress granules in glial cells.     

Isolation of new polar granule components in Drosophila reveals P body and ER associated proteins

Germ plasm, a specialized cytoplasm present at the posterior of the early Drosophila embryo, is necessary and sufficient for germ cell formation. Germ plasm is rich in mitochondria and contains electron dense structures called polar granules. To identify novel polar granule components we isolated proteins that associate in early embryos with Vasa (VAS) and Tudor (TUD), two known polar granule associated molecules. We identified Maternal expression at 31B (ME31B), eIF4A, Aubergine (AUB) and Transitional Endoplasmic Reticulum 94 (TER94) as components of both VAS and TUD complexes and confirmed their localization to polar granules by immuno-electron microscopy. ME31B, eIF4A and AUB are also present in processing (P) bodies, suggesting that polar granules, which are necessary for germ line formation, might be related to P bodies. Our recovery of ER associated proteins TER94 and ME31B confirms that polar granules are closely linked to the translational machinery and to mRNP assembly.     

Changes in the localization and levels of starch and lipids in cambium and phloem during cambial reactivation by artificial heating of main stems of Cryptomeria japonica trees

Background and Aims

Cambial reactivation in trees occurs from late winter to early spring when photosynthesis is minimal or almost non-existent. Reserve materials might be important for wood formation in trees. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules in cambium and phloem were examined from cambial dormancy to the start of xylem differentiation in locally heated stems of Cryptomeria japonica trees in winter.

Methods

Electric heating tape was wrapped on one side of the stem of Cryptomeria japonica trees at breast height in winter. The localization and approximate levels of starch and lipids (as droplets) and number of starch granules were determined by image analysis of optical digital images obtained by confocal laser scanning microscopy.

Key Results

Localized heating induced earlier cambial reactivation and xylem differentiation in stems of Cryptomeria japonica, as compared with non-heated stems. There were clear changes in the respective localizations and levels of starch and lipids (as droplets) determined in terms of relative areas on images, from cambial dormancy to the start of xylem differentiation in heated stems. In heated stems, the levels and number of starch granules fell from cambial reactivation to the start of xylem differentiation. There was a significant decrease in the relative area occupied by lipid droplets in the cambium from cambial reactivation to the start of xylem differentiation in heated stems.

Conclusions

The results showed clearly that the levels and number of storage starch granules in cambium and phloem cells and levels of lipids (as droplets) in the cambium decreased from cambial reactivation to the start of xylem differentiation in heated stems during the winter. The observations suggest that starch and lipid droplets might be needed as sources of energy for the initiation of cambial cell division and the differentiation of xylem in Cryptomeria japonica.     

Phagocytosis of Streptococcus pyogenes by All-Trans Retinoic Acid-Differentiated HL-60 Cells: Roles of Azurophilic Granules and NADPH Oxidase

Background

New experimental approaches to the study of the neutrophil phagosome and bacterial killing prompted a reassessment of the usefulness of all-trans retinoic acid (ATRA)-differentiated HL-60 cells as a neutrophil model. HL-60 cells are special in that they possess azurophilic granules while lacking the specific granules with their associated oxidase components. The resulting inability to mount an effective intracellular respiratory burst makes these cells more dependent on other mechanisms when killing internalized bacteria.

Methodology/Principal Findings

In this work phagocytosis and phagosome-related responses of ATRA-differentiated HL-60 cells were compared to those earlier described in human neutrophils. We show that intracellular survival of wild-type S. pyogenes bacteria in HL-60 cells is accompanied by inhibition of azurophilic granule–phagosome fusion. A mutant S. pyogenes bacterium, deficient in M-protein expression, is, on the other hand, rapidly killed in phagosomes that avidly fuse with azurophilic granules.

Conclusions/Significance

The current data extend our previous findings by showing that a system lacking in oxidase involvement also indicates a link between inhibition of azurophilic granule fusion and the intraphagosomal fate of S. pyogenes bacteria. We propose that differentiated HL-60 cells can be a useful tool to study certain aspects of neutrophil phagosome maturation, such as azurophilic granule fusion.     

Regulation of Drosophila Vasa In Vivo through Paralogous Cullin-RING E3 Ligase Specificity Receptors

In Drosophila species, molecular asymmetries guiding embryonic development are established maternally. Vasa, a DEAD-box RNA helicase, accumulates in the posterior pole plasm, where it is required for embryonic germ cell specification. Maintenance of Vasa at the posterior pole requires the deubiquitinating enzyme Fat facets, which protects Vasa from degradation. Here, we found that Gustavus (Gus) and Fsn, two ubiquitin Cullin-RING E3 ligase specificity receptors, bind to the same motif on Vasa through their paralogous B30.2/SPRY domains. Both Gus and Fsn accumulate in the pole plasm in a Vasa-dependent manner. Posterior Vasa accumulation is precocious in Fsn mutant oocytes; Fsn overexpression reduces ovarian Vasa levels, and embryos from Fsn-overexpressing females form fewer primordial germ cells (PGCs); thus, Fsn destabilizes Vasa. In contrast, endogenous Gus may promote Vasa activity in the pole plasm, as gus females produce embryos with fewer PGCs, and posterior accumulation of Vas is delayed in gus mutant oocytes that also lack one copy of cullin-5. We propose that Fsn- and Gus-containing E3 ligase complexes contribute to establishing a fine-tuned steady state of Vasa ubiquitination that influences the kinetics of posterior Vasa deployment.Establishment and maintenance of polarity is essential for multicellular development. Asymmetric distribution of proteins within cells is often realized by localizing specific mRNAs to distinct positions and by tightly regulating their translation (1). During Drosophila oogenesis, polarized deployment of key mRNAs is crucial for the maternal determination of the embryonic body axes (17). However, although asymmetric mRNA localization within cells is widespread (1, 22), some proteins localize directly. An example is Vasa (Vas), which accumulates in a highly polarized fashion in Drosophila oocytes from a uniformly distributed mRNA that is not believed to be under translational control (8, 20, 21). Vas accumulates in the pole plasm of the oocyte, where it is necessary for embryonic posterior patterning and primordial germ cell (PGC) formation (26). Accumulation of high levels of Vas in the pole plasm requires the deubiquitinating enzyme (DUB) Fat facets (Faf) (25). In faf mutants, levels of posterior Vas are reduced, and polyubiquitinated forms of Vas accumulate. This indicates that Vas stability in the pole plasm is regulated by ubiquitin-dependent pathways.Ubiquitination culminates in the E3 ligase-catalyzed formation of a covalent bond between the C terminus of ubiquitin and a lysine residue of the ubiquitinated protein (12). Target proteins can be ubiquitinated simultaneously and/or sequentially on different lysine residues, and the presence of seven internal lysine residues in ubiquitin itself allows for the formation of topologically distinct polyubiquitin chains (9, 12, 27). Different forms of ubiquitination usually produce different effects on the target protein, and modulation of the steady-state dynamics of ubiquitin conjugation can strongly influence a target''s activity and/or stability. The regulatory logic governing the steady state of target ubiquitination can consist of nonlinear pathways that involve feedback mechanisms, responses to cellular stimuli such as phosphorylation, and complex cross-regulation between individual components of the ubiquitin conjugation machinery and corresponding DUBs.Cullin-RING ubiquitin E3 ligases (CRLs) comprise the largest class of ubiquitin E3 ligases (30). CRLs contain a substrate specificity receptor that binds the ubiquitinated target and a RING protein that is involved in recruiting an E2-conjugating enzyme, which catalyzes transfer of ubiquitin to the associated substrate through the E3 ligase. RING proteins and particular substrate specificity receptors are brought together by scaffold proteins called Cullins, often through small adaptor proteins that link the Cullin with the receptor. Cullin-1 (Cul-1) CRLs recruit their substrate through F-box proteins, with a Skp family adaptor protein forming a bridge between the Cullin and the F-box. In contrast, CRLs containing Cullin-5 (Cul-5) recognize their substrates through receptor proteins that contain a SOCS-box, which are linked to the Cullin by the Elongin B/Elongin C (EloBC) adaptor complex.In this study we identified the F-box protein Fsn and the SOCS-box protein Gus as in vivo regulators of Vas. Fsn and its Caenorhabditis elegans orthologue are required for normal synaptic development and associate with RING proteins encoded by highwire and rpm-1, respectively (24, 46). Gus was previously shown to interact with Vas and was implicated in its posterior localization (37). Gus features a B30.2/SPRY domain through which it directly interacts with a five-amino-acid motif on Vas (DINNN), and it can be cocrystallized with the EloBC complex (44, 45). Fsn has a B30.2/SPRY domain that is very similar to that of Gus (40% identity). We show experimentally that Fsn also binds the DINNN motif and that Gus associates more stably with Vas than does Fsn. Using genetic methods, we investigated the contributions of Gus and Fsn to regulating Vas activity and deployment.     

Isolation of polar granules and the identification of polar granule-specific protein.

During early embryogenesis in Drosophila melanogaster the posterior polar plasm has the capacity to induce the formation of primordial germ cells. Polar granules, organelles located exclusively in this polar plasm, have been implicated in this determinative capacity. Using cell populations enriched for pole cells as starting material, we have obtained a particulate subcellular fraction that by EM analysis consists predominately of polar granules. The chemical nature of the polar granule has been defined by establishing a strict correlation between the morphological entity and the presence of specific chemical components. We have identified a basic protein with a molecular weight on SDS-polyacrylamide gels of 95,000 daltons that is unique to embryonic cell populations containing pole cells. This protein is enriched specifically in particulate subcellular fractions containing polar granules and is the only major protein species present in preparations in which polar granules are the major morphological constituent. Based on these data, polar granules appear to be composed primarily of one major basic protein species.     

Drosophila valois encodes a divergent WD protein that is required for Vasa localization and Oskar protein accumulation

valois (vls) was identified as a posterior group gene in the initial screens for Drosophila maternal-effect lethal mutations. Despite its early genetic identification, it has not been characterized at the molecular level until now. We show that vls encodes a divergent WD domain protein and that the three available EMS-induced point mutations cause premature stop codons in the vls ORF. We have generated a null allele that has a stronger phenotype than the EMS mutants. The vlsnull mutant shows that vls+ is required for high levels of Oskar protein to accumulate during oogenesis, for normal posterior localization of Oskar in later stages of oogenesis and for posterior localization of the Vasa protein during the entire process of pole plasm assembly. There is no evidence for vls being dependent on an upstream factor of the posterior pathway, suggesting that Valois protein (Vls) instead acts as a co-factor in the process. Based on the structure of Vls, the function of similar proteins in different systems and our phenotypic analysis, it seems likely that vls may promote posterior patterning by facilitating interactions between different molecules.     

Auxin transport in maize roots in response to localized nitrate supply

Background and Aims

Roots typically respond to localized nitrate by enhancing lateral-root growth. Polar auxin transport has important roles in lateral-root formation and growth; however, it is a matter of debate whether or how auxin plays a role in the localized response of lateral roots to nitrate.

Methods

Treating maize (Zea mays) in a split-root system, auxin levels were quantified directly and polar transport was assayed by the movement of [³H]IAA. The effects of exogenous auxin and polar auxin transport inhibitors were also examined.

Key Results

Auxin levels in roots decreased more in the nitrate-fed compartment than in the nitrate-free compartment and nitrate treatment appeared to inhibit shoot-to-root auxin transport. However, exogenous application of IAA only partially reduced the stimulatory effect of localized nitrate, and auxin level in the roots was similarly reduced by local applications of ammonium that did not stimulate lateral-root growth.

Conclusions

It is concluded that local applications of nitrate reduced shoot-to-root auxin transport and decreased auxin concentration in roots to a level more suitable for lateral-root growth. However, alteration of root auxin level alone is not sufficient to stimulate lateral-root growth.