Rolf Reuter

Evidence for the existence of snRNAs U4 and U6 in a single ribonucleoprotein complex and for their association by intermolecular base pairing.

Small nuclear ribonucleoprotein particles (snRNPs) from eucaryotic cells can be fractionated on affinity columns prepared with antibodies of high affinity for 2,2,7‐trimethyl‐guanosine (m3G), which is present in the 5′‐terminal caps of the snRNAs. While the snRNPs U1, U2 and U5 are eluted with the nucleoside m3G in the presence of 0.1 M salt, the snRNP species U4 and U6 are only desorbed when the salt concentration is increased. The same fractionation pattern was likewise observed for snRNPs from HeLa or Ehrlich ascites tumor cells. Since U6 RNA lacks the m3G residue and its RNA does not react with anti‐m3G, its co‐chromatography with U4 RNP on anti‐m3G affinity columns suggests either that discrete snRNPs U4 and U6 are intimately associated in nuclear extracts or that both RNAs are organized in one ribonucleoprotein particle. Further evidence for a U4/U6 RNP particle is obtained by sedimentation studies with purified snRNPs in sucrose gradients. Gel fractionation of RNAs shows identical distributions of snRNAs U4 and U6 in the gradient, and the U4/U6 RNP particle sediments faster than the snRNPs U1 or U2. Physical association between snRNPs U4 and U6 during sedimentation is shown by their co‐precipitation with anti‐m3G IgG from the gradient fractions. Finally, experimental evidence is provided that snRNAs U4 and U6 are associated by intermolecular base pairing in the U4/U6 RNP particle, as demonstrated by our finding that anti‐m3G IgG co‐precipitates U6 RNA with U4 RNA following phenolization of U4/U6 RNPs at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Serpent regulates Drosophila immunity genes in the larval fat body through an essential GATA motif

Insects possess a powerful immune system, which in response to infection leads to a vast production of different antimicrobial peptides. The regulatory regions of many immunity genes contain a GATA motif in proximity to a κB motif. Upon infection, Rel proteins enter the nucleus and activate transcription of the immunity genes. High levels of Rel protein‐mediated Cecropin A1 expression previously have been shown to require the GATA site along with the κB site. We provide evidence demonstrating that the GATA motif is needed for expression of the Cecropin A1 gene in larval fat body, but is dispensable in adult fat body. A nuclear DNA‐binding activity interacts with the Cecropin A1 GATA motif with the same properties as the Drosophila GATA factor Serpent. The GATA‐binding activity is recognized by Serpent‐specific antibodies, demonstrating their identity. We show that Serpent is nuclear in larval fat body cells and haemocytes both before and after infection. After overexpression, Serpent increases Cecropin A1 transcription in a GATA‐dependent manner. We propose that Serpent plays a key role in tissue‐specific expression of immunity genes, by priming them for inducible activation by Rel proteins in response to infection.

Purification of snRNPs U1, U2, U4, U5 and U6 with 2,2,7-trimethylguanosine-specific antibody and definition of their constituent proteins reacting with anti-Sm and anti-(U1)RNP antisera.

Small nuclear ribonucleoprotein particles (snRNPs) of the U‐snRNP class from Ehrlich ascites tumor cells were purified in a one‐step procedure by affinity chromatography with antibodies specific for 2,2,7‐trimethylguanosine (m23.2.7G), which is part of the 5′‐terminal cap structure of snRNAs U1‐U5. Antibody‐bound snRNPs are desorbed from the affinity column by elution with excess nucleoside m23.2.7G; this guarantees maintenance of their native structure. The snRNPs U1, U2, U4, U5 and U6 can be recovered quantitatively from nuclear extracts by this procedure. Co‐isolation of U6 snRNP must be due to interactions between this and other snRNPs, as anti‐m23.2.7G antibodies do not react with deproteinized U6 snRNA. We have so far defined nine proteins of approximate mol. wts. 10 000, 12 000, 13 000, 16 000, 21 000, 28 000, 32 000, 34 000 and 75 000. Purified snRNPs react with anti‐(U1)RNP and with anti‐Sm antisera from patients with mixed connective tissue disease and from MRL/l mice. As determined by the protein blotting technique, six of the snRNP polypeptides, characterized by apparent mol. wts. 13 000, 16 000, 21 000, 28 000, 34 000 and 75 000, bear antigenic determinants for one or the other of the above autoantibody classes. This suggests strongly that the U‐snRNPs produced by the procedure described here are indeed representative of the snRNPs in the cell. With highly purified snRNPs available, investigation of possible enzymic functions of the particles may now be undertaken.

The PDZ-GEF dizzy regulates cell shape of migrating macrophages via Rap1 and integrins in the Drosophila embryo.

In Drosophila embryos, macrophages originate from the cephalic mesoderm and perform a complex migration throughout the entire embryo. The molecular mechanisms regulating this cell migration remain largely unknown. We identified the Drosophila PDZ G-nucleotide exchange factor (PDZ-GEF) Dizzy as a component essential for normal macrophage migration. In mutants lacking Dizzy, macrophages have smaller cellular protrusions, and their migration is slowed down significantly. This phenotype appears to be cell-autonomous, as it is also observed in embryos with a dsRNA-induced reduction of dizzy function in macrophages. In a complementary fashion, macrophages overexpressing Dizzy are vastly extended and form very long protrusions. These cell shape changes depend on the function of the small GTPase Rap1: in rap1 mutants, Dizzy is unable to induce the large protrusions. Furthermore, forced expression of a dominant-active form of Rap1, but not of the wild-type form, induces similar cell shape changes as Dizzy does overexpression. These findings suggest that Dizzy acts through Rap1. We propose that integrin-dependent adhesion is a Rap1-mediated target of Dizzy activity: in integrin mutants, neither Dizzy nor Rap1 can induce cell shape changes in macrophages. These data provide the first link between a PDZ-GEF, the corresponding small GTPase and integrin-dependent cell adhesion during cell migration in embryonic development.

Localization and structure of snRNPs during mitosis. Immunofluorescent and biochemical studies.

The distribution of U snRNAs during mitosis was studied by indirect immunofluorescence microscopy with snRNA cap-specific anti-m3G antibodies. Whereas the snRNAs are strictly nuclear at late prophase, they become distributed in the cell plasm at metaphase and anaphase. They re-enter the newly formed nuclei of the two daughter cells at early telophase, producing speckled nuclear fluorescent patterns typical of interphase cells. While the snRNAs become concentrated at the rim of the condensing chromosomes and at interchromosomal regions at late prophase, essentially no association of the snRNAs was observed with the condensed chromosomes during metaphase and anaphase. Independent immunofluorescent studies with anti-(U1)RNP autoantibodies, which react specifically with proteins unique to the U1 snRNP species, showed the same distribution of snRNP antigens during mitosis as was observed with the snRNA-specific anti-m3G antibody. Immunoprecipitation studies with anti-(U1)RNP and anti-Sm autoantibodies, as well as protein analysis of snRNPs isolated from extracts of mitotic cells, demonstrate that the snRNAs remain associated in a specific manner with the same set of proteins during interphase and mitosis. The concept that the overall structure of the snRNPs is maintained during mitosis also applies to the coexistence of the snRNAs U4 and U6 in a single ribonucleoprotein complex. Particle sedimentation studies in sucrose gradients reveal that most of the snRNPs present in sonicates of mitotic cells do not sediment as free RNP particles, but remain associated with high molecular weight (HMW) structures other than chromatin, most probably with hnRNA/RNP.

A Vertex Model of Drosophila Ventral Furrow Formation

Ventral furrow formation in Drosophila is an outstanding model system to study the mechanisms involved in large-scale tissue rearrangements. Ventral cells accumulate myosin at their apical sides and, while being tightly coupled to each other via apical adherens junctions, execute actomyosin contractions that lead to reduction of their apical cell surface. Thereby, a band of constricted cells along the ventral epithelium emerges which will form a tissue indentation along the ventral midline (the ventral furrow). Here we adopt a 2D vertex model to simulate ventral furrow formation in a surface view allowing easy comparison with confocal live-recordings. We show that in order to reproduce furrow morphology seen in vivo, a gradient of contractility must be assumed in the ventral epithelium which renders cells more contractile the closer they lie to the ventral midline. The model predicts previous experimental findings, such as the gain of eccentric morphology of constricting cells and an incremental fashion of apical cell area reduction. Analysis of the model suggests that this incremental area reduction is caused by the dynamical interplay of cell elasticity and stochastic contractility as well as by the opposing forces from contracting neighbour cells. We underpin results from the model through in vivo analysis of ventral furrow formation in wildtype and twi mutant embryos. Our results show that ventral furrow formation can be accomplished as a “tug-of-war” between stochastically contracting, mechanically coupled cells and may require less rigorous regulation than previously thought. Summary For the developmental biologist it is a fascinating question how cells can coordinate major tissue movements during embryonic development. The so-called ventral furrow of the Drosophila embryo is a well-studied example of such a process when cells from a ventral band, spanning nearly the entire length of the embryo, undergo dramatic shape change by contracting their tips and then fold inwards into the interior of the embryo. Although numerous genes have been identified that are critical for ventral furrow formation, it is an open question how cells work together to elicit this tissue rearrangement. We use a computational model to mimic the physical properties of cells in the ventral epithelium and simulate the formation of the furrow. We find that the ventral furrow can form through stochastic self-organisation and that previous experimental observations can be readily explained in our model by considering forces that arise when cells execute contractions while being coupled to each other in a mechanically coherent epithelium. The model highlights the importance of a physical perspective when studying tissue morphogenesis and shows that only a minimal genetic regulation may be required to drive complex processes in embryonic development.

A dual role for the βPS integrin myospheroid in mediating Drosophila embryonic macrophage migration.

Summary Throughout embryonic development, macrophages not only act as the first line of defence against infection but also help to sculpt organs and tissues of the embryo by removing dead cells and secreting extracellular matrix components. Key to their function is the ability of embryonic macrophages to migrate and disperse throughout the embryo. Despite these important developmental functions, little is known about the molecular mechanisms underlying embryonic macrophage migration in vivo. Integrins are key regulators of many of the adult macrophage responses, but their role in embryonic macrophages remains poorly characterized. Here, we have used Drosophila macrophages (haemocytes) as a model system to address the role of integrins during embryonic macrophage dispersal in vivo. We show that the main &bgr;PS integrin, myospheroid, affects haemocyte migration in two ways; by shaping the three-dimensional environment in which haemocytes migrate and by regulating the migration of haemocytes themselves. Live imaging revealed a requirement for myospheroid within haemocytes to coordinate the microtubule and actin dynamics, and to enable haemocyte developmental dispersal, contact repulsion and inflammatory migration towards wounds.

Expression, function and regulation of Brachyenteron in the short germband insect Tribolium castaneum.

T-domain transcription factors are involved in many different processes during embryogenesis, such as mesoderm, heart or gut development in vertebrates and in invertebrates. In insects, the following five types of T-box genes are known: brachyenteron (byn), optomotor-blind (omb), optomotor-blind-related-gene-1 (org-1), dorsocross (doc) and H15. As all these classes are present in the genome of the fruit fly Drosophila melanogaster and the flour beetle Tribolium, the multiplicity of the five types of genes varies from dipterans to the beetle. In higher dipterans, a small cluster of three doc genes (doc1–doc3) exists, while the Tribolium genome contains a single Tc-doc gene only. Two H15 genes, Tc-H15a and Tc-H15b, are present in the Tribolium genome compared to a single H15 gene in Drosophila. We have analysed the expression and function of the Tribolium brachyenteron ortholog (Tc-byn). During embryogenesis, Tc-byn is exclusively expressed in the growth zone of the extending germband and later becomes confined to the distal proctodeum and the hindgut, a situation that parallels the expression pattern of byn in Drosophila. Tc-byn-RNAi treated embryos phenocopy Drosophila byn mutants and form no hindgut. In addition, we have characterised a regulatory element upstream of the Tc-byn transcription start site that confers specific gene expression in the developing hindgut of the Drosophila embryo. Our results demonstrate a highly conserved role for Brachyury-type transcriptional regulators in posterior gut development of insects at the level of expression, function and regulation.

The PDZ-GEF protein Dizzy regulates the establishment of adherens junctions required for ventral furrow formation in Drosophila

Summary The PDZ-GEF protein Dizzy (Dzy) and its downstream GTPase Rap1 have pleiotropic roles during development of the Drosophila embryo. Here, we show that maternally provided Dzy and Rap1 first function during ventral furrow formation (VFF) where they are critical to guarantee rapid apical cell constrictions. Contraction of the apical actomyosin filament system occurs independently of Dzy and Rap1, but loss of Dzy results in a delayed establishment of the apical adherens junction (AJ) belt, whereas in the absence of Rap1 only a fragmentary apical AJ belt is formed in the epithelium. The timely establishment of apical AJs appears to be essential for coupling actomyosin contractions to cell shape change and to assure completion of the ventral furrow. Immediately after VFF, the downregulation of Dzy and Rap1 is necessary to allow normal mesodermal development to continue after the epithelial-to-mesenchymal transition, as overexpression of Dzy or of constitutively active Rap1 compromises mesodermal migration and monolayer formation. We propose that Dzy and Rap1 are crucial factors regulating the dynamics of AJs during gastrulation.

The T-related gene (Trg), a Brachyury homologue in insects

The T-related gene (Trg) of Drosophila is a member of the T-box gene family and displays a high degree of similarity to the vertebrate Brachyury genes. Trg acts down-stream of the terminal gap genes tailless and huckebein and is required for the development of a particular organ, the hindgut. It is expressed throughout embryogenesis, first at the end of the blastoderm stage in the primordium of the hindgut close to the posterior pole of the embryo, then in the differentiating hindgut during gastrulation and organogenesis. A corresponding expression pattern of Trg homologues in the developing hindgut of short germ insects implies that the function of Trg in gut development has been highly conserved during evolution. This conservation in insects and the high similarity between Trg and the chordate Brachyury genes allows speculations about a link between gut parts of insects and notochord of chordates.